ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

Abbildung auf dem Umschlag:
Das Titelbild zeigt eine symbolische Darstellung des
“funktionalen Genomik-Ansatzes” am Beispiel von
Mycoplasma pneumoniae. Kolorierte elektronenmikroskopische
Aufnahmen von Mycoplasma pneumoniae
bilden den Hintergrund. Die Genkarte im Zentrum
mit farbkodierten Genfunktionen und deren zirkuläre
Darstellung in der rechten oberen Ecke bilden
den Ausgangspunkt für die Transkriptionsanalyse mittels
Mikro-Array-Technik (mitte rechts) und die Proteomik
(unten rechts). Ein Ausschnitt aus einem zweidimensionalen
Gel, integriert in ein Massenspektrum,
stellen den Ansatz der Proteom-Analyse dar.
Cover illustration:
The cover depicts an illustrative representation of the
functional genomics approach on Mycoplasma pneumoniae.
The background is showing electromicrographs
of Mycoplasma pneumoniae cells. The genemap
in the center with a colour coding for gene functions
and in the right corner a segment from a circular
gene-map are both serving as basis for transcription
analysis by micro-array (center right) and for proteomics
(bottom right). The proteomics approach is
represented by a two-dimensional gel integrated in a
mass spectrum, showing partial sequence of a tryptic
peptide.
J. Regula, C.-U. Zimmermann, Y. Cully
and R. Herrmann
Zentrum für Molekulare Biologie • Universität Heidelberg (ZMBH)
ZMBH
Im Neuenheimer Feld 282
Postfach 10 62 49
D-69120 Heidelberg
Telefon (0 62 21) 54 68 00
Telefax (00 49 - 62 21) 54 55 07
Internet http://www.zmbh.uni-heidelberg.de
Report 2000

ZMBH Report 2000
Verantwortlich: Prof. Dr. Dr. h.c. Konrad Beyreuther
Redaktion: Prof. Dr. Dr. h.c. Konrad Beyreuther
Dr. H. P. Blaschkowski
Gerlind Güth-Köhler
Gesamtherstellung: Yves Cully
2
Inhaltsverzeichnis - Table of Contents
Forschungsgruppenleiter, Direktorium 6
Wissenschaftlicher Beirat - Scientific Advisory Board 7
Vorbemerkungen des Direktors - Introductory Remarks of the Director 8
Research Reports 23
Konrad Beyreuther Molecular Neurobiology of Alzheimer's Disease 25
Gerd Multhaup 1 Ligand-Dependent Functions of the Amyloid Precursor Protein 40
(APP) and Ligand-Associated Conformational Changes
Hermann Bujard I. Expanding the Applicability of the Tet Regulatory Systems 48
II. The Merozoite Surface Protein 1 of the Human Malaria
Parasite Plasmodium falciparum
Christine Clayton Molecular Cell Biology of Trypanosomes 54
Bernhard Dobberstein Protein Targeting and Intracellular Sorting 61
Dirk Görlich Nucleocytoplasmic Transport 67
Richard Herrmann Molecular Biology of the Bacterium Mycoplasma pneumoniae 74
Ralf-Peter Jansen Asymmetric Cell Division and RNA Transport in Yeast 80
Stefan Jentsch Ubiquitin-Dependent Proteolysis 84
Jörg Höhfeld 1 Function and Regulation of the Mammalian Chaperone Hsc70 88
Klaus-Armin Nave Myelin Genetics and Developmental Neurobiology 91
Renato Paro Chromatin-Controlled Epigenetic Regulation of Transcription 97
3

Frank Sauer Mechanisms of Transcriptional Regulation 104
Hans Ulrich Schairer I. Stigmatella aurantiaca, a Prokaryotic Organism for Studying 109
Intercellular Signalling and Morphogenesis
II. Molecular Biology of the Infection Process by the Entomo-
pathogenic Fungus Beauveria bassiana
Heinz Schaller Regulation of Hepatitis B Virus Replication 116
Percy Knolle 1 Regulation of the Immune Response in the Liver 125
Blanche Schwappach Quality Control of Ion Channels and ABC Proteins 129
Dominique Soldati-Favre Cell and Molecular Biology of the Obligate Intracellular Parasite 131
Toxoplasma gondii
_____________
1 Project Group
Central Facilities 139
ZMBH-Lehrprogramm im Grund- und Hauptstudium 148
Studienprogramm für Graduierte des ZMBH 152
Anhang - Appendix 157
ZMBH-Colloquia, Seminars and Cell Biology Lectures 1998/99 – Invited Speakers 158
Administrative and Technical Staff 164
ZMBH-Budget 1999 165
4

Forschungsgruppenleiter
Konrad Beyreuther, Prof. Dr. Dr. h.c.
Tel.: 06221-546845, Fax: 06221-545891
Hermann Bujard, Prof. Dr. Dr. h.c
Tel.: 06221-548215, Fax: 06221-545892
Christine Clayton, Prof. Dr.
Tel.: 06221-546876, Fax: 06221-545894
Bernhard Dobberstein, Prof. Dr.
Tel.: 06221-546825, Fax: 06221-545892
Dirk Görlich, Dr.
Tel.: 06221-545884, Fax: 06221-545892
Richard Herrmann, Prof. Dr.
Tel.: 06221-546827, Fax: 06221-545893
Ralf-Peter Jansen, Dr.
Tel.: 06221-546869, Fax: 06221-545894
Stefan Jentsch, Prof. Dr. 1)
Klaus-Armin Nave, Prof. Dr. 2)
Renato Paro, Prof. Dr.
Tel.: 06221-546878, Fax: 06221-545891
Frank Sauer, Dr.
Tel.: 06221-546858, Fax: 06221-545894
Hans Ulrich Schairer, Prof. Dr.
Tel.: 06221-546880, Fax: 06221-545893
Heinz Schaller, Prof. Dr.
Tel.: 06221-546885, Fax: 06221-545893
Dominique Soldati-Favre, Dr.
Tel.: 06221-546870, Fax: 545892
Projektgruppenleiter
Jörg Höhfeld, PD Dr. 3)
Percy Knolle, PD Dr.
Tel.: 06221-546815, Fax: 06221-545893
Gerd Multhaup, PD Dr.
Tel.: 06221-546849, Fax: 06221-545891
Direktorium
Prof. Dr.Dr. h.c. Konrad Beyreuther (Direktor)
Prof. Dr. Klaus-Armin Nave (1. Stellvertreter)
Prof. Dr. Bernhard Dobberstein (2. Stellvertreter)
Sekretariat: Dr. Hans Peter Blaschkowski
Gerlind Güth-Köhler
Tel.: 06221-546850, Fax: 06221-545507
1) Neue Adresse: Max-Planck-Institut für Biochemie,
Am Klopferspitz 18a, 82152 Martinsried,
Tel.: 089-8578-3000/9, Fax: 089-8578-3011/3022
2) Neue Adresse: Max-Planck-Institut für Experimentelle
Medizin, Abt. Neurogenetik, Herrmann-Rein-
Str. 3, 37075 Göttingen, Tel.: 0551-3899757,
Fax: 0551-3899758
3) Neue Adresse: Max-Planck-Institut für Biochemie,
Abt. Molekulare Zellbiologie, Am Klopferspitz
18a, 82152 Martinsried, Tel. : 089-8578-3027,
Fax: 089-8578-3022
Wissenschaftlicher Beirat – Scientific Advisory Board
Prof. Dr. Volkmar Braun
Institut für Biologie II
Universität Tübingen
Prof. Dr. Walter Doerfler
Institut für Genetik
Universität Köln
Prof. Dr. Kurt von Figura
Abteilung Biochemie II
Universität Göttingen
Prof. Dr. Ari Helenius
Lab. for Biochemistry
Swiss Federal Institute
of Technology Zürich
Prof. Dr. Peter Herrlich
Institut für Genetik und Toxikologie
von Spaltstoffen
Forschungszentrum Karlsruhe
Prof. Dr. Harvey Lodish
Whitehead Institute
for Biomedical Research
Cambridge, MA
Prof. Dr. Richard Losick
Dept. of Cellular & Developmental Biology
Harvard University
Cambridge, MA
Prof. Dr. Siegfried Neumann (Industry)
Fa. E. Merck
Forschung DIAG
Darmstadt
Prof. Dr. Christiane Nüsslein-Volhard
Max-Planck-Institut
für Entwicklungsbiologie
Tübingen
Prof. Dr. Klaus Rajewsky
Institut für Genetik
Universität Köln
Prof. Dr. Martin Schwab
Institut für Hirnforschung
Universität Zürich
6 7

Vorbemerkungen des Direktors
In den fünfzehn Jahren seines Bestehens hat das
ZMBH gezeigt, daß es möglich ist, erfolgreich die
Aufgaben zu übernehmen, gleichzeitig Forschung
auf hochkompetitiven Gebieten der Molekular- und
Zellbiologie zu betreiben, Studenten aus- und junge
Wissenschaftler weiterzubilden und unabhängigen
Nachwuchsgruppen jede Förderung zu geben. Als Universitätsinstitut
gegründet, liegt der Schwerpunkt der
Aufgaben des ZMBH in der Verbindung von exzellenter
Grundlagenforschung mit ebensolchen Lehrprogrammen
für Studierende auf allen Stufen ihrer
Ausbildung. Die Symbiose zwischen Forschung und
Lehre, glaube ich heute feststellen zu können, haben
wir früh erreicht. Bereits drei Jahre nach seiner Gründung
führte das ZMBH ein eigenes für seine Diplomanden
und Doktoranden obligatorisches Studienprogramm
für Graduierte ein.
Wer das Treppenhaus zu den Labors des ZMBH
betritt, wird an der Tür oftmals ein Poster finden, mit
dem einer unserer Wissenschaftler einen Methodenkurs
für unsere Graduierten ankündigt. Da das ZMBH
als Department eingerichtet wurde, in dem alle seine
Einrichtungen von den Wissenschaftlern gleichberechtigt
benutzt werden können, ist die Vermittlung von
Technologien an unsere Studenten und Mitarbeiter
eine unserer Aktivitäten, um der Herausforderung in
der Biologie, Biomedizin und Biotechnologie beim
Start in das neue Jahrtausend zu begegnen. Die Entwicklung
der modernen Biologie hängt in steigendem
Maße von Methoden ab und steht auch in steigendem
Maße im Austausch mit der Biotechnologie und Medizin,
nicht nur mit Bezug auf die Anwendung neuer
grundlegender Erkenntnisse, sondern auch mit neuen
Methoden und Fragestellungen. Als Vorsitzender
8
des Vereins „BioRegion Rhein-Neckar-Dreieck, e.V.“
erfahre ich beinahe täglich die Bedeutung dieses engen
Dialogs, der für die Förderung des Transfers zwischen
Grundlagenforschung und ihrer innovativen Anwendung
in der Medizin und Biotechnologie notwendig
ist.
Die größte Herausforderung in der Biologie und Biomedizin
besteht derzeit darin, daß die vollständigen
Genomsequenzen von mehr als einem Dutzend Prokaryonten,
von Hefe, von Caenorhabditis elegans und
von Drosophila melanogaster aufgeklärt wurden, aber
die darin enthaltene Information damit noch nicht
dekodiert ist. Die vollständigen Genomsequenzen weiterer
multizellulärer Eukaryonten, einschließlich der
des Menschen, werden bald bekannt sein. Der erste
deutsche Beitrag, die vollständige Genomsequenz von
Mycoplasma pneumoniae, kam übrigens von unserem
Kollegen Richard Herrmann. Die Genomik wird von
ihrer weitgehend deskriptiven Phase des Sequenzierens,
die wohl nur noch fünf bis zehn Jahre andauern
wird, zur nächsten Ebene des Verstehens, wie Gene
funktionieren und zusammenwirken, übergehen. Hierfür
müssen Methoden entwickelt werden, mit denen
aufgeklärt werden kann, wie die im Genom kodierte
Information in biologische Prozesse in Zellen, Organen
und ganzen Organismen übersetzt wird. Um diesen
Herausforderungen auf der Ebene der Technologien
und Methodologien zu begegnen, wird das ZMBH als
flexible Organisation seinen Wissenschaftlern wichtige
neue Dienstleistungen in Form der bewährten
Service-Einheiten zur Verfügung stellen. Gegenwärtig
werden folgende neue Technologien und Methoden in
unserem Institut etabliert: Hochleistungs-Mikroskopie
als neue zentrale Einrichtung, Massenspektroskopie
Introductory Remarks of the Director
In the fifteen years of its existence, the ZMBH has
proven that it is possible to successfully address the
mission to conduct research in the forefront in highly
competitive fields of molecular and cellular biology, to
educate students, to train young scientists and to give
full support to independent junior groups. Because it
was designed as a university institute, the focus of the
ZMBH is to combine basic research with excellent
education programs for students at all levels. Three
years after its inauguration, the ZMBH introduced its
own graduate program obligatory for its diploma and
doctoral students.
If you enter the staircase leading to the laboratories,
you may encounter at the door a poster advertising
a practical course held for our graduate students by
one of our faculty members. Because the ZMBH was
designed as a department in which resources are open
to the ZMBH faculty, transferring technology to our
students and coworkers is just one of our activities to
meet with the challenge in biology, biomedicine and
biotechnology at the start of this new millennium. The
development of modern biology increasingly depends
on methods and is also increasingly in exchange with
biotechnology and medicine, not only in regard to the
application of discoveries in basic science but also
by the provision of methods and scientific problems.
As chairman of the BioRegion Rhein-Neckar-Triangle
association, I experience almost daily the importance
of having a close dialogue to exchange information
between fundamental research and its innovative
application in medicine and biotechnology industry.
One of the major discoveries of the past years driving
the progress in biology and biomedicine is the fact that
we now know the complete genomic sequences for
more than a dozen prokaryotic organisms as well as
that of yeast, Caenorhabditis elegans and Drosophila
melanogaster. The first contribution of Germany, the
complete genomic sequence of Micrococcus pneumoniae,
came from our colleague Richard Herrmann.
The full genomic sequences of further multicellular
eukaryotes, including humans will soon be known.
Genomics will be passing from a largely descriptive
phase of genomic sequencing, that may only last for
another five to ten years, to the next level of understanding
of how genes function and interact. For this,
methods need to be developed which allow to uncover
how the information encoded in the genome is translated
into biological processes within cells, organs and
complete organisms. To meet the challenges at the
level of technologies and methodologies, the ZMBH
as a flexible organization continues to provide to its
faculty essential novel services through its powerful
infrastructure of central service units. New technologies
and methods are being presently set up at our
institute: advanced light microscopy as a new central
service unit, mass spectrometry for protein identification
and sequencing within the existing biomolecular
chemistry unit, advanced cell sorting for the selection
and isolation of rare genotypes of transfected eukaryotic
cells and a transgenic Drosophila service as part
of our animal house facility. We are currently discussing
the instalment of a DNA microarray technology
service unit as part of biomolecular chemistry. Microarray
technology that detects mRNA promises to measure
expression of genomes in cells in response to
internal or external signals and is therefore of interest
to most of the ZMBH faculty. To meet with the laboratory
space demand caused by the new central services,
we will expand and build space for offices on top of
9

zur Proteinidentifizierung und -sequenzierung in der
bestehenden Service-Einheit Biomolekulare Chemie,
hochentwickeltes Cell Sorting zur Anreicherung und
Isolierung seltener Genotypen in transfizierten Zellen,
und der Service transgener Drosophila als Abteilung
unserer Versuchstierhaltung. Aktuell diskutieren wir
die Einrichtung der DNA-‘Microarray‘-Technologie
als Teil unserer zentralen Einheit Biomolekulare
Chemie. Mit der vielversprechenden ‚Microarray‘-
Technologie zur Detektion von mRNA können die
Expressionsmuster von Genomen in Zellen als Antwort
auf interne und externe Signale gemessen werden.
Dies ist von zentralem Interesse für die meisten Forschungsgruppen
des ZMBH. Um dem Laborflächenbedarf
der neuen zentralen Dienste zu entsprechen,
werden wir das Institutsgebäude mit einem Dachpavillon
aufstocken, um Platz für Büros zu schaffen. Das
Büro des Direktors und solche zentralen Einheiten,
die keine voll installierten Laborflächen brauchen, die
bisher aber noch in solchen untergebracht sind (Biocomputing
und Dokumentation), werden in das neue
fünfte Stockwerk hinaufziehen - hoffentlich im Herbst
nächsten Jahres. Das ZMBH ist glücklicherweise in
der Lage, mit der Unterstützung von zwei seiner Professoren,
den größten Teil der Baukosten mit Mitteln
seines ‚overheads‘ zu finanzieren.
In Hinsicht auf seine wissenschaftliche Produktivität,
seine Lehraktivitäten und seine Berufungspolitik im
Zeitraum dieses Berichts hat das ZMBH bewiesen,
daß es eine flexible Universitätseinrichtung mit kompromißlosen
Qualitätsstandards ist, daß es fähig ist,
aktuelle Forschungsthemen und Lehrinhalte zu implementieren.
Das ZMBH als ‚center of exzellence‘ kann
sicherlich auch als eine universitäre Modelleinrichtung
bezeichnet werden, das in der Lage ist, den Herausforderungen
der modernen Biologie wissenschaftlich
zu entgegnen. Bisherige Bilanz:
10
Wissenschaftliche Leistungen – wie von unseren
Forschungsgruppenleitern in diesem und den vorausgegangenen
Jahresberichten dargestellt, waren viele
Höhepunkte und wirkliche Durchbrüche zu verzeichnen.
Für mich wurden die bemerkenswertesten erreicht
auf den Gebieten der Kontrolle der eukaryontischen
Genexpression unter physiologischen und artifiziellen
Bedingungen (Tetrazyklin-System), der Chromatin
induzierten epigenetischen Kontrolle der Genexpression
in Drosophila und Mammalia, des Targeting und
innerzellulären Sortings von Molekülen einschließlich
des Transports zwischen Kern und Cytoplasma, der
Ubiquitin-abhängigen Proteolyse, der Funktion und
Regulation von Chaperonen, der Neurobiologie der
erregenden Neurotransmission, der Myelin-Genetik
und der Neurodegeneration sowie der Wirt-Parasiten/
Virus Interaktionen.
Modell ZMBH – mit dem Einführen neuer Strukturen
für ein Universitätsinstitut vor 15 Jahren – in dem
Grundlagenforschung in kleinen Gruppen betrieben
wird, die effizient durch eine Infrastruktur gemeinsam
genutzter leistungsfähiger wissenschaftlicher, technischer
und administrativer Dienste unterstützt werden
(rund 50% unserer Finanzmittel vom Land Baden-
Württemberg werden hierfür verwendet und nur die
andere Hälfte dieser Mittel wird den einzelnen Forschungsgruppen
direkt zur Verfügung gestellt) – jetzt
übernommen von einem Zentrum der Universität
Tübingen und zwei neuen Zentren in Heidelberg
Umfassende Reform des Curriculums der Fakultät
für Biologie, die vom ZMBH initiiert und entwickelt
wurde, und an die sich jetzt eine erfolgreiche Initiative
zur Einführung eines internationalen Bachelor- und
Master-Studienganges für Molekulare und Zellbiologie
anschließt
Hervorragende Ausbildung und Unterstützung
unserer Diplomanden, Doktoranden, Postdocs, For-
the ZMBH. The office of the director and of those
central units that do not need but presently use fully
equipped lab space (biocomputing and documentation)
will be moved to the new fifth floor, hopefully by
next fall. The ZMBH is fortunate to be able to cover
most of the building costs using its overhead money
and with the financial help of two senior faculty members.
Regarding its scientific output, teaching activities and
hiring policy for the period covered by this report, the
ZMBH has proven to be a flexible university institution
with uncompromising standards of quality, continuing
adaptation to changing needs and international
recognition as a center of excellence and for its added
value that include:
Scientific achievements, as documented by the
research reports of our group leaders in this issue
and previous reports, we had many highlights and
real break-throughs. For me, the most notable being
achieved in the field of control of eucaryotic gene
expression under physiological and artificial conditions
(tetracyclin system), of chromatin-induced epigenetic
control of gene expression in Drosophila and
mammals, protein targeting and intracellular sorting
including transport between nucleus and cytoplasm,
ubiquitin-dependent proteolysis, function and regulation
of chaperons, neurobiology of excitatory neurotransmission,
myelin genetics and neurodegeneration
and of host-parasite/virus interactions.
Conceptual leadership in introducing a new structure
for an university institute 15 years ago - in which the
research done by small groups is efficiently supported
by an efficient infrastructure with jointly used scientific,
technical and administrative service (approximately
50% of our funds from the State of Baden-
Wuerttemberg is allocated to it and only the other half
of these funds is passed on directly to the individual
groups) - now adopted by one center at the University
of Tuebingen and two new centers in Heidelberg
Initiation of a distinct, revised curriculum of the
faculty for biology, conceived by the ZMBH is now
being successfully followed by plans to inaugurate an
international curriculum of a bachelors and masters
program in Molecular and Cellular Biology
Outstanding training and career possibilities of
diploma students, doctoral students, and postdoctoral
fellows, group leaders and visitors – alone five former
group leaders of the ZMBH have moved to Max-
Planck-Institutes as directors
The period of 1998 – 2000
In the past 32 months the ZMBH has seen an unprecedented
turnover among its group leaders. Stefan
Jentsch and Klaus Nave moved to Max-Planck-Institutes
in Munich and Goettingen, respectively. Our
“founding members” Hermann Bujard and Heinz
Schaller reached the official retirement age and are
now no longer allowed to sign official documents, a
discrimination that has been overcome some time ago
in other countries. As documented by their reports,
Hermann Bujard and Heinz Schaller have both been
extremely successful with their projects and publications.
We highly appreciate that both scientists continue
doing their research and teaching at the ZMBH
as usual. To enable this, we changed our bylaws and
created what we call the “Emeritus Regelung”, which
allows retired group leaders to continue research as
long as they obtain external funding and their projects
are accepted by the ZMBH’s scientific advisory board
and faculty. Contracts are for one to three years and
renewable.
Another pleasant development is that Renato Paro was
11

schungsgruppenleiter und Gastwissenschaftler - allein
fünf frühere Gruppenleiter des ZMBH sind als Direktoren
an Max-Planck-Institute übergewechselt.
Zum Berichtszeitraum 1998 - 2000
In den vergangenen 32 Monaten sah das ZMBH einen
beispiellosen Personalwechsel bei seinen Gruppenleitern.
Stefan Jentsch und Klaus-Armin Nave wechselten
zu Max-Planck-Instituten in München und Göttingen.
Unsere „Gründungsmitglieder“ Hermann Bujard
und Heinz Schaller erreichten das offizielle Pensionsalter
und dürfen jetzt keine offiziellen Dokumente mehr
unterzeichnen, eine Diskriminierung, die in anderen
Ländern vor einiger Zeit abgeschafft wurde. Wie in
ihren Forschungsberichten dokumentiert, waren Hermann
Bujard und Heinz Schaller beide weiterhin
außerordentlich erfolgreich mit ihren Forschungsprojekten
und Veröffentlichungen. Wir schätzen es
sehr, daß Beide ohne weitere Einschränkungen ihre
Forschung und Lehre am ZMBH fortsetzen. Hierfür
haben wir unsere Institutssatzung geändert und eine
spezielle, wie wir sie nennen „Emeritus-Regelung“
geschaffen. Letztere ermöglicht es Gruppenleitern im
Ruhestand, ihre Forschungsarbeiten weiterzuführen,
solange sie hierfür Drittmittel einwerben können und
vom Wissenschaftlichen Beirat und vom Kollegium
des ZMBH hierfür die Zustimmung erhalten. Diese
wird befristet für 1 - 3 Jahre gegeben und ist verlängerbar.
Eine zukunftsweisende erfreuliche Entwicklung wurde
vergangenen Dezember mit der Berufung von Renato
Paro als Nachfolger von Stefan Jentsch eingeleitet.
Zum Zeitpunkt, an dem dieser Bericht geschrieben
wird, haben wir noch drei freie Professuren. Wir rechnen
damit, daß zwei dieser Professuren noch im Jahr
2000 wiederbesetzt werden, und wir sind optimistisch,
daß die Dritte kurz darauf besetzt werden kann.
12
Im Jahre 2000 ist Blanche Schwappach von der Universität
Californien, San Francisco einem Ruf als neue
Nachwuchsgruppenleiterin ans ZMBH gefolgt. Ihre
innovativen Arbeiten über ER-Trafficking-Signale, die
eine essentielle Kontrollfunktion für ein Plasmamembran-Kanalprotein
haben, überzeugten uns bei der Auswahl
unter mehr als 50 Bewerbern. Blanche Schwappachs
Forschungsthematik auf dem Gebiet der Regulation
des intrazellulären Transports paßt gut in unser
Forschungsprogramm. Wir sind davon überzeugt, daß
ihre experimentellen Strategien von unserer Expertise
im Bereich der molekularen Zellbiologie profitieren
werden und daß wir ihre Expertise bei der Anwendung
ausgefeilter Methoden des ‚Cell Sortings‘ zur Selektion
interessanter, selten vorkommender Genotypen/
Phenotypen transfizierter eukaryontischer Zellen gut
nutzen werden.
appointed as successor of Stefan Jentsch last December.
At the time of writing, we have three open tenure
positions. We expect two of the three positions to be
filled during 2000 and we are optimistic that the third
professorship will follow soon after.
In July 2000, we hired Blanche Schwappach from the
University of California, San Francisco as a new junior
group leader. Her innovative studies of ER trafficking
signals serving an essential quality control function
for a plasma membrane channel protein made us select
her from over 50 applicants. Blanche Schwappach’s
research focus on the regulation of intracellular transport
mechanism fits well into our research program.
We are confident that her experimental strategies will
benefit from our expertise in molecular cell biology,
and that we will benefit from her expertise in using
sophisticated cell sorting for the selection of interesting
rare genotypes/phenotypes of transfected eukaryotic
cells.
Due to their outstanding achievements in research and
teaching, Gerd Multhaup and Percy Knolle were promoted
to “project group leader”. The host for both
is currently the director. The status of “project group
leader” at the ZMBH as anchored to our bylaws,
allowing the ZMBH to advance successful young scientists
to formal independence, when justified by their
achievements. Further requirements are independent
research, grant support, the German “habilitation” and
formal approval by the ZMBH‘s faculty. The appointed
project group leader receives lab space and financial
support from our state budget but stays associated with
one of the permanent groups for organizational purposes.
Considerable financial investments have again been
allocated to our infrastructure. We are proud of the
achievements of our transgenic unit under its experienced
and skilled head Juergen Weiss. The remarkable
success of Frank Zimmermann and Domenico Basta to
produce transgenic founders is acknowledged not only
in the ZMBH. The latest addition to the animal facility
is the service for transgenic flies that was installed
to meet the increasing demands of several groups. The
biomedical chemistry unit received a new Q-TOF mass
spectrometer suited for proteomics from the Deutsche
Forschungsgemeinschaft. We are indebted to Richard
Herrmann for acting as interim head of this unit
after Rainer Frank‘s departure. Thomas Ruppert will
become the new head effective October 2000. He is
experienced in proteomics and worked previously at
the Institute for Biochemistry, Charité, Berlin. The
unit for high-resolution microscopy is currently being
set up on the third floor with the assistance of Axel
Baumm. It will be moved to laboratories on the first
floor, currently being used as the director‘s office as
13

In Anerkennung ihrer hervorragenden Forschung und
Lehre wurden Gerd Multhaup und Percy Knolle zum
„Projektgruppenleiter“ ernannt. Der ‚host‘ für beide
ist gegenwärtig der Direktor. Der Status eines „Projektgruppenleiters“,
wie er in den Institutsregelungen
festgelegt ist, erlaubt es dem ZMBH, erfolgreichen
jungen Wissenschaftlern formale Unabhängigkeit zu
geben, wenn der Fortschritt ihrer wissenschaftlichen
Arbeiten dies zuläßt. Weitere Voraussetzungen sind
unabhängige Forschungsvorhaben, projektbezogene
Drittmittel, die Habilitation und die formelle Zustimmung
des ZMBH-Kollegiums. Der ernannte Projektgruppenleiter
erhält eigenen Laborraum und eigene
Institutsmittel, bleibt aus organisatorischen Gründen
aber mit einer der permanenten Gruppen assoziiert.
Erhebliche Investitionsmittel wurden wieder für unsere
Infrastruktur aufgewendet. Wir sind stolz auf die
exzellenten Leistungen unserer Transgenen Einheit
unter der erfahrenen und fachmännischen Leitung von
Jürgen Weiß. Die bemerkenswerten Erfolge von Frank
Zimmermann und Domenico Basta bei der Herstellung
transgener Stämme werden nicht nur vom ZMBH
hoch geschätzt. Die jüngste Erweiterung der Versuchstierhaltung
ist der Service für transgene Fliegen, der
etabliert wurde, um dem anwachsenden Bedarf mehrerer
Gruppen nachzukommen. Die Einheit Biomolekulare
Chemie erhielt von der Deutschen Forschungsgemeinschaft
ein spezielles Q-TOF-Massenspektrometer
für die Proteomik. Wir sind Richard Herrmann dankbar,
daß er die zentrale Einheit vertretungsweise leitet,
seit Rainer Frank ausgeschieden ist. Thomas Ruppert
wird im Oktober 2000 die Leitung übernehmen; er hat
zuvor am Institut für Biochemie, Charité, Berlin gearbeitet
und bringt große Erfahrung im Bereich der Proteomik
mit. Die Einheit für hochauflösende Mikroskopie
wird jetzt zunächst im 3. Stockwerk mit der
Assistenz von Axel Baumm eingerichtet. Sie wird in
14
Laborräume im 1. Stockwerk, die zur Zeit noch als
Sekretariat des Direktors genutzt werden, umziehen,
sobald dieses in den neuen Dachpavillon einziehen
kann. Wie zuvor bemerkt, sind weitere Service-Einheiten
für DNA-‘Microarray‘-Technologie und für quantitative
PCR geplant. Der Leiter der Verwaltung, Jürgen
Auer und sein Team haben erfolgreich ein den spezifischen
Anforderungen der Buchhaltung des ZMBH
angepaßtes EDV-Programm eingeführt. Hiermit konnte
die Effizienz und Geschwindigkeit der Verwaltungsdienste
weiter erhöht werden. Ohne die EDV-Abteilung
unter der Leitung von Raphael Mosbach gäbe es
keine effektive und aktuelle Bioinformatik im ZMBH.
In diesem Zusammenhang sind mit Anerkennung die
essentielle Zuarbeit unserer von Matthias Pawlitschko
und Gert Stegmüller geleiteten Elektro- und Mechanikwerkstätten
aufzuführen. Die hohe Priorität, die
das Biocomputing bei uns hat, erlaubt es dem ZMBH
jetzt, mit einem Lehrprogramm für Bioinformatik zu
beginnen, das unsere Studenten in die „Exploration“
von Datenbanken einführt, um Struktur und Funktion
aus Sequenzen vorherzusagen, oder Einsicht in die
Evolution von intra- und interspezies Sequenzvariationen
und ihren Folgen zu gewinnen. Die exzellenten
graphischen Arbeiten von Yves Cully, von der Dokumentationsabteilung
bereitgestellt, haben entscheidend
zum Erfolg bei vielen Vorträgen und bei der Präsentation
von Postern durch ZMBH-Mitglieder auf
Tagungen und Ausstellungen beigetragen. Abschließend
möchte ich Frau Gerlind Güth-Köhler, Frau Heidemarie
Demuth und Dr. Hans Peter Blaschkowski für
ihre effiziente und kreative Unterstützung des Direktors
danken.
In diesem Jahr haben unsere Labors im 1. Stockwerk
eine Klimaanlage bekommen. Diejenigen von uns, die
auf diesem Stock arbeiten, können endlich auch im
Sommer mit Drosophila experimentieren und müssen
soon as the office can be moved to the new fifth floor.
As already indicated, services for DNA chip technology
and quantitative PCR are in the planning stage.
The head of the administration Juergen Auer and his
team have successfully implemented software programs
tailored for the special needs of bookkeeping at
the ZMBH. This has further increased the efficiency
and speed of our administrative support. The biocomputing
unit headed by Raphael Mosbach constantly
increases our computing power and network support.
In this context, we acknowledge the essential support
of our electrical and mechanical workshop led by Matthias
Pawlitschko and Gert Stegmueller. With the hardware
support of biocomputing at the ZMBH a teaching
program in bioinformatics can now be launched
this year to prepare our students for the “mining” of
databases and for predicting both structure and function
from sequence, as well as evolution of intra- and
interspecies sequence variation and its consequences.
The artwork provided by Yves Cully of our documentation
service has been critical for the success of
the many talks, presentations of posters presented by
ZMBH members at meetings and exhibitions. Last
but not least, I am grateful to Gerlind Gueth-Koehler,
Heidemarie Demuth and Dr. Hans Peter Blaschkowski
for their very efficient and creative support of the
director.
This year our laboratories of the first floor have
received air-conditioning. Those of us working in this
floor can now continue with our experiments using
Drosophila as a model during summer time and do not
have to worry any longer about losing valuable strains.
We very much hope that the laboratories in the other
floors will receive the same air-conditioning standard
in the coming two years. To meet with the orders of the
fire brigade and the very limited lab space allocated to
each research group, the ZMBH had to invest in freez-
ers, suitable for using in the floors connecting the laboratories.
This investment was again made possible by
the overhead brought in by those groups having EC,
HFSP and industrial support.
After fourteen years of provisional delivery and waste
management, the ZMBH received a new building for
that purpose. We are grateful to the University for the
improvement. In order to make life easier for our busy
caretaker Michael Konrad, we hope that the waste
containers will soon be moved into this shelter.
The ZMBH itself shall become a building site this fall.
As mentioned earlier, we are looking forward to the
new office space on the fifth floor. If everything works
according to plan, we shall move into the new offices
next fall and be able to set free the desperately needed
lab space for our new service units.
We very much regret that the new building for physics
which is presently under construction next to
State Minister von Trotha visits the mechanical workshop
of the ZMBH.
15

nicht mehr den Verlust wichtiger Stämme befürchten.
Wir hoffen jetzt sehr, daß auch die Labors in den anderen
Stockwerken den selben Klimatisierungs-Standard
in den nächsten Jahren bekommen. Um den Anordnungen
der Feuerpolizei nachzukommen und um die
knappen Laborflächen, die jeder Forschungsgruppe
zur Verfügung stehen, optimal zu nutzen, mußte das
ZMBH umfangreiche Investitionen in Spezialkühlschränke
tätigen, die in den Verbindungsfluren zwischen
den Labors aufgestellt werden dürfen. Diese
Investitionen werden wiederum durch den ‚overhead‘
von den Gruppen ermöglicht, die über Drittmittel der
EU, des HFSP und der Industrie verfügen.
Nach vierzehn Jahren Improvisation bei der Lagerhaltung
und dem Abfall-Management hat das ZMBH
einen Anbau für diese Zwecke erhalten. Um die Arbeit
unseres eifrigen Hausmeisters Michael Konrad zu
erleichtern, hoffen wir, daß die Abfallcontainer hier
endlich ordnungsgemäß untergebracht werden.
Das ZMBH-Gebäude selbst wird in diesem Herbst
eine Baustelle werden. Wie zuvor geschildert, sehen
wir erwartungsvoll dem Dachpavillon entgegen. Wenn
alles nach Plan verläuft, werden wir die neuen Büroräume
im nächsten Jahr beziehen und damit die Laborflächen
räumen, die dringend für unsere neuen Service-Einheiten
benötigt werden.
Wir bedauern außerordentlich, daß das neue Institut
für Physik, das jetzt direkt neben dem ZMBH errichtet
wird, uns sein Technikum zuwendet und nicht, wie
wir wünschten, seine Hörsäle. Der einzige Kompromiß,
den wir in Verhandlungen erreichen konnten, war
ein größerer Abstand zwischen dem Physik-Technikum
und dem ZMBH. Nach den ursprünglichen Planungen
wäre der Physik-Neubau so nahe an unser Institut
herangesetzt worden, daß wir erhebliche Störungen
bei unseren erschütterungsempfindlichen mikroskopischen
Untersuchungen befürchten mußten.
16
Vor vier Jahren wurde das eigene Studienprogramm
für Doktoranden am ZMBH durch zwei „Graduierten-Kollegs“
der Deutschen Forschungsgemeinschaft
erweitert. Wir sind stolz darauf, daß diese „Graduierten-Kollegs“
verlängert und weiterhin von Christine
Clayton und Bernhard Dobberstein organisiert
werden.
Zur speziellen und flexiblen Förderung von Postdocs
und Gastwissenschaftlern hat das ZMBH-Kollegium
vor vier Jahren das „ZMBH-Stipendium“ gestiftet, das
inzwischen an mehr als ein Dutzend junger Mitarbeiter
und Gastwissenschaftler vergeben wurde.
Die Tradition des ZMBH, wissenschaftlichen Austausch
zu fördern, wurde im vergangenen Zeitraum
ebenfalls weiterverfolgt. Im Jahr 1998 fand das
ZMBH-Forum ‚Genetic Basis of Brain Function‘ statt,
es wurde von Klaus-Armin Nave organisiert. Das
ZMBH-Forum 1999 mit dem Titel ‚Pathogenetic Protozoa:
Molecules, Structures & Mechanims‘ organisierte
Christine Clayton. Beide Foren waren mit exzellenten
Rednern besetzt und wieder sehr gut besucht.
Heidelberg ist das Herz der BioRegion Rhein-Neckar-
Dreieck. Aus einem Wettbewerb mit 17 anderen deutschen
Regionen vor vier Jahren ging die BioRegion
Rhein-Neckar-Dreieck als eine der drei Gewinner
hervor. Der größte Teil der 50 Mio. DM, die bei diesem
Wettbewerb erhalten wurden, wurde jetzt für Projekte
15 junger Biotechnologie-Firmen vergeben. Der Beitrag
des ZMBH wird durch die Tatsache unterstrichen,
daß Bernhard Dobberstein bei der Gründung der
BioRegion den Wissenschaftsbereich koordinierte und
daß ich zur Zeit der Vorsitzende des neu gegründeten
Vereins „BioRegion Rhein-Neckar-Dreieck, e.V.“ bin.
Neben den vielen günstigen Ereignissen der letzten
Jahre und der fortlaufenden Unterstützung durch
Rektor, Kanzler und Kanzlerin der Universität und
the ZMBH faces us with its workshop and not, as
we wished, with its lecture theater. The only compromise
that we were able to negotiate was the distance
between the physics workshop and the ZMBH. The
original plan would have placed the two buildings in
such a close proximity that we expected to encounter
severe problems with our vibration sensitive microscopic
studies.
Four years ago the ZMBH‘s own study program for
doctoral students was extended by two “Graduierten
Kollegs” of the Deutsche Forschungsgemeinschaft.
We are proud that these “Graduierten Kollegs” were
renewed and continue to be organized by Christine
Clayton and Bernhard Dobberstein.
To permit special and flexible support for postdoctoral
fellows and visitors the ZMBH faculty implemented
the “ZMBH fellowship” four years ago which
has meanwhile been awarded to more than a dozen fellows.
The tradition at the ZMBH to promote scientific
exchange was also continued in the recent past. In
1998 the ZMBH Forum on “Genetic Basis of Brain
Function” was organized by Klaus-Armin Nave. In
the following year 1999, Christine Clayton organized
the ZMBH Forum under the theme “Pathogenic Protozoa:
Molecules, Structures & Mechanisms”. Both
were again best-received and well attended ZMBH
meetings.
Heidelberg is in the heart of the BioRegion Rhine-
Neckar-Triangle. Two years ago in competition with 17
other German regions the BioRegion Rhine-Neckar-
Triangle emerged as one of the three winners. The
major part of the 50 million DM received from this
competition has now been awarded to projects in fifteen
start-up biotech companies. The contribution of
the ZMBH is underlined by the fact that Bernhard
Dobberstein initially coordinated the scientific part of
the application and I am presently chairman of the
newly found BioRegion Rhine-Neckar-Triangle association.
MdL Pfisterer und MdB Lamers (1. und 2. von links)
besichtigen gentechnische Labors des ZMBH.
Besides many favorable events from the last years and
the continuing support by the Rector and Chancellor
of the University and Minister von Trotha, there are
developments which make us worry. Of major concern
for us is that the position for a successor of Hermann
Bujard is still pending. Furthermore, given the high
turnover rate of the ZMBH faculty members, the time
it takes to fill a tenured position, at present up to two
years, is much too long in a highly competitive situation.
In contrast, it takes the ZMBH only six to nine
months from the advertisement to the start of a new
junior group leader. This is not due to a less careful
selection process. The selection of a junior group
leader by the ZMBH faculty is done according to international
academic standards and by seeking advice
from our scientific advisory board.
As repeatedly mentioned by the former director and
cofounder of the ZMBH Hermann Bujard we must
17

durch Minister von Trotha, gibt es einige Sorge bereitende
Entwicklungen. Mit wachsender Beunruhigung
sehen wir, daß die Stelle für den Nachfolger von Hermann
Bujard immer noch nicht bewilligt ist. Außerdem
ist, in Anbetracht der fortdauernd hochkompetitiven
Personalsituation im Bereich der molekularen
Biowissenschaften und bei dem gegebenen großen
Wechsel der Mitglieder des ZMBH-Kollegiums, der
Zeitraum von gegenwärtig zwei Jahren für die Wiederbesetzung
frei gewordener Professuren viel zu groß.
Das dies auch anders geht, zeigen die Besetzungen
von Stellen für Nachwuchsgruppenleiter. Das ZMBH
benötigt lediglich sechs bis neun Monate von der Ausschreibung
einer freien Stelle bis zum Arbeitsbeginn
eines neuen Nachwuchsgruppenleiters. Dies hat seinen
Grund nicht in einem weniger aufwendigen Auswahlverfahren.
Die Berufung eines Nachwuchsgruppenleiters
durch das ZMBH wird internationalen akademischen
Standards entsprechend und mit Beteiligung
unseres Wissenschaftlichen Beirats durchgeführt.
Wie wiederholt von dem früheren Direktor und Mitgründer
des ZMBH Hermann Bujard dargelegt, müssen
wir nachdrücklich darauf bestehen, daß die Institute
der Universitäten in die Lage versetzt werden, die
wichtigsten Orte der Forschung zu sein. Hier, an diesen
Institutionen erhält die große Mehrzahl der jungen
Wissenschaftler ihre erste und damit wegweisende
Ausbildung, und die Qualität dieser Ausbildung wird
durch die Qualifikation ihrer Lehrer bestimmt. Deshalb
muß die Universität ihre Attraktivität für die Besten
ihres Faches behalten und darf sie nicht an außenstehende
Einrichtungen verlieren. Wir sind Minister von
Trotha außerordentlich dankbar für die offene Diskussion
unserer Besorgnis, das dieser essentielle Synergismus
höchst gefährdet ist und hoffen, daß das neue
Universitätsgesetz zu Änderungen in die richtige Richtung
führt. In einer Zeit, in der wir die höchst erfreu-
18
liche Zunahme der Zahl der biotechnischen Gründerfirmen
vor uns sehen, müssen wir realisieren, daß die
Zukunft dieser Unternehmen von breitester Grundlagenforschung
und einer großen Zahl bestens ausgebildeter
Graduierter und promovierter Wissenschaftler
abhängt. Beide Ziele können nur mit gut ausgestatteten
und hochflexiblen Universitätseinrichtungen
Minister von Trotha im Lehrlabor des ZMBH.
erreicht werden. Es bleibt darauf hinzuweisen, daß
die biomedizinische Forschung in Deutschland wieder
hinter die der USA zurückfällt, wenn die beteiligten
Institutionen keine entsprechende Unterstützung
bekommen. Daß die wissenschaftliche Grundlagenforschung
an den Universitäten höhere Priorität bekommen
muß, als ihr heute eingeräumt wird, veranlaßte
das ZMBH konsequenterweise, einen weitgreifenden
Dialog mit der Politik und Öffentlichkeit zu führen.
Wir waren dankbar, daß der Ministerpräsident von
Baden-Württemberg Erwin Teufel unsere Einladung
annahm, anläßlich seiner Bereisung der BioRegion und
des Technologieparks Heidelberg am 21. Januar 1999
auch das ZMBH zu besuchen. Der Wirtschaftsminister
des Landes Walter Döring besuchte das ZMBH am
7. April 1998, um mit uns Aspekte des Technologie-
stress and repeat that university institutes need to be
able to maintain the essential sites for research. It is at
these institutions where the vast majority of young scientists
receive their first and thus life-deciding train-
Fotis C. Kafatos, Director-General at EMBL, acknowledged
Hermann Bujard's leading role in bringing the
EMBL to Heidelberg (celebration of H. Bujard's 65th
birthday).
ing with the level of training being determined by the
quality of teachers. Hence the university must hold
attraction for the best people in a field and is not to
lose them to outside institutions. We are most grateful
to Minister von Trotha for the open discussion of our
concerns and hope that the new Hochschulgesetz will
lead to changes in the right direction. At a time where
we witness an unprecedented increase in the number
of biotech start-up companies in Germany, we have
to realize that their future depends on basic research
being as broad as possible and a great number of welltrained
graduates and PhDs. Both goals can only be
achieved with well-funded and highly flexible university
institutions. It remains to be emphasized that basic
biomedical research of the postgenome area in Germany
will fall again behind the USA if the support
of the institutions involved is not alike. The demand
that basic biosciences at universities has to be given a
higher priority than it receives at present has made it
essential for the ZMBH to develop a mature dialogue
with politicians and the public. We were very pleased
that the Prime Minister of Baden-Wuerttemberg Erwin
Teufel accepted our invitation to visit the ZMBH January
on 21 st , 1999, on the occassion of his visit to the
BioRegion and the Technology Park Heidelberg. Our
State Minister for Economic Affairs Walter Doering
visited the ZMBH on April 7 th , 1998 and discussed
with us the aspects of know-how transfer and issues
regarding start-up companies. Minister von Trotha discussed
with us the above-mentioned issues during his
visit of the ZMBH on July 23 rd , 1998. We discussed
issues of the regulation regarding animal experimentation
and gene technology with Members of the State
Parliament Pfisterer and Hildebrand and Members of
Hermann Bujard at the party of his 65 th birthday.
19

transfers und Probleme von Gründerfirmen zu diskutieren.
Minister von Trotha diskutierte mit uns die
oben erwähnten Themen während seines Besuches
am 23.07.1998. Über aktuelle Fragen der gesetzlichen
Regelungen von Tierversuchen und Gentechnik diskutierten
wir mit den Mitgliedern des Landtags Hildebrand
und Pfisterer und den Mitgliedern des Bundestags
Binding und Dr. Lamers bei ihren Besuchen
des ZMBH. Verschiedene Aspekte, die die Priorität
der Förderung biowissenschaftlicher Grundlagenforschung
betreffen, wurden auch beim Besuch des Mitglieds
des Vorstandes der BASF für den Bereich Forschung
Dr. Marcinowski am 19.11.198 behandelt.
Der zweite Bereich der Kommunikation, um unserer
Gesellschaft Themen der Biowissenschaften näher
zu bringen, wurde mit Tagen der „Offenen Tür“,
mit öffentlichen Vorträgen und Laborführungen sowie
mit speziellen Veranstaltungen und Laborpraktika
für Schüler angegangen. Wissenschaftler des ZMBH
Nobelpreisträger Bert Sakmann auf der Party zum 65.
Geburtstag von Hermann Bujard.
20
waren auch an der Einrichtung der Ausstellung „Genwelten“
im Landesmuseum für Technik und Arbeit in
Mannheim, der Landesmesse „Wirtschaft trifft Wissenschaft“
sowie der Landesinitiative „Zukunftswerkstatt
Baden-Württemberg“ beteiligt.
Das lebendige gesellschaftliche Leben im ZMBH
reichte wiederum vom Karneval bis zu Sommer- und
Weihnachtsfeiern; nicht vergessen werden dürfen die
von Axel Baumm wie immer sorgfältig geplanten
jährlichen ZMBH-Ausflüge. Schwungvoll mit einer
ZMBH-Party feierten wir auch den 65-jährigen
Geburtstag von Hermann Bujard.
Am 1. Januar 1998 endete die Amtszeit von Bernhard
Dobberstein als Direktor des ZMBH. Als sein Nachfolger
danke ich Bernhard Dobberstein, daß er dieses
Amt zwei Jahre auf sich genommen und sich jetzt
als zweiter stellvertretender Direktor zur Verfügung
gestellt hat. Ebenso bin ich Renato Paro dankbar, daß
er bereits kurz nach seiner Berufung zustimmte, erster
stellvertretender Direktor zu werden und die vielen
damit verbundenen Verwaltungsaufgaben zu übernehmen.
Meinen Kollegen danke ich für ihre Unterstützung
und das mir entgegengebrachte Vertrauen in den vergangenen
32 Monaten meiner Direktorenschaft.
Konrad Beyreuther
the Bundestag Binding and Dr. Lamers during their
visit of the ZMBH. Various aspects of the issue regarding
the priority of basic biosciences at universities
were also brought up during the visit of the Chief
Scientific Officer of the BASF Dr. Marcinowski who
came to the ZMBH on November 19 th , 1998. The
second major area of communication to bring scientific
issues to our community was addressed with
“open days” with lectures and guided lab visits for
the general public and with special meetings as well
as lab courses for high school students. Scientists of
the ZMBH were also actively involved in making the
Exhibition “Gene World”, shown at the State Museum
for Technology and Labour in Mannheim, a success.
The social life at the ZMBH was again alive with
annual highlights ranging from carneval to summer
to Christmas parties; not to forget the annual ZMBH
excursion carefully organized by Axel Baumm. We
also celebrated the 65 th birthday of Hermann Bujard
with a ZMBH party.
On January 1 st of 1998, Bernhard Dobberstein stepped
down as director of the ZMBH. As his successor I
thank Bernhard Dobberstein for shouldering this duty
for two years and for carrying on as second deputy
director. I am grateful also to Renato Paro for having
accepted to become first deputy shortly after his
appointment and for his commitment to take over the
many administrative responsibilities.
I thank my colleagues for their support and trust over
the past 32 months of my directorship.
Konrad Beyreuther
21

Research reports
23

Konrad Beyreuther
Molecular Neurobiology of Alzheimer's
Disease
We study key aspects of Alzheimer’s disease related to
synaptic and neuronal function, genetic and biochemical
control mechanisms and how these are regulated
in neural cells by the amyloid precursor protein (APP)
supergene family, the presenilin (PS) gene family and
other genes associated with neuronal function and the
disease. The information that we gained is used to
create cellular and animal models to study further the
physiological and pathogenic role of the APP supergene
family, and genes associated with cholesterol
biosynthesis, transport, transmembrane signaling and
metabolism in the brain.
To understand synaptic loss and neurodegeneration in
Alzheimer’s disease we have tried to consider what
are the physiological functions of the amyloid precursor
protein (APP), its Aß-amyloid domain and of free
Aß peptide. The latter is a normal metabolic product
of APP and the principle subunit of amyloid plaques
that are characteristic of Alzheimer‘s disease.
From studies in transgenic Drosophila melanogaster
and primary neurons, we suggest that in neurons
APP’s physiological function is related to the regulation
of synaptic strength whereas in nonneuronal
cells APP appears to regulate cell-cell and cell-matrix
adhesion.
Since the axonal transport of APP is dependent on its
Aß domain, this suggests that the Aß sequence could
function as axonal sorting signal of APP. It also indicates
that the Aß region could bind to molecules that
control the recruitment of APP into axonally transported
vesicles.
In neurons, metabolism of APP releasing the Aß
peptide was found to occur at all sorting stations of
APP such as at the ER/cisGolgi and TGN/endosomes
that gives rise to intracellular Aß peptide as well as
at the cell surface leading to secretory Aß peptide.
Regarding the Aß species generated in the different
neuronal compartments, the long form of Aß (Aß42) is
produced in the ER/cis Golgi and at or near to the cell
surface and short Aß (Aß40) in the TGN/endosomal
compartment and also at or near to the cell surface.
Given an Aß function as axonal sorting signal of APP,
release of Aß from APP may regulate the axonal transport
of APP. Not only the removal of the Aß sequence
from APP abolishes axonal APP transport but also free
Aß could - by blocking the APP binding sites of the
axonal transport machinery of APP - serve such a regulatory,
physiological function. Excess intracellular
and extracellular Aß may convert the latter physiological
function of Aß to a pathogenic one by inhibiting
the axonal transport of those proteins that use the
same transport system as APP.
Because the apoEε4 allele may be associated with
higher cholesterol levels in neurons and higher risk
of developing Alzheimer‘s disease and because the
axonal transport of membrane proteins is cholesterol
dependent, we studied the influence of cholesterol on
neuronal Aß generation. By lowering the cholesterol
level in neuronal cultures with statins (HMG-CoA
reductase inhibitors), the formation of secretory and
intracellular Aß is drastically reduced. Since the
amount of Aß produced by neurons is cholesterol
dependent, both the physiological and pathogenic regulation
of APP transport by Aß appears to be controled
in neurons by cholesterol, this implies a link
between brain cholesterol, APP transport, Aß production
and the risk of developing Alzheimer‘s disease.
These intriguing relationships open new strategies to
25

influence the progression of Alzheimer‘s disease by
modulating cholesterol biosynthesis of neurons with
statins.
I. APP supergene family
S. Eggert, S. Kreger, K. Paliga, A. Weidemann
The Alzheimer‘s disease amyloid protein precursor
(APP) gene is part of a multi-gene super-family from
which sixteen homologous amyloidprecursor-like proteins
(APLP) and APP species homologues are known.
Comparison of exon structure (including the uncharacterised
APL-1 gene), construction of phylogenetic
trees, and analysis of the protein sequence alignment
of known homologues of the APP super-family were
performed to reconstruct the evolution of the family
and to assess the functional significance of conserved
protein sequences between homologues. This analysis
supports an adhesion function for all members
of the APP super family, with specificity determined
by those sequences which are not conserved between
APLP lineages, and provides evidence for an increasingly
complex APP superfamily during evolution. The
analysis also suggests that Drosophila APPL and Caenorhabditis
elegans APL-1 may be a fourth APLP lineage
indicating that these proteins, while not functional
homologues of human APP, are similarly likely to regulate
cell adhesion. Furthermore, the Aß sequence is
highly conserved only in APP orthologues, strongly
suggesting this sequence is of significant functional
importance in this lineage.
II. Physiological function of the Aß domain
of APP and sites of production of Aß40
and Aß42
C. Bergmann, T. Hartmann, H. Grimm, P. J.
Tienari, I. Tomic
26
Cleavage of APP by ß- and γ-secretase results in the
release of the Aß domain. Cleavage occurs after residue
40 of Aß (Aß40) and after residue 42 (Aß42).
It is believed that even slightly increased amounts of
Aß42 might be sufficient to cause Alzheimer‘s disease.
What is the role of APP cleavage by these secretases.
To understand the physiological function of APP
processing centered around its Aß domain, deletion
analyses were preformed which showed that the Aß
domain of APP is essential for the axonal transport of
APP (Fig. 1). This suggests that the Aß region of APP
interacts with a sorting receptor or a sorting platform
for delivery to the axonal membrane. Removal of the
Aß domain or free Aß peptide is therefore expected
to regulate the axonal transport of APP and other proteins
utilizing the same transport machine as APP.
Using immunogold electron microscopy and cell fractionation
we have identified in neurons the endoplasmic
reticulum/cis Golgi as the site for generation of
Aß 42 and the trans-Golgi network (TGN) as the site
for Aß 42 generation. It is interesting that intracellular
generation of Aß seemed to be high in neurons,
because we found that nonneuronal cells produced
significant amounts of Aß40 and Aß42 only at the cell
surface. This shows that neurons which are able to
decode axonal sorting signals produce Aß at all sorting
stations of APP. Aß has thus the potential to control
neuronal APP transport. The specific production
of the critical Aß42 isoform in the ER/cis Golgi of
neurons links this compartment with APP transport
and the generation of Aß. It also explains why primarily
ER/cis Golgi localized (mutant) proteins such as
the presenilins could induce Alzheimer’s disease. We
suggest that the earliest event taking place in Alzheimer’s
disease might be the generation of Aß42 in the
ER.
Figure 1: Aß is produced within neurons at the ER/cisGolgi as Aß42, at the TGN/endosomal compartment as Aß40 and at the
cell surface/synapse as secreted Aß40 and Aß42. Secretory forms of Aß42 are aggregating to amyloid plaques. The Aß domain
of APP is essential for axonal sorting of APP. Deletion of the extracellular part of Aß leads to somato-dendritic sorting and
abolishes axonal sorting. Familial mutations at sites designated as FAD Swedish and FAD London increase Aß42 production
but do not alter axonal sorting of APP.
III. Mechanism of the cleavage of APP within
its transmembrane domain by γ-secretase
H. Grimm, B. Grziwa,T. Hartmann, S. Lichtenthaler
Proteolytic processing of the amyloid precursor protein
by ß-secretase yields A4CT (C99), which is
cleaved further by the as yet unknown γ-secretase,
yielding Aß40 and Aß42. We therefore used A4CT
as a model to study the specificity of the cleavage of
APP within its transmembrane domain by γ-secretase.
Because the position of γ-secretase cleavage is cru-
cial for the pathogenesis of Alzheimer‘s disease, we
individually replaced all membrane-domain residues
of A4CT outside the Aß domain with phenylalanine,
stably transfected the constructs in COS7 cells, and
determined the effect of these mutations on the
cleavage specificity of γ-secretase (Aß42/Aß40 ratio).
Assuming an alpha-helical conformation of the transmembrane
domain of APP, mutations of residues
superimposed at one helical face (residues 44, 47, and
50) led to decreased Aß42/Aß40 ratios, whereas mutations
affecting superimposed residues at the opposite
site (residues 43, 45, 46, 49, and 51) led to increased
27

Aß42/Aß40 ratios (Figure 2). A massive effect was
observed for A4CT-I45F (34-fold increase) making
this construct important for the generation of animal
models for Alzheimer‘s disease. Unlike the other mutations,
A4CT-V44F was processed mainly to Aß38, as
determined by mass spectrometry. Our data provide a
detailed model for the active site of γ-secretase (Figure
2). According to this model Aß40 is produced when
γ-secretase interacts with APP by binding to one side
of the alpha-helical transmembrane domain of APP.
Alternatively, Aß42 arises by binding of γ-secretase
to the opposite side (Fig. 2). Mutations in the transmembrane
domain of APP interfere with the interaction
between γ-secretase and APP and, thus, alter the
cleavage specificity of γ-secretase (Fig. 2).
Figure 2: Schematic representation of the amino acid positions
(P) of the ß-secretase product A4CT of APP relative to
the cleavage site of γ-secretase. Top: linear arrangement of the
residues relative to the cleavage site after residue 40 (Aß40)
and residue 42 (Aß42). The scissile peptide bond is constituted
by residues P1 and P1‘. Bottom: helical wheel arrangement
of amino acids 40 to 49 of A4CT with respect to the cleavage
sites after residues 40 and 42. Binding of γ-secretase to the
transmembrane domain giving rise to Aß40 or Aß42 has to
occur at opposite sides of the helix.
28
IV. Regulation of exon 15 splicing of APP
premRNA
C. Bergsdorff, S. Kreger, K. Paliga
Alternative splicing of exon 15 of the amyloid precursor
protein (APP) pre-mRNA generates two APP
isoform groups APP(ex15) (containing exon 15) and
L-APP (without exon 15), which show a cell-specific
distribution in non-neuronal cells and neurons of rat.
Both APP isoforms differ in regard to functional properties
like post-translational modification, APP secretion,
and proteolytic production of Aß peptide from
APP molecules. Since Aß generation is an important
factor in the development of Alzheimer‘s disease, one
could anticipate that these major APP isoforms might
contribute differentially to the mechanisms underlying
neurodegeneration in Alzheimer‘s disease. We
established an APP minigene system in a murine cell
system to identify cis-acting elements controlling exon
15 recognition. A 12.5-kilobase pair genomic fragment
of the murine APP gene contained all cis-regulatory
elements to reproduce the splicing pattern of the
endogenous APP transcripts. By using this approach,
two intronic cis-elements flanking exon 15 were identified
that block the inclusion of exon 15 in APP transcripts
of non-neuronal cells. Point mutation analysis
of these intronic regions indicated that pyrimidinerich
sequences are involved in the splice repressor
function. Finally, grafting experiments demonstrated
that these regulatory regions cell-specifically enhance
the blockage of a chimeric exon in the non-neuronal
splicing system.
V. Physiological function of APP
A. Fossgreen, F. Reinhard, S. Scheurermann, P.
Soba, J. Stumm
In collaboration with B. Brückner and R. Paro, ZMBH,
we used Drosophila melanogaster as a model system
to analyze the function of APP by expressing wildtype
and various mutant forms of human APP in fly
tissue culture cells as well as in transgenic fly lines.
After expression of full-length APP forms, secretion
of APP but not of Aß was observed in both systems. By
using SPA4CT, a short APP form in which the signal
peptide was fused directly to the Aß region, transmembrane
domain, and cytoplasmic tail, we observed
Aß release in flies and fly-tissue culture cells. Consequently,
we showed a γ-secretase activity in flies.
Interestingly, transgenic flies expressing full-length
forms of APP have a blistered-wing phenotype. As
the wing is composed of interacting dorsal and ventral
epithelial cell layers, this phenotype suggests that
human APP expression interferes with cell adhesion/
signaling pathways in Drosophila, independently of
Aß generation. Wing morphogenesis in Drosophila is
characterized by the apposition of two epithelial cell
layers, the dorsal and ventral layer and may thus be
used as a model to analyze the role of APP in the interaction
of presynaptic and postsynaptic membranes at
the synapse. We presume that APP negatively interacts
with factors involved in the adhesion of the two wing
epithelia and the two synaptic membranes, respectively.
In Drosophila, some mutations deficient for the expression
of specific integrin subunits develop distinct wing
blisters whereas another one was shown to be involved
in short term memory. This provides an interesting
link between physiological APP functions, integrinmediated
adhesion processes and memory mechanisms.
Since integrins are heterodimeric receptors that
may be ligand-bound to extracellular matrix molecules
and may have a signaling function, integrins are
especially required for the function of the cell-matrixcell
junction, where one surface of a cell such as a
synaptic density adheres to the other. This function
of integrins supports our suggestion of APP’s involvement
in cell adhesion. We envisage that APP might
act as an antagonist for integrins, both in the developing
wing and at plasticity of the synapse. APP could
bind, either directly or via other molecules to the same
extracellular matrix sites as integrins. Indeed, in rat
primary neurons APP and integrins were colocalized
in structures that are reminiscent of synapses. However,
APP might be involved in other pathways resulting
in a deterioration of the adhesion or signaling
pathways supported by the integrins. For example,
expression of a mutant form of G protein involved in
cAMP signaling or the blistered gene encoding a protein
related to human serum response factors result
in Drosophila a comparable wing. Taken together,
Drosophila seems to be a valuable animal-model
system for studying the physiological and pathogenic
functions of human APP since it may be suited for
unraveling APP transmembrane signaling mechanisms
potentially related to Alzheimer‘s disease. By using
this fly model, testing genetic interactions between
APP-expressing lines producing the blistered phenotype
and other mutants engaged in wing morphology
offers the perspective to unravel those physiological
and pathogenic pathways linked to human APP.
VI. Regulation of APP-Aß metabolism by cholesterol
C. Bergmann, T. Hartmann, H. Runz, P. Semenfeld,
I. Tomic
Cholesterol metabolism and Alzheimer‘s disease are
genetically linked. The apoEε4 allele of the apolipoprotein
E gene is associated with higher cholesterol
levels and was shown to increase the risk of develop-
29

ing the disease. To study the influence of cholesterol
on APP metabolism we collaborated with M. Simons,
K. Simons and C.G. Dotti (EMBL) and used primary
cultures of rat hippocampal neurons infected with
recombinant Semliki Forest virus carrying APP and
APP mutants. This system has been used previously
by us to study the intracellular transport and processing
of human APP. Inhibition of cholesterol biosynthesis
and extraction of cholesterol from neuronal membranes
was achieved by a combination of statin treatment
and methyl-ß-cyclodextrin extraction. Statins
such as lovastatin or simvastatin allow in the presence
of low amounts of mevalonate the control of the synthesis
of cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) reductase. Mevalonate is
required for the synthesis of non-steroidal products
and essential to prevent inhibition of the proteasome
by lovastatin. ß-Cyclodextrin has been shown to very
efficiently and selectively extract cholesterol from the
plasma membrane.
The combined treatment of rat hippocampal neurons
with statins and methyl-ß-cyclodextrin resulted in
reduced intracellular Aß production and Aß secretion
(Fig. 3), however APP levels and cell viability
remained unaffected in both treated and control cells
during the time course of the experiments.
Since Aß generation occurs in two steps, we employed
the two APP constructs APP695 and A4CT (SPC99)
to analyze which Aß cleavage is inhibited by cholesterol
lowering drugs. The first cleavage of APP by
ß-secretase generates the 10-kDa fragment C99 that is
further cleaved within the transmembrane domain by
γ-secretase to produce Aß. When antibodies recognizing
the C-terminal domain of APP were used to immunoprecipitate
APP fragments from the cell lysates of
cholesterol-depleted and control rat hippocampal neurons
expressing human APP695 a dramatic inhibition
30
of ß-cleavage was detected while the production of the
fragment generated by α-secretase was unperturbed.
To analyze the effect of lovastatin treatment on the
cleavage of γ-secretase, neurons expressing SPC99
were used since removal of the transient signal
sequence of SPC99 generates C99 which is identical
with the C-terminal ß-secretase product of APP. Conversion
of C99 to Aß requires γ-secretase. Both lovastatin
treatment and methyl-ß-cyclodextrin treatment
- alone and together - of neurons expressing SPC99
inhibited Aß production as well suggesting that cholesterol-lowering
regiments inhibit also γ-secretase
cleavage. Thus our combined results suggest that cholesterol
is required for APP cleavage by ß-secretase
and γ-secretase but not for α-cleavage that produces
αAPPsec. We conclude that lowering of cholesterol
affects amyloidogenic processing of APP while allowing
nonamyloidogenic cleavage to proceed.
These findings raise the question how cholesterol
affects Aß formation. One possibility is that reduc-
Figure 3: Cholesterol-lowering of hippocampal neurons
reduces intracellular (lysate) production and secretion
(media) of Aß40 and Aß2. Neurons were grown for 4 days
in the presence of lovastatin/mevalonate and after infection
with Semliki Forest virus/APP-expression vectors were
treated with 5mM methyl-ß-cyclodextrin for 20 min.
tion in membrane cholesterol changes the intracellular
transport of APP so that the protein does not reach the
cellular sites where ß- and γ-secretase cleavage takes
place. If this is the case, cholesterol is required as a
sorting platform for inclusion of APP protein cargo
destined for delivery to the apical membrane in nonneuronal
cells and to the axonal membrane in neurons.
Alternatively, the ß- and γ-secretase require cholesterol
for their activity.
VII. Aß as biological marker of Alzheimer’s
disease
A. Diehlmann, T. Hartmann, N. Ida
All mutations known to cause familial Alzheimer‘s
disease act by increasing the levels of soluble ß-amyloid
peptide, especially the longer form, Aß42. However,
in vivo elevation of soluble Aß in sporadic
Alzheimer‘s disease has so far not been shown. In collaboration
with M. Jensen and L. Lannfelt (Karolinska),
we used our monoclonal antibodies specific for
Aß40 and Aß42 in an enzyme-linked immunosorbent
assay to investigate cerebrospinal fluid from sporadic
Alzheimer‘s disease at different stages of disease
severity, to clarify the roles of Aß42 and Aß40 during
disease progression. We also evaluated three other
groups, one group of patients with mild cognitive
impairment who were at risk of developing dementia,
a cognitively intact, nondemented reference group
diagnosed with depression, and a perfectly healthy
control group. We found that Aß42 is strongly elevated
in early and mid stages of AD, and thereafter
it declines with disease progression. On the contrary,
Aß40 levels were decreased in early and mid stages
of AD. The group of cognitively impaired patients
and the depression reference group had significantly
higher levels of Aß42 than the healthy control group,
implying that Aß42 is increased not only in AD, but
in other central nervous system conditions as well.
Our data also point out the importance of having thoroughly
examined control material. The initial increase
and subsequent decrease of Aß42 adds a new biochemical
tool to follow the progression of AD and
might be important in the monitoring of therapeutics.
Acknowledgements: We acknowledge the stimulating
and productive collaboration with among others
the group of Gerd Multhaup (ZMBH), Colin L. Masters
(The University of Melbourne, Australia), Renato
Paro (ZMBH), Kai Simons and Carlos G. Dotti
(EMBL), Thomas A. Bayer (Psychiatry, University of
Bonn, Germany), Michael Hennerici and Klaus Fassbender
(Neurology, University Heidelberg at Mann–
heim), Lars Lannfelt (Karolinska Institute, Stockholm,
Sweden); Joachim Schröder (Psychiatry, University
Heidelberg), Bart De Strooper (University Leuven,
Belgium), Rudolph E. Tanzi and Ashley I. Bush
(Harvard University, Cambridge, USA), Hans Peter
Schmidt (Neuropathology, University Heidelberg),
Hans Förstl (Psychiatry, TU Munich, Germany) and
Christian Haass (Biochemistry, University Munich).
External Funding
Our research summarized in this report would not
have been possible without the following grants: an
institutional grant of the Minister for Science and Arts
of the State of Baden-Württemberg, by grants and project
grants of the Deutsche Forschungsgemeinschaft,
of the Landes Forschungsschwerpunktprogramm of
Baden-Württemberg, the European Community, of the
German-Israelic-Foundation, of the Humboldt-Foundation,
of the Fonds of the Chemical Industry of Germany
and by generous donations.
31

PUBLICATIONS
Bayer, T., Cappai, R., Masters, C.L., Beyreuther, K.,
and Multhaup, G. (1999). It all sticks together - the
APP-related family of proteins and Alzheimer‘s disease.
Molecular Psychiatry 4, 524-528.
Bayer, T.A., Jäkälä, P., Hartmann, T., Egensperger,
R., Buslei, R., Falkai, P., and Beyreuther, K. (1999).
Neural expression profile of alpha-synuclein in developing
human cortex. Neuroreport 10, 2799-2803.
Bayer, T.A., Jäkälä, P., Hartmann, T., Havas, L.,
McLean, C., Culvenor, J.G., Li, Q.X., Masters, C.L.,
Falkai, P., and Beyreuther, K. (1999). Alpha-synuclein
accumulates in Lewy bodies in Parkinson’s disease
and dementia with Lewy bodies but not in Alzheimer’s
disease beta-amyloid plaque cores. Neurosci.
Lett. 266, 213-216.
Beher, D., Elle, C., Underwood, J., Davis, J.B., Ward,
R., Karran, E., Masters, C.L., Beyreuther, K., and
Multhaup, G. (1999). Proteolytic fragments of Alzheimer’s
disease-associated presenilin 1 are present in
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Bergsdorf, C., Paliga, K., Kreger, S., Masters, C.L.
and Beyreuther, K. (2000). Identification of cis-Elements
Regulating Exon 15 Splicing of the Amyloid
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2046-2056.
Borchert, T., Camakaris, J., Cappai, R., Masters, C.L.,
Beyreuther, K., and Multhaup, G. (1999). Copper
inhibits ß-amyloid production and stimulates the nonamyloidogenic
pathway of amyloid-precursor-protein
32
secretion. Biochem. J. 344, 461-467.
Capell, A., Grünberg, J., Pesold, B., Diehlmann, A.,
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and Haass, C. (1998). The proteolytic fragments of
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Cappai, R., Mok, S.S., Galatis, D., Tucker, D.F.,
Henry, A., Beyreuther, K., Small, D.H., and Masters,
C.L. (1999). Recombinant human amyloid precursor
like-protein2 (APLP2) expressed in the yeast Pichia
pastoris can stimulate neurite outgrowth. FEBS Lett.
442, 95-98.
Cappai, R., Stewart, L., Jobling, M.F., Thyer, J.M.,
White, A.R., Beyreuther, K., Collins, S.J., Masters,
C.L., Barrow, C.J. (1998). Familial prion disease
mutation alters the secondary structure of recombinant
mouse prion protein. Biochemistry 99, 3053-3058.
Caswell, M.D., Mok, S.S., Henry, A., Cappai, R.,
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D.H. (1999). The amyloid beta-protein precursor of
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Kunitz protease inhibitor domain-sensitive trypsinlike
serine protease in cultures of chick sympathetic
neurons. Eur. J. Biochem. 266, 509-516.
Cherny, R.A., Legg, J.T., McLean, C.A., Fairlie, D.P.,
Huang, X., Atwood, C.S., Beyreuther, K., Tanzi,
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deposits by biometal depletion. J. Biol. Chem. 274,
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Coulson, E.J., Paliga, K., Beyreuther, K., and Masters,
C.L. (2000). What the evolution of the amyloid protein
precursor supergene family tells us about its function.
Neurochem. Int. 36, 175-184.
Christie, G., Markwell, R.E., Gray, C.W., Smith, L.,
Godfrey, F., Mansfield, F., Wadsworth, H., King, R.,
McLaughlin, M., Cooper, D.G., Ward,R.V., Howlett,
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Connop, B.P., Thies, R.L., Beyreuther, K., Ida, N.,
and Reiner, P.B. (1999). Novel effects of FCCP [carbonyl
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Culvenor, J., Henry, A., Hartmann, T., Evin, G., Galatis,
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Culvenor, J.G., McLean, C.A., Cutt, S., Campbell,
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with Abeta Amyloid. Am. J. Pathol. 155,
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Culvenor, J.G., Evin, G., Cooney, M.A., Wardan, H.,
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(2000). Presenilin 2 expression in neuronal cells:
induction during differentiation of embryonic carcinoma
cells. Exp. Cell Res. 255, 192-206.
Diehlmann, A., Ida, N., Weggen, S., Grunberg, J.,
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Egensberger, R.,Weggen, S., Ida, N., Multhaup, G.,
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Emilien, G., Beyreuther, K., Masters, C.L., and Maloteaux,
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Kwok, J.B., Li, Q.X., Hallupp, M., Whyte, S., Ames,
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(1999). The hydrophobic core sequence modulates the
neurotoxic and secondary structure properties of the
prion peptide 106-126. J. Neurochem. 4, 1557-1565.
Multhaup, G., Hesse, L., Borchardt, T., Ruppert, T.,
Cappai, R., Masters, C.L., and Beyreuther, K. (1998).
Autoxidation of amyloid precursor protein and formation
of reactive oxygen species. In Copper Transport
and Its Disorders: Molecular and Cellular Aspects. A.
Leone and J.F.B Mercer eds. (New York: Plenum), pp
75-94.
LeBrocque, D., Henry, A., Cappai, R., Li, Q.X.,
Tanner, J.E., Galatis, D., Gray, C., Holmes, S., Underwood,
J.R., Beyreuther, K., Masters, C.L., and Evin,
G. (1998). Processing of the Alzheimer’s disease amyloid
precursor protein in Pichia pastoris: α-, β- and
γ-secretase products. Biochemistry 37, 14958-14965.
Li, Q.-X., Cappai, R., Evin, G., Tanner, J.E., Gray,
C.W., Beyreuther, K., and Masters, C.L. (1998). Products
of the amyloid precursor protein generated by
ß-secretase are present in human platelets, and secreted
upon degranulation. American Journal of Alzheimer’s
Disease. 15, 236-244.
Li, Q.-X., Fuller, S.J., Beyreuther, K., and Masters, C.L.
(1999). The amyloid precursor protein of Alzheimer’s
disease in human brain and blood. J. Leukoc. Biol. 66,
567-574.
Li, Q.X., Maynard, C., Cappai, R., McLean, C.A.,
Cherny, R.A., Lynch, T., Culvenor, J.G., Trevaskis,
J., Tanner, J.E., Bailey, K.A., Czech, C., Bush, A.I.,
Beyreuther, K., and Masters, C.L. (1999). Intracellular
accumulation of detergent-soluble amyloidogenic
A beta fragment of Alzheimer’s disease precursor protein
in the hippocampus of aged transgenic mice. J.
Neurochem. 72, 2479-2487.
Li, Q.-X., Whyte, S., Tanner, J.E., Evin, G., Beyreuther,
K., and Masters, C.L. (1998). Release of the amyloidogenic
Aß fragment of Alzheimer’s disease from
human platelets. Lab. Invest. 78, 461-469.
Lichtenthaler, S.F., Multhaup, G., Masters, C.L., and
Beyreuther, K. (1999). A novel substrate for analyzing
Alzheimer’s disease gamma-secretase. FEBS Lett.
453, 288-292.
Lichtenthaler, S.F., Wang, R., Masters, C.L., and
Beyreuther, K. (1999). Mechanism of the cleavage
specificity of Alzheimer’s disease γ-secretase revealed
by phenylalanine-scanning mutagenesis of the transmembrane
domain of APP. Proc. Natl. Acad. Sci. USA
96, 3053-3058.
Masters, C.L., and Beyreuther, K. (1998), Science,
medicine and the future. Alzheimer’s disease. British
35

Medical Journal 316, 446-448.
Masters, C.L., and Beyreuther, K. (1999). Amyloid
Nomenclature Committee. Amyloid 6, 151-152.
Masters, C.L., and Beyreuther, K. (2000). Genomic
neurology. Arch. Neurol. 57, 53.
McLean, C.A., Cherry, R.A., Fraser, F.W., Fuller, S.J.,
Smith, M.J., Beyreuther, K., Bush, A.I., and Masters,
C.L. (1999). Soluble pool of Aß as a determinant of
severity of neurodegeneration in Alzheimer’s disease.
New Engl. J. Medicine, Ann. Neurol. 46, 860-866.
Moir, R.D., Lynch, T., Bush, A.I., Whyte, S., Henry,
A., Portbury, S., Tanzi, R.E., Multhaup, G., Small,
D.H., Beyreuther, K., and Masters, C.L. (1998). Relative
increase in Kunitz protease inhibitory forms of
cerebral Aß amyloid protein precursor in Alzheimer’s
disease. J. Biol. Chem. 273, 5013-5019.
Multhaup, G., Hesse, L., Borchardt, T., Ruppert, T.,
Cappai, R., Masters, C.L., Beyreuther, K., and Masters,
C.L. (1999). Autoxidation of amyloid precursor
protein and formation of reactive oxygen species. Adv.
Exp. Med. Biol. 448, 183-192.
Multhaup, G., Masters, C.L., and Beyreuther, K.
(1998). Oxidative stress in Alzheimer’s disease. Alzheimer’s
Reports 1, 147-154.
Multhaup, G., Masters, C.L., Beyreuther, K., and
Cappai, R. (1998). The biological activities and function
of the amyloid precursor protein of Alzheimer’s
disease. In The Molecular Biology of Alzheimer’s Disease
- Genes and Mechanisms Involved in Amyloid
Generation. C. Hasss, ed. (Harwood Academic Publishers,
Amsterdam), pp 75-94.
36
Multhaup, G., Ruppert, T., Schlicksupp, A., Hesse, L.,
Bill, E., Pipkorn, R., Masters, C.L., and Beyreuther,
K. (1998). Copper-binding amyloid precursor protein
undergoes a site-specific fragmentation in the reduction
of hydrogen peroxide. Biochem. 37, 7224-7230.
Postuma, R.B., Martins, R.N., Cappai, R., Beyreuther,
K., Masters, C.L., Strickland, D.K., Mok, S.S., and
Small, D.H. (1998). Effects of the amyloid protein
precursor of Alzheimer’s disease and other ligands of
the LDL receptor-related protein on neurite outgrowth
from sympathetic neurons in culture. FEBS Lett. 428,
13-16.
Postuma, R.B., He, W., Nunan, J., Beyreuther, K.,
Masters, C.L., Barrow, C.J., and Small, D.H. (2000).
Substrate-bound beta-amyloid peptides inhibit cell
adhesion and neurite outgrowth in primary cultures. J.
Neurochem. 74, 1122-1130.
Ren, R.F., Lah, J.J., Diehlmann, A., Kim, E.S., Hawver,
D.B., Levey, A.I., Beyreuther, K., and Flanders, K.C.
(1999). Differential effects of transforming growth
factor-beta(s) and glial cell line-derived neurotrophic
factor on gene expression of presenilin-1 in human
post-mitotic neurons and astrocytes. Neuroscience 93,
1041-1049.
Rondot, S., Anthony, K.G., Duebel, S., Ida, N., Wiemann
S., Beyreuther, K., Frost, L.S., Little, M., and
Breitling, F. (1998). Epitopes fused to F-pilin are
incorporated into functional recombinant pili. J. Mol.
Biol. 279, 589-603.
Rossjohn, J., Cappai, R., Feil, S.C., Henry, A., McKinstry,
W.J., Galatis, D., Hesse, L., Multhaup, G.,
Beyreuther, K., Masters, C.L., and Parker, M.W.
(1999). Crystal structure of the N-terminal, growth
factor-like domain of Alzheimer amyloid precursor
protein. Nat. Struct. Biol. 6, 327-331.
Sberna, G., Sáez-Valero, Li, Q.-X., Czech, C.,
Beyreuther, K., Masters, C.L., McLean, C.A., and
Small, D.H. (1998). Acetylcholinesterase is increased
in the brain of transgenic mice expressing the C-terminal
fragment (CT100) of the ß-amyloid protein precursor
of Alzheimer’s disease. J. Neurochemistry 71,
723-731.
Simons, M., Keller, P., De Strooper, B., Beyreuther,
K., Dotti, C.G., and Simons, K. (1998). Chostersterol
depletion inhibits the generation of ß-amyloid in hippocampal
neurons. Proc. Natl. Acad. Sci. USA 95,
6460-6464.
Smith, M.J., Gardner, R.J., Knight, M.A., Forrest,
S.M., Beyreuther, K., Storey, E., McLean, C.A.,
Cotton, R.G., Cappal, R., and Masters, C.L. (1999).
Early-onset Alzheimer’s disease caused by a novel
mutation at codon 219 of the presenilin-1 gene. Neuroreport
10, 503-507.
Storey, E., Katz, M., Brickman, Y., Beyreuther, K.,
and Masters, C.L. (1999). Amyloid precursor protein
of Alzheimer’s disease: evidence for a stable, fulllength,
trans-membrane pool in primary neuronal cultures.
Eur. J. Neurosci. 11, 1779-1788.
Tesco, G., Kim, T.-W., Diehlmann, A., Beyreuther,
K., and Tanzi, R.E. (1998). Abrogation of the presenilin
1/ß-catenin interaction and preservation of the
heterodimeric presenilin 1 complex following caspase
activation. J. Biol. Chem. 273, 33909-33914.
Weggen, S., Diehlmann, A., Buslei, R., Beyreuther,
K., and Bayer, T. (1998). Prominent Expression of
Presenilin-1 in Senile Plaques and Reactive Astrocytes
in Alzheimer’s Disease Brain. Neuroreport 9,
3279-3283.
Weggen, S., Tienari, P.J., Beyreuther, K., and Bayer
T.A. (1999). Retroviral shuttle vectors in Alzheimer’s
disease research. Neurol. Psych. Brain Res. 6,
213-218.
Weidemann, A., Paliga, K., Durrwang, U., Reinhard,
F.B.M., Evin, G., Masters, C.L., and Beyreuther, K.
(1999). Cleavage of the Alzheimer’s disease amyloid
precursor protein during apoptosis by activated caspases.
In Alzheimer’s Disease and Related Disorders.
K. Iqbal, D.F. Swaab, B. Winblad, and H.M. Wisniewski
eds. (John Wiley & Sons Ltd., Chichester), pp
391-396.
White, A.R., Bush, A.I., Beyreuther, K., Masters, C.L.,
and Cappai, R. (1999). Exacerbation of copper toxicity
in primary neuronal cultures depleted of cellular
glutathione. J. Neurochem. 72, 2092-2098.
White, A.R., Collins, S.J., Maher, F., Jobling, M.F.,
Stewart, L.R., Thyer, J.M., Beyreuther, K., Masters,
C.L., and Cappai, R. (1999). Prion protein-deficient
neurons reveal lower gluthione reductase activity and
increased susceptibility to hydrogene peroxide toxicity.
Am. J. Pathol. 155, 1723-1730.
White, A.R., Multhaup, G., Maher, F., Bellingham,
S., Camakaris, J., Zheng, H., Bush, A.I., Beyreuther,
K., Masters, C.L., and Cappai, R. (1999). The Alzheimer’s
disease amyloid precursor protein modulates
copper-induced toxicity and oxidative stress in primary
neuronal cultures. J. Neurosci. 19, 9170-9179.
37

White, A.R., Reyes, R., Mercer. K-F-B-. Camacaris,
J., Zheng, H., Bush, A.I., Multhaup, G., Beyreuther,
K., Masters, C.L., and Cappai, R. (1999). Copper
levels are increased in the cerebral cortex and liver
of APP and APLP2 knockout mice. Brain Res. 842,
439-444.
White, A.R., Zheng, H., Galatis, D., Maher, F., Hesse,
L., Multhaup, G., Beyreuther, K., Masters, C.L., and
Cappai, R. (1998). Survival of cultured neurons from
amyloid precursor protein knock-out mice against
Alzheimer’s amyloid-ß toxicity and oxidative stress.
J. Neurosci. 18, 6207-6217.
THESES
Diploma
Großkreutz, Yannik (1999). Herstellung und Charakterisierung
von APP-GFP Fusionsproteinen.
Scheuermann, Stefan (1999). Nachweis und Charakterisierung
der Dimerisierung des Amyloid Precursor
Proteins.
Dissertations
Bergsdorf, Christian (1999). Molekularbiologische
Charakterisierung der Regulation des alternativ gespleißten
Exons 15 des Amyloid Vorläuferproteins.
Picard, Martin (1998). Untersuchungen zur Alzheimer
Krankheit: Neuroinflammation und Glucose Konjugate
entzündungshemmender Wirkstoffe.
Uhrig, Rainer (1999). Glycokonjugate von Antioxidantien
– Synthese, Transport und Bedeutung für ein
hirnspezifisches Drug Targeting bei Morbus Alzheimer.
38
STRUCTURE OF THE GROUP
E-mail: beyreuther@zmbh.uni-heidelberg.de
Group leader Beyreuther, Konrad, Prof.
Dr. rer. nat. Dr. med. h. c.
Assistant Hartmann, Tobias, Dr. rer. nat.
Senior research
associates Prior, Peter, Dr. rer. nat.
Weidemann, Andreas, Dr. rer. nat.
Postdoctoral
fellows Bergmann, Christine, Dr. med.
Bergsdorf, Christian, Dr. rer. nat.
Foßgreen, Anke, Dr. rer.nat.*
Ishii, Kazuhiro, M.D.
Lichtenthaler, Stefan, Dr. rer. nat.*
Paliga, Krzysztof, Dr. rer. nat.
Peraus, Gisela, Dr. rer.nat*
Picard, Martin, Dr. rer. nat.*
Uhrig, Rainer, Dr. rer. nat.*
Zhao, Liyun, Ph.D.
Visiting
scientist Jäkkälä, Pekka, M.D.*
Graduate
students Grimm, Heike, Dipl. Biol.
Grziwa, Beate, Dipl. Biol.
Kinter, Jochen, Dipl. Biol.*
Reinhard, Friedrich, Dipl. Chem.
Runz, Heiko, cand. med.*
Scheuermann, Stefan, Dipl. Biol.
Soba, Peter, Dipl. Chem.*
Stumm, Joachim, Dipl. Biol.
Staff scientist Diehlmann, Anke, Dipl. Biol.*
Techn.
assistants Kreger, Sylvia
Samenfeld, Petra
Tomic, Inge
Administration
and secretary Demuth, Heidemarie
*part of the time reported
39

Project Group Gerd Multhaup
Ligand-Dependent Functions of the Amyloid
Precursor Protein (APP) and Ligand-Associated
Conformational Changes
APP is a transmembrane glycoprotein that undergoes
extensive alternative splicing. APP belongs to a multigene
family that contains at least two other homologs
known as amyloid precursor-like proteins (APLP1 and
APLP2) (Fig. 1). APP and APLPs share most of the
domains and motifs of APP, but only APP contains the
Aß region and can be cleaved by ß- and γ-secretase
to generate Aß. Thus, APLPs cannot contribute to Aß
deposition in Alzheimer’s disease but may compensate
for the function of APP.
The normal functions of APP and APLPs are not
well understood. There exists at least some evidence
for neuritic and protective roles. APP binds Zn(II) at
higher nanomolar concentrations and an altered APP
metabolism or expression level is believed to result in
neurotoxic processes. APP can reduce Cu(II) to Cu(I)
in a cell-free system potentially leading to increased
oxidative stress in neurons. The domain that contributes
to such activities is the copper binding domain
residing between residues 135 and 158 of APP, a
region that shows strong homology to APLP2 but not
to APLP1.
Potentially, APP-Cu(I) complexes are involved that
reduce hydrogen peroxide to form an APP-Cu(II)hydroxyl
radical intermediate. APP residues 135 to
158 consisting of cysteine and Cu-coordinating histidine
residues can modulate copper-mediated lipid
peroxidation and neurotoxicity in culture of APP
knockout (APP 0/0 ) and wild-type (wt) neurons. Wt
neurons were found to be more susceptible than APP 0/0
neurons to physiological concentrations of copper but
not other metals.
Figure 1: Summary of the structural domains and binding motifs of the APP-family. The broken vertical lines denote APP
sequences conserved in the APLP molecules. Abbreviations used are: α, alpha-secretase site; “α”, homologous to alphasecretase
cleavage site for release of the ectodomain; β, beta-secretase site; CD, cytoplasmic domain; CBD, collagen-binding
domain; CHO, N-linked carbohydrate site; CS-GAG, chondroitin sulfate glycosaminglycan; CuBD-1, copper-binding domain
1; CuBD-2, copper-binding domain 2; cys, cysteine; δ, delta-secretase site; γ, gamma-secretase site; GPD, growth promoting
domain; HBD-1, heparin-binding domain 1; HBD-2, heparin-binding domain 2; KPI, Kunitz-type protease inhibitor; NPXY,
asparagine-proline-any amino acid-tyrosine; OX-2, OX-2 homology domain; Poly-T, poly-threonine; P, phosphorylation site;
SP, signal peptide; ZnBD-1, zinc-binding domain 1; ZnBD-2, zinc-binding domain 2.
40
APP 0/0 mice have significantly increased copper but
not zinc or iron levels in the cerebral cortex and liver
as compared to age and genetically matched wt mice.
APLP2 0/0 mice also revealed increases in copper in
cerebral cortex and liver. These findings suggest that
the APP family can modulate copper homeostasis
and that APP/APLP2 expression may be involved in
copper efflux from liver and cerebral cortex. Most
importantly, copper was found to influence APP processing
in a cell culture model system when copper
was observed to greatly reduce the levels of amyloid
Aß peptide and copper also caused an increase in
the secretion of the APP ectodomain. An increase in
intracellular APP levels which paralleled the decrease
in Aß generation suggested that additional copper was
acting on two distinct regulating mechanisms, one on
Aß production and the other on APP synthesis. Taken
together, APP and APLP2 are most likely involved in
copper homeostasis.
Our work is funded by grants of the DFG (through
SFB317, 1991-1999; Mu-901), by the BMBF, by the
AFI-foundation (Alzheimer Forschung Initiative), by
the Graduiertenkolleg (C. Elle), by the International
Copper Association (ICA) and by the Fonds der Chemischen
Industrie.
We collaborate with Prof. Dr. Dr. K. Beyreuther
(ZMBH, Heidelberg), Prof. Dr. C.L. Masters (University
of Melbourne, Dep. of Pathology), Prof. Dr. C.
Haass (Ludwig-Maximilians-University, Adolf-Butenandt-Institute,
München), Prof. Dr. W. Stremmel (University
of Heidelberg, Dep. of Gastroenterology), Prof.
Dr. J. Camakaris (University of Melbourne, Dep. of
Genetics), Prof. Dr. J. Mercer (Deakin University,
Melbourne), Prof. Dr. A. Bush (Harvard University,
USA), Dr. T. Bayer (University of Bonn) and Prof. Dr.
K. Wieghardt (MPI, Mülheim).
I. The role of copper in the pathological
function of the amyloid precursor protein
(APP)
C. Schmidt, M. Strauss, A. Simons, A. Schlicksupp,
G. Multhaup (in collaboration with T.
Bayer, R. Cappai, A. White, E. Bill, R. Pipkorn,
C.L. Masters and K. Beyreuther)
The extracellular domain of transmembrane Aß amyloid
precursor protein (APP) has a Cu(II) reducing
activity upon Cu(II) binding associated with the formation
of a new disulfide bridge. The complete assignment
of the disulfide bond revealed the involvement
of cysteines 144 and 158 around copper-binding histidine
residues ( Fig. 2).
Figure 2: The APP copper binding motif corresponds to
type-II copper binding sites and is encompassed by copper
coordinating histidine residues 147, 149 and 151. The
reduction of copper by APP results in a corresponding oxidation
of cysteines 144 and 158. The zinc binding motif
maps to exon 5 of APP between residues 181-200 with the
neighboring cysteines involved in chelation.
41

APP catalyzed the reduction of H 2 O 2 and oxidation of
Cu(I) to Cu(II) in a „peroxidative“ reaction in vitro.
The resulting bound copper-hydroxyl radical intermediate
[APP-Cu(II) (•OH)] then likely participated in
a Fenton type of reaction with radical formation as a
prerequisite for APP-Cu(I) complex degradation. Evidence
from two observations suggests that the reaction
takes place in two phases. Bathocuproine, a trapping
agent for Cu(I), abolished the initial fragmentation
of APP, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic
acid) interrupted the reaction
cascade induced by H 2 O 2 at later stages. WT cortical,
cerebellar and hippocampal neurons were significantly
more susceptible than their respective APP 0/0 neurons
to toxicity induced by physiological concentrations
of copper but not by zinc or iron. There was no difference
in copper toxicity between APLP2 0/0 and WT
neurons, demonstrating specificity for APP-associated
copper toxicity. Treatment of neuronal cultures with a
peptide corresponding to the human APP copper binding
domain (APP142-166) potentiated copper but not
iron or zinc toxicity. Incubation of APP142-166 with
low density lipoprotein (LDL) and copper resulted
in significantly increased lipid peroxidation compared
to copper and LDL alone. Substitution of the
copper co-ordinating histidine residues with asparagines
(APP142-166 H147N, H149N, H151N ) abrogated the
toxic effects. A peptide corresponding to the zinc binding
domain (APP181-208) failed to induce copper or
zinc toxicity in neuronal cultures. These data support a
role for the APP copper binding domain in APP-mediated
Cu(I) generation and toxicity in primary neurons.
To test the hypothesis that biometals such as copper
can contribute to the amyloid pathology in AD we are
currently investigating the dietary exposure of copper
and its chelators to APP transgenic mice and to APP
42
knock-out mice. The effects of dietary supplementation
is studied by ICP-MS (inductively coupled plasma
mass-spectrometry) to determine the levels of Cu, Zn
and Fe. The AD pathology is analyzed by conventional
methods. We expect that Cu has an effect on the
AD pathology and metal-chelation can delay amyloid
Aß deposition in APP transgenic mice.
II. The role of copper and zinc in the normal
function of the amyloid precursor protein
(APP)
C. Schmidt, A. Simons, M. Strauss, A. Schlicksupp,
G. Multhaup (in collaboration with D.
Ha-Hao, R. Cappai, A. White, C.L. Masters and
K. Beyreuther)
The expression of APP and APLP2 in the brain
suggests they could have an important direct or indirect
role in neuronal metal homeostasis. In APP and
APLP2 knockout mice copper levels were significantly
elevated in both APP 0/0 and APLP2 0/0 cerebral
cortex (40% and 16%, respectively) and liver (80% and
36%, respectively) compared with matched wild-type
(WT) mice. These findings indicate APP and APLP2
expression specifically modulates copper homeostasis
in the liver and cerebral cortex, the latter being a
region of the brain particularly involved in AD. Perturbations
to APP metabolism and in particular, its secretion
or release from neurons may alter copper homeostasis
and explain a disturbed metal-ion homeostasis
observed in AD.
Zinc up to concentrations of 50µM or the presence of
1,10-phenanthroline specifically increased the level of
secreted APP in APP transfected CHO-K1 cells. By
contrast, the level of secreted APP in copper-resistant
CHO-CUR3 cells remained unaffected. APP holoprotein
increased dramatically in CHO-CUR3 cells com-
pared with CHO-K1 cells. The large decrease of Aß
release seen in both cell lines at elevated extracellular
zinc levels was due to specific inhibition of secretion.
These results indicate that a disturbed zinc-homeostasis
may be an important factor influencing APP production,
transport and processing.
Figure 3: The α- and β-secretase protease activities cleave
APP within its ectodomain. The remaining membranebound
C-terminal fragment p3CT (after α-secretase cleavage
of APP) is further cleaved by γ-secretase in the middle
of the putative transmembrane domain, yielding p3 (α- and
γ-secretase activities). Amyloid Aβ is produced by β- and
γ-secretase activities; CuBD-I, copper binding domain 1.
Adding copper to APP-transfected CHO cells greatly
reduced the levels of ß-amyloid (Aß) peptide in both
parental CHO-K1 and in copper resistant CHO-CUR3
cells which have lower intracellular copper levels.
Copper also caused an increase in the secretion of the
APP ectodomain indicating that the large decrease in
Aß release was not due to a general inhibition in protein
secretion. There was an increase in intracellular
full-length APP levels which paralleled the decrease
in Aß generation suggesting the existence of two distinct
regulating mechanisms, one acting on Aß production
and the other on APP synthesis. Thus, our
findings suggest that copper or copper agonists might
be useful tools to discover novel targets for anti-
Alzheimer drugs since copper promoted the non-amyloidogenic
pathway of APP.
Our current understanding is that copper and/or zinc
binding is central to the normal cellular function of
APP. We therefore want to identify agonists of APP
ligand binding sites (e.g. of the copper-binding site)
that are able to inhibit amyloidogenic proteolytic processing
of APP. Lead compounds will be discovered
followed by a further screen for small APP ligands.
Our research program combines biochemical, immunohistochemical
and cellular approaches to start a
risk-benefit analysis of divalent metal binding agonists
of APP in vitro.
III. Dimerization and stability of APP isoforms:
influence of APP ligands on relative
stability and metabolism
M. Strauss, C. Schmidt, A. Simons, A. Schlicksupp,
C. Elle, G. Multhaup (in collaboration with
C. Haass, C.L. Masters and K. Beyreuther)
The highly conserved nature and tissue specificity of
the eight APP isoforms provide circumstantial evidence
that functional differences among isoforms may
exist in vitro and in vivo. When APP and Aß are central
to AD pathogenesis, then molecules which influence
the conformation and the stability of APP isoforms
and that interact with specific APP forms could
differentially alter their metabolism/activity and thus
represent risk factors for AD.
For example, heparin and Zn(II) were found to augment
the ability of full-length and secreted KPI-APP to
inhibit FXIa. In contrast, both compounds heparin and
43

Zn(II) failed to have an effect on C-terminally truncated
recombinant KPI-APP (APP residues 18-350)
from Pichia pastoris and on the inhibition of trypsin
or chymotrypsin. Together with the observation that
native KPI-APP was required for the potentiation of
inhibition by Zn(II) these data indicate that inhibitory
effects were enhanced by other domains of APP than
KPI. Further structural investigations of the N-terminal
heparin binding domain of APP (residues 28-123)
revealed a highly charged basic surface and an abutting
hydrophobic surface immediately adjacent to the
low-affinity N-terminal heparin binding site (residues
96-110) that is proposed to play an important function
in dimerization and/or ligand binding. A second heparin
binding site of high affinity was identified within
a region conserved in rodent and human APP, APLP1
and APLP2. This binding site was located between
residues 316-337 of APP695 which is the carbohydrate
domain of APP (Figure 1). The affinity for heparin
is increased two- to four-fold in the presence of
micromolar Zn(II).
The current model of APP dimerization is complex
and the information about the nature of APP-APP
interaction is indirect and inferred. Indeed, APP
released into the cell culture supernatant has been
found to be secreted as dimers and also to exist intracellularly
as dimerized transmembrane APP. Most
likely, both sites, the APP-APP interaction domain
encoded by residues 448-465 and the large hydrophobic
patch of N-terminal APP residues 96-116 (N-terminal
interaction site, (NIS)) are involved in the generation
of dimers (Fig. 4). The N-terminal site is sufficient
for oligomerization since dynamic light scattering
analysis of recombinant APP18-350 from Pichia
pastoris showed that the C-terminal truncated protein
is secreted as a homodimer (Fig. 4).
44
Figure 4: The three-dimensional structure of an N-terminal
fragment (APP residues 28-123) has been determined
(Rossjohn et al., 1999) and resembles a growth factor. The
large hydrophobic patch of residues 96-116 is most likely
involved in the generation of dimers (arrows). The structure
and the contribution to this activity of the metalbinding
domains, carbohydrate domain and the C-terminal
domains (residues 124-770) remain unknown. The APP
N-terminal domain consists of nine β-strands and one
α-helix which fold into a compact, globular domain. The
helix represents the longest stretch of secondary structure,
being 13 residues long.
The overall aim of this project is to understand how
monomer-to-dimer transition controls the activity and
the metabolism of APP. We believe that the elucidation
of biological regulatory mechanisms in APP can
lead to the design of new approaches for manipulating
the APP-system. This will help to better understand its
function and to achieve new outcomes for a possible
therapy and diagnosis.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 317 “Molekulare Biologie neuraler
Mechanismen und Interaktionenen”, Graduiertenkolleg
“Molekulare Zellbiologie”, Graduiertenkolleg
“Molekulare und zelluläre Neurobiologie” and Projekt-’Sachbeihilfen’),
from the BioRegion Rhein-
Neckar (BMBF ), from the Alzheimer Forschung Initiative
e.V. and the International Copper Association
(ICA).
PUBLICATIONS
Moir, R.D., Lynch, T., Bush, A.I., Whyte, S., Henry,
A., Portbury, S., Tanzi, R.E., Multhaup, G., Small,
D.H., Beyreuther, K. and Masters, C.L. (1998). Relative
increase in Alzheimer‘s disease of soluble forms
of cerebral Aß amyloid protein precursor containing
the kunitz protease inhibitory domain. J. Biol. Chem.
273, 5013-9.
Multhaup, G., Ruppert, T., Schlicksupp, A., Hesse, L.,
Bill, E., Pipkorn, R., Masters, C.L. and Beyreuther, K.
(1998). APP-copper complexes undergo site-specific
fragmentation in the reduction of hydrogen peroxide.
Biochemistry 37, 7224-7230.
Ilg, T., Craik, D., Currie, G., Multhaup, G., and Bacic,
A. (1998). Stage-specific proteophosphoglycan from
leishmania mexicana amastigotes. Structural characterization
of novel mono-, di-, and triphosphorylated
phosphodiester-linked oligosaccharides. J. Biol.
Chem. 273, 13509-23.
Henry, A., Li, Q-X., Galatis, D., Hesse, L., Multhaup,
G., Beyreuther, K., Masters, C.L. and Cappai, R.
(1998). Inhibition of platelet activation by the Alzheimer‘s
disease amyloid precursor protein. Br. J. Haematol.
103, 402-415.
Rossjohn, J., Cappai, R., Feil, S.C., Henry, A., McKinstry,
W.J., Galatis, D., Hesse, L., Multhaup, G.,
Beyreuther, K., Masters, C.L. and Parker, M.W.
(1999). Crystal structure of the N-terminal, growth
factor-like domain of Alzheimer‘s amyloid precursor
protein. Nature Struc. Biol. 6, 327-331.
Urban, S., Kruse, C. and Multhaup, G. (1999). A
soluble form of the avian hepatitis B virus receptor.
Biochemical characterization and functional analysis
of the receptor ligand complex. J. Biol. Chem. 274,
5707-5715.
Beher, D., Elle, C., Underwood, J., Davis, J.B., Ward,
R., Karran, E., Roberts, G., Masters C.L., Beyreuther
K. and Multhaup, G. (1999). Proteolytic fragments of
Alzheimer’s disease-associated Presenilin 1 are present
in synaptic organelles and growth cone membranes
of rat brain. J. Neurochem. 72, 1564-1573.
Multhaup, G., Hesse, L., Borchardt, T., Ruppert, T.,
Cappai, R., Masters, C. L., and Beyreuther, K. (1999).
Autoxidation of amyloid precursor protein and formation
of reactive oxygen species. Adv. Exp. Med. Biol.
448, 183-92.
Huang, X., Atwood, C. S., Hartshorn, M. A., Multhaup,
G., Goldstein, L. E., Scarpa, R. C., Cuajungco, M. P.,
Gray, D. N., Lim, J., Moir, R. D., Tanzi, R. E., and
Bush, A. I. (1999). The Abeta peptide of Alzheimer‘s
disease directly produces hydrogen peroxide through
metal ion reduction. Biochemistry 38, 7609-16.
45

Multhaup, G., Hesse, L., Borchardt, T., Ruppert, T.,
Cappai, R., Masters, C.L. and Beyreuther, K. (1999).
Autoxidation of amyloid precursor protein and formation
of reactive oxygen species. In Copper Transport
and Its Disorders: Molecular and Cellular Aspects,
16, 183-192 (A. Leone and JFB Mercer, eds) Plenum,
New York.
Egensperger, R., Weggen, S., Ida, N., Multhaup, G.,
Schnabel, R., Beyreuther, K., and Bayer, T. A. (1999).
Reverse relationship between beta-amyloid precursor
protein and beta-amyloid peptide plaques in Down‘s
syndrome versus sporadic/familial Alzheimer‘s disease.
Acta Neuropathol. (Berl) 97, 113-8.
Multhaup, G., and Masters, C. L. (1999). Metal binding
and radical generation of proteins in human neurological
diseases and aging. Met. Ions Biol. Syst. 36,
365-87.
Lichtenthaler, S. F., Multhaup, G., Masters, C. L., and
Beyreuther, K. (1999). A novel substrate for analyzing
Alzheimer‘s disease gamma-secretase. FEBS Lett.
453, 288-92.
White, A.R., Reyes, R., Mercer, J.F.B., Camakaris, J.,
Zheng, H., Bush, A.I., Multhaup, G., Beyreuther, K.,
Masters, C.L. and Cappai, R. (1999). Copper levels
are increased in the cerebral cortex and liver of APP
and APLP2 knockout mice. Brain Res. 842, 439-444.
Bayer, T. A., Cappai, R., Masters, C. L., Beyreuther,
K., and Multhaup, G. (1999). It all sticks together - the
APP-related family of proteins and Alzheimer‘s disease.
Mol. Psychiatry 4, 524-528.
Borchardt, T., Camakaris, J., Cappai, R., Masters, C.
46
L., Beyreuther, K., and Multhaup, G. (1999). Copper
inhibits ß-amyloid production and stimulates the nonamyloidogenic
pathway of amyloid precursor protein
(APP) secretion. Biochem. J. 344, 461-467.
White, A. R., Multhaup, G., Maher, F., Bellingham,
S., Camakaris, J., Zheng, H., Bush, A. I., Beyreuther,
K., Masters, C. L., and Cappai, R. (1999). The
Alzheimer‘s disease amyloid precursor protein (APP)
modulates copper-induced toxicity and oxidative
stress in primary neuronal cultures. J. Neurosci. 19,
9170-9179.
Huang, X., Cuajungco, M.P., Atwood, C.S., Hartshorn,
M.A., Tyndall, J.D.A., Hanson, G.R., Stokes,
K.C., Leopold, M., Multhaup, G., Goldstein, L.E.,
Scarpa, R.C., Saunders, A.J., James Lim, Moir, R.D.,
Glabe, C., Bowden, E.F., Masters, C.L., Fairlie, D.P.,
Tanzi, R.E., and Bush, A.I. (1999). Cu(II) potentiation
of Alzheimer Aß neurotoxicity. Correlation with cellfree
hydrogen peroxide production and metal reduction.
J. Biol. Chem. 274, 3711-6.
Urban, S., Schwarz, C., Marx, U., Zentgraf, W. and
Multhaup, G. (2000). Receptor recognition by a hepatitis
B virus reveals a novel mode of high affinity
virus-host interaction. EMBO J. 19, 1217-1227.
Kulic, L., Walter, J., Multhaup, G., Teplow, D.B.,
Romig, H., Capell, A., Steiner, H. and Haass, C.
(2000). Separation of presenilin function in endoproteolysis
of the ß-amyloid precursor protein and notch.
Proc. Natl. Acad. Sci. USA, 97, 5913-5918.
Borchardt, T., Schmidt, C., Camakaris, J., Cappai,
R., Masters, C.L., Beyreuther, K. and Multhaup, G.
(2000). Differential effects of zinc on amyloid precur-
sor protein (APP) processing in copper-resistant variants
of cultured Chinese hamster ovary cells. Cell.
Mol. Biol, 46, 785-795.
Capell, A., Steiner, H., Willem, M., Kaiser, H., Meyer,
C., Walter, J., Lammich, S., Multhaup, G. and Haass,
C. (2000). Maturation and pro-peptide cleavage of
ß-secretase (BACE). J Biol Chem, (in press).
THESES
Dissertation
Hesse, Lars (1998): Funktionelle Analyse von extrazellulären
Domänen des Alzheimer Vorläuferproteins
und anderer Mitglieder der APP-Genfamilie.
Awards
1998 ‚Award‘ der ‚International Copper Association‘
(ICA)
1999 Alzheimer Forschung Initiative (AFI) ‚Grant
award‘
STRUCTURE OF THE GROUP
E-mail: g.multhaup@zmbh.uni-heidelberg.de
Group leader Multhaup, Gerd, Priv.-
Doz. Dr.
Research associate Strausak. Daniel, Dr.*
Postdoctoral fellows Hesse, Lars, Dr.*
Schmidt, Carsten, Dr.*
Ph.D. students Simons, Andreas*
Strauss, Markus*
Elle, Christine
Techn. assistant Schlicksupp, Andrea
*part of the time reported
47

Herrmann Bujard
I Expanding the Applicability of the
Tet Regulatory Systems
U. Baron, S. Freundlieb, R. Löw, Ch. Schirra-Müller
Though very tight regulation of gene expression may
be achieved with the Tet regulatory system - demonstrated
by the successful control of the diphteria
toxin gene in transgenic mice most impressively - it
is sometimes difficult to initially establish cell lines or
transgenic animals where genes encoding potentially
toxic products are under Tet control. The main reason
for such failures is the transient state of the transferred
DNA. During this period when chromatin suppression
is missing, background expression from multiple
copies of the target gene may be sufficient to kill
the cell. We have deviced two approaches that help
to overcome this limitaton. By fusing transcriptional
silencing domains to the Tet repressor (TetR), tetracycline
(Tc) controlled silencers (tTS) were developed
that bind to the operator sequences within P tet (Fig. 1)
48
and shield the promoter from outside activation. Addition
of doxycycline (Dox) will dissociate tTS from P tet
and at the same time cause its activation via rtTA. The
second approach makes use of gene transfer via retroviruses
where single genomes can be delivered to
the nucleus. Several vehicles including lentiviral vectors
(collaboration with L. Naldini, Candiolo/Torino,
Italy) were composed, which function well. Together,
these components should not only abrogate present
limitations, they offer themselves also for the development
of tightly controllable gene delivery systems that
may become useful in gene therapy. Several collaborations
in this area are ongoing.
Figure 1: The family of Tc-controlled regulatory proteins.
TetR (red) was originally fused to a portion of the Herpes
simplex virus activator protein VP16 (green) resulting in
tTA (Tc controlled transactivator). Replacing the VP16
moiety by multiples of 13 amino acid long minimal activation
domains (upper left) has increased the specificity of
tTA. A quadruple mutant of TetR exhibiting a reverse phenotype
(violet) was fused to activation domains resulting in
rtTA that requires Dox for operator binding. Modifying the
DNA recognition specificity of tTA and rtTA (blue) as well
as the dimerization surface of tTA (grey) led to tTA and rtTA
versions that can be used to control two genes in a mutually
exclusive way by Dox. Fusion of TetR with an altered
dimerization surface to silencing domains led to Tc-controlled
transcriptional silencers (tTS) that, in concert with
rtTA, establish a repression/ activation system where in the
„OFF“ state, i.e. in absence of Dox, P tet - a minimal promoter
fused to an array of 7 tet operators - is shielded from
outside activation.-
Probing the sequence space of TetR
T. Baldinger, U. Baron, M. Hasan, Ch. Schirra-Müller
Most of the TetR mutants that have advantageously
modified the Tet regulatory system resulted from powerful
genetic approaches in E.coli. However, the phenotypes
observed in the prokaryotic cell do frequently
not correlate with those seen in the eukaryotic environment.
Therefore, two eukaryotic genetic systems
were conceived; one developed by W. Hillen and his
group (Universität Erlangen) is based on S.cerevisiae,
the other on mammalian cells. With these systems,
the sequence space of fusions between TetR and functional
domains can be directly explored e.g. by screening
or selecting for activation or silencing of marker
functions. First screens revealed a number of interesting
TetR mutants of which one - identified in the lab in
Erlangen - exhibits a striking reverse phenotype that
is superior to the originally described rtTA. Furthermore,
we are interested in whether tTA/rtTA mutants
can be identified which discriminate between different
tetracycline derivatives or which may even be susceptible
to non-tetracycline compounds as inducers.
If successful, this search could lead to well defined
and largely homologous regulatory circuits that would
allow to control several genes independently from
each other by just using different inducer molecules
(collaboration with W. Hillen and B. Berkhout).
Controlling genes in vivo
S. Berger, M. Hasan, R. Löw, K. Schönig
Our transgenic mouse lines that synthesize tTA or
rtTA specifically in hepatocytes and in specific areas
of the brain, respectively, were used to measure kinet-
ics of induction and shut-off of gene activities in these
two compartments of the animal. Using a non-invasive
approach that monitores luciferase activity in live
animals, repeated cycles of gene activation and deactivation
can be followed in individual mice (Fig. 2).
These studies have provided not only insights into the
time course of switching between active and inactive
states of a gene via Dox in live animals but also into
the longevity of the expression system. Thus, independent
of whether the luciferase reporter gene was kept
in the „ON“ or in the „OFF“ state, regulatory cycles
could be re-initiated after 6 months in all individuals
tested. As any target gene may be coregulated together
with the luciferase gene as indicator via the bidirectional
P tet promoters, monitoring luciferase activity in
live animal will be indicative for the activity of the
gene under study (Fig. 2). In summary, the newly
developed rtTA‘s together with some novel doxycycline
derivatives that are presently being characterized
and the induction studies have made us confident that
genes of interest can be controlled in the mouse brain
tightly and reliably over long periods of time. In
this context, it is our aim to control gene functions
indirectly by interferring at the level of transcription
initiation and/or in post-transcriptional processes, an
approach which would leave the endogeneous gene
of interest untouched in its genomic setting. Therefore,
we explore several experimental options which
include padlock RNA‘s, ribozymes and zinc finger
proteins. We have chosen the NRI subunit of the
NMDA receptor as primary target for this work. Our
technology is used in several rather challenging collaborations
to generate animal models for human diseases.
One exciting example is a mouse model for
prion disease that is being developed by S. Prusiner
and P. Tremblay, UCSF, San Francisco, USA.
49

Figure 2: Monitoring
gene activation and
inactivation in live animals.
Mice double transgenic
for tTA and the
luciferase gene under
Ptet control are injected
with luciferin and anesthesized
for 5 min.
before they are placed
into the Hamamatsu
photon counting device
Argus 20 (left upper part
of fig.). Photons emitted
by luciferase active in
liver (left most animal)
and in brain (right
animal) are collected as
imaged below. The single
transgenic animal in the
middle serves as control.
Feeding Dox to the
“liver mouse” abolishes
luciferase activity within
5 days. Removal of Dox
in the drinking water reestablishes
luciferase activity within 5 days (“brain mouse” as control). The cycle of switching was repeated with the same
animals after 3 months with identical results, except that 2 days of exposure to Dox were sufficient to shut off luciferase activity
in the liver. The luciferase gene is driven by a bidirectional Ptet which coregulates the Cre recombinase gene. Luciferase
activity correlates with Cre activity. Whole body images of the animals are shown.
II The Merozoite Surface Protein 1 of
the Human Malaria Parasite Plasmodium
falciparum
Malaria is one of the most widely spread infectious
diseases with around 40 % of the world‘s population
living in areas at risk. The hope for an effective vaccine
candidate against infection of P.falciparum, the
most lethal one among the human malaria parasites,
relies on the subunit concept where individual components
of the parasite are utilized to elicit a protective
immune response. Our studies focus on a 190 kDa
protein which is the major surface protein of the mero-
50
zoite (MSP-1), one of the parasite‘s blood forms. This
protein is believed to play a role during invasion of
erythrocytes by the parasite. It is a target of the human
immune response at the humoral as well as at the cellular
level. Such findings suggest that MSP-1 may
elicit a protective immune response when used as a
vaccine in humans. The aim of our work is to develop
an experimental vaccine that is suitable for human
trials. Moreover, we are interested in the function of
MSP-1 and its structure at the parasite‘s surface.
Synthetic msp-1 genes provide material for
immunological, structural and functional studies
C. Epp, C. Fernandez-Becerra, C. Schmid, I. Türbachova,
N. Westerfeld
P.falciparum DNA has an exceptionally high AT content
which amounts to 76 % in the coding area of
MSP-1. The high AT content has prevented the stable
cloning of large genes of this parasite and thus severely
hampered their study. We have synthesized the genes
of both MSP-1 prototypes (together around 10 000
bp) based on human codon frequencies and are now in
a position to produce MSP-1 and derivatives thereof
in various heterologous systems. During maturation
of merozoites, MSP-1 undergoes proteolytic cleavage
resulting in five major fragments that can be isolated
as one complex from the merozoite surface. The proteolytic
cleavage sites were reconciled in the design of
our synthetic genes, and accordingly expression vehicles
encoding the various fragments are available as
well. Efficient expression of the intact genes as well
as of DNA encoding the processing products has been
achieved in E.coli. Full size MSP-1 is also produced
and secreted into the medium by bacterial L-forms
(collaboration with M. Kujau, IMB, Jena) from where
it can be isolated in soluble form. We now concentrate
on devicing a production procedure for MSP-1
that is suitable for „good manufacturing practice“.
Our highly purified preparations will also be used
for structural studies and for exploring interactions
between the individual processed domains as well as
between MSP-1 and the erythrocyte surface. A second
focus of this project is the integration of msp-1 into
viral systems that are suitable as vaccine carriers in
humans, such as vaccinia and measles viruses.
A MSP-1 based ELISA for sero-epidemiological
surveys
C. Epp, I. Idler, I. Türbachova, S. Weerasuriya
The availability of the various MSP-1 processing products
in apparently native conformation has enabled us
to develop an ELISA that can be used for serum analysis.
In a first approach, we have examined the sera
from two trials in which Aotus monkeys were immunized
with MSP-1 and challenged with merozoites
(collaboration with S. Herrera, Cali, Colombia). Full
protection was achieved in 60 % of the animals. Interestingly,
protection correlated well with the humoral
response against certain areas of MSP-1. Using the
new ELISA, we are now re-examining the sera collected
during our earlier epidemiological studies in
West Africa. Our previous analysis has indicated a
correlation between antibody titers towards certain
regions of MSP-1 and a reduced risk of reinfection
by P.falciparum.We hope that our new analysis will
reveal more clearly which regions of MSP-1 may be
involved in eliciting a protective humoral response.
The ELISA may eventually permit the development of
a diagnostic tool with predictive properties.
Attempts to identify interactions between
MSP-1 and the surface of erythrocytes
P. Burghaus, I. Türbachova
As the major protein at the surface of merozoites,
MSP-1 has been implicated in early association events
during the erythrocyte invasion. Following the successful
strategy described for the Duffy binding protein,
we have exposed full length MSP-1 as well as
stepwise truncated versions of the protein on the surface
of HeLa cells, fixed, as in the parasite, by a
51

GPI-anchor. Moreover, in collaboration with D. Soldati
(ZMBH), we have generated several recombinant
Toxoplasma gondii strains that also expose at the
surface full size MSP-1, the p42 or the p19 processing
product, respectively. In both, the HeLa cell and
the T.gondii system, monoclonal antibodies directed
towards conformational epitopes of MSP-1 interact
efficiently with the protein at the surface of the respective
cells. Nevertheless, so far we were neither able
to unequivocally demonstrate an interaction between
MSP-1 and erythrocytes nor can we definitely rule out
such interactions. The failure to reveal the putative
affinity may be due to a number of reasons. We, therefore,
will further pursue these studies.
External Funding
During the period reported, our research was supported
by grants from the BMBF (Forschungsschwerpunkt
Tropenmedizin), the Volkswagen-Stiftung, the
Deutsche Forschungsgemeinschaft (SFB 544 “Kontrolle
tropischer Infektionskrankheiten” and Projekt-
’Sachbeihilfen’), from the EU, the BioRegion Rhein-
Neckar (BMBF - Fa. Knoll AG) and the Fonds der
Chemischen Industrie Deutschlands.
PUBLICATIONS
Tremblay, P., Meiner, Z., Galou, M., Heinrich, C.,
Petromilli, C., Lisse, T., Cayateno, J., Torchia, M.,
Mobley, W., Bujard, H., DeArmond, S.J. and Prusiner,
S.B. (1998). Doxycycline control of prion protein
transgene expression modulates prion disease in mice.
Proc. Natl. Acad. Sci. USA 95, 12580-12585.
Baron, U., Schnappinger, D., Helbl, V., Gossen, M.,
Hillen, W. and Bujard, H. (1999). Generation of conditional
mutants in higher eukaryotes by switching
52
between the expression of two genes. Proc. Natl.
Acad. Sci. USA 96, 1013-1018
Freundlieb, S., Schirra-Müller, C. and Bujard, H.
(1999). A tetracycline controlled activation /repression
system for mammalian cells. J. Gene Med. 1, 4-12.
Pan, W., Ravot, E., Tolle, R., Frank, R., Mosbach, R.,
Türbachova, I. and Bujard, H. (1999). Vaccine candidate
MSP-1 from Plasmodium falciparum: a redesigned
4917 bp polynucleotide enables synthesis and
isolation of full length protein from E.coli and mammalian
cells. Nucl. Acids Res. 27, 1094-1103.
Redfern, C.H., Coward, P., Degtyarev, M.Y., Lee, E.K.,
Kwa, A., Hennighausen, L., Bujard, H., Fishman, G.I.
and Conklin, B.R. (1999). In vivo conditional expression
and signaling of a specifically designed G i -coupled
receptor. Nature Biotech. 17, 165-169.
Burghaus, P.A., Gerold, P., Pan, W., Schwarz, R.T.,
Lingelbach, K. and Bujard, H. (1999). Analysis of
recombinant merozoite surface protein-1 of Plasmodium
falciparum expressed in mammalian cells. Mol.
Biochem. Parasitol. 104, 171-183.
Lavon, I., Goldberg, I., Amit, S., Landsman, L., Jung,
S., Tsuberi, B., Barshack, I., Kopolovic, J., Galun, E.,
Bujard, H. and Ben-Neriah, Y. (2000) High susceptibility
to bacterial infection, but no liver dysfunction,
in mice compromised for hepatocyte NF-κB activation.
Nat. Med. 6, 573-577.
Baron, U. and Bujard, H. (2000) The Tet repressor
based system for regulated gene expression in eukaryotic
cells: principles and advances. Methods Enzymol.
327, 659-686.
Mansuy, I. and Bujard, H. (2000) Tetracycline-regulated
gene expression in the brain. Curr. Op. Neurobiol.,
in press.
Urlinger, S., Baron, U., Thellmann, M., Hasan, M.,
Bujard, H. and Hillen, W. (2000). Exploring the
sequence space for tetracycline dependent transcriptional
activators: novel mutations yield expanded
range and sensitivity. Proc. Natl. Acad. Sci. USA, 97,
7963-68.
THESES
Diploma
Epp, Christian (1998): Analyse der humoralen Immunantwort
von Aotus-Affen gegen eine Immunisie–
rung mit MSP-1 aus Plasmodium falciparum
Schönig, Kai (1998): Konstruktion und Analyse von
„integrierten Regulationseinheiten“ zur Tetrazyklin
kontrollierten Genexpression in Säugerzellen
Schmid, Christina (1999): Untersuchungen zum Oberflächenprotein
1 von Merozoiten (MSP-1) des Mala–
riaerregers Plasmodium falciparum: Kontrollierte Synthese
des 42kDa-Fragments in Säugerzellen
Dissertation
Baron, Udo (1998): Weiterentwicklung der Methodik
der Tetrazyklin-kontrollierten Genexpression zur Ana–
lyse der Funktion von Genen in komplexen eukaryotischen
Systemen.
Awards
Ruperto Carola Preis 1998 der Universität Heidelberg,
for outstanding PhD Thesis to Udo Baron
STRUCTURE OF THE GROUP
E-mail: bujardh@sun0.urz.uni-heidelberg.de
Group leader Bujard, Hermann, Prof. Dr.
Guest scientist del Portillo, Hernando, Prof. Dr.*
Research
Associate Freundlieb, Sabine, Dr.
Postdoctoral Baron, Udo, PhD
fellows Fernandez-Becerra, Carmen, PhD*
Hasan, Mazahir, PhD*
Löw, Rainer, PhD*
Weerasuriya, Siromi, PhD*
Ph.D. students Berger, Stefan, Dipl.Chem.
Epp, Christian, Dipl.Biol.
Idler, Irina, Dipl.Biol.*
Kolster, Kathrin, Dipl.Biol.*
Schönig, Kai, Dipl.Biol.
Türbachova, Ivana, Dipl.Biol.
Westerfeld, Nicole, Dipl.Biol.
Diploma
students Baldinger, Tina*
Schmid, Christina*
Techn. assistants Rittirsch, Melanie*
Schirra-Müller, Christiane
Wahedy, Soraya*
* part of the time reported
53

Christine Clayton
Molecular Cell Biology of Trypanosomes
Introduction
Salivarian trypanosomes are unicellular parasites that
live in the blood and tissue fluids of mammals. They
cause sleeping sickness in humans and also infect
domestic animals and wildlife, mainly in subSaharan
Africa. The geographical restriction is determined by
the fact that the parasites are usually transmitted from
one mammal to the next by Tsetse flies. They develop
and multiply within the insect midgut and can undergo
a sexual cycle before infection of another mammal
via the insect salivary glands. Salivarian trypanosomes
do not multiply inside cells, unlike the closely
related tropical disease parasites Trypanosoma cruzi
and Leishmania. At present about 300 000 people are
infected with the sleeping sickness trypanosomes Trypanosoma
brucei rhodesiense and Trypanosoma gambiense.
The disease is invariably fatal unless treated,
and very few drugs are available, especially to combat
the late stages of the disease when the parasites invade
the brain. Moreover, most of the available drugs are
either too expensive or unacceptably toxic and drug
resistance is developing.
Trypanosomes and Leishmanias belong to the genus
Kinetoplastida, which branched extremely early in
eucaryotic evolution. As a consequence they exhibit
a number of remarkable deviations from standard
eucaryotic paradigms. In the realm of gene expression,
most transcription is polycistronic, all mRNAs are
trans spliced, there is almost no developmental regulation
of transcription, and the mitochondrial RNAs
are extensively edited. Energy and redox metabolism
also exhibit unique features: for example, the glycolytic
enzymes are compartmentalised in an organelle,
54
Figure 1: Electron micrographs of trypanosomes. On the left
are normal trypanosomes with many round glycosomes (Gly).
On the right are cells with a 90% reduction in PEXII (Evelyn
Baumgart, Anatomy Dept.).
the glycosome. At the same time, the parasites show
many conserved features. For example, the glycosome
is a specialised type of peroxisome, and trans splicing
is mechanistically related to cis splicing.
Our work concentrates on Trypanosoma brucei brucei,
a close relative of the sleeping sickness trypanosomes
which is not infective for humans. The parasites grow
in laboratory rodents or in vitro and life-cycle related
differentiation can be mimicked in culture. They are
diploid, carrying about 60 Mb of DNA on 11 chromosome
pairs; a sequencing project is in progress. A full
palette of genetic manipulation methods is available,
including gene replacement by homologous recombination
and inducible gene expression. Conditional
knockouts can be made by placing the gene of interest
under control of a tetracycline-inducible promoter,
then deleting the remaining gene copies via homologous
recombination.
In our research on trypanosomes, we try to keep two
aims in view. Firstly, we concentrate on aspects of
the parasites that uniquely distinguish them from their
mammalian hosts, and may therefore represent suitable
targets for specific anti-parasitic chemotherapy.
Secondly, by examining the similarities and differences
between fundamental processes in trypanosomes,
mammals and yeast we can clearly distinguish
those aspects that are conserved throughout eucaryotic
evolution and therefore likely to be essential from
those that are required for specialised lifestyles.
Control of gene expression
Maciej Drodzd, Antonio Gonzales, Claudia Hartmann,
Henriette Irmer, Luis Quilada
Specialised surface molecules are essential for the
survival and virulence of pathogens. In the case of
trypanosomes, the forms that grow in the mammal
(bloodstream forms) wear a uniform coat of variant
surface glycoprotein (VSG), expressed from a single
gene chosen from a repertoire of up to 1000 different
VSG genes. The VSG expressed can be changed either
through recombination or by transcriptional switches.
This enables the parasites to change their surface coat,
thereby evading the immune response. In the Tsetse
midgut, the VSG is replaced by the EP and GPEET
repetitive, acidic proteins. All these surface proteins
are retained on the plasma membrane by a glycosyl
phosphatidylinositol (GPI) lipid anchor.
Normally, protein-coding genes can only be functionally
expressed by RNA polymerase II. This is because
the mRNAs have to be modified at the 5‘-end by
enzymes that are associated exclusively with RNA
polymerase II. The Kinetoplastids are not subject to
this restriction because the 5‘ spliced leader is capped.
The VSG and the EP/GPEET genes are transcribed
by RNA polymerase I as part of polycistronic tran-
scription units. Nearly all other protein-coding genes
are transcribed by RNA polymerase II, but again transcription
is polycistronic. Although the expression of
many genes has to be strongly regulated to enable
survival in the disparate environments of Tsetse and
mammal, there is no evidence for any developmental
control of RNA polymerase II transcription. Instead,
regulation usually requires sequences which are found
in the 3‘-untranslated regions and control RNA degradation
and/or translation. Polymerase I-mediated transcription
of the VSG genes is, exceptionally, subject to
strong developmental regulation, but the EP/GPEET
gene transcription is only weakly regulated.
To compensate for the lack of transcriptional regulation,
the EP/GPEET mRNAs are extremely unstable
and very poorly translated in bloodstream forms,
so that no protein product is detectable. A uridinerich
26 nt sequence in the 3‘-untranslated region of
EP/GPEET mRNA is essential for this control. We
have examined the mechanism of RNA degradation
in detail. Relatively stable RNAs such as the actin
mRNA appear to be degraded by the standard mechanism
seen in eucaryotes: initial destruction of the
poly(A) tail at the 3‘-end is followed by degradation
of the rest of the mRNA. The EP/GPEET mRNAs
are in contrast rapidly degraded in bloodstream forms
without prior degradation of the poly(A) tail. Detailed
characterisation of the EP RNA secondary structure
indicated that the 26mer is in an exposed, extended
conformation, so it should be accessible to RNA-binding
proteins or specific endonucleases. Searches for
specific 26mer-binding proteins by several methods
have however proved fruitless.
Searches of the available genome sequence have
revealed a number of potential trypanosome proteins
that are very similar to yeast enzymes involved in
mRNA degradation and processing of stable RNAs, or
55

to proteins involved in regulation of RNA processing.
The relevant trypanosome genes and proteins are currently
being functionally characterised in order to pin
down their roles in regulated and constitutive RNA
degradation.
The Glycosome
Christina Guerra-Giraldez, Sandra Helfert, Alexander
Maier, Sebastian Schulreich
Bloodstream trypanosomes compartmentalise the first
nine enzymes of glucose and glycerol metabolism in
a peroxisome-related microbody, the glycosome. Glucose
is metabolised mostly to pyruvate, with a very
low energy yield of only 2 molecules of ATP per molecule
of glucose. In procyclic trypanosomes, the major
energy sources are amino acids; although glycolysis
is still present, the products are much more efficiently
used in a well-developed mitochondrion, yielding up
to ten times as much ATP.
Normally, bloodstream trypanosome glycolysis involves
an oxidation step. To maintain this oxidation,
the parasites have two alternatives. Under aerobic conditions
they can indirectly use oxygen, via a mitochondrial
oxidase. Alternatively, at least in short-term
incubations, they can recover oxidising power by a
corresponding reduction of triose phosphate, converting
each molecule of glucose to one molecule each of
pyruvate and glycerol, generating only one molecule
of ATP per glucose. It has therefore generally been
assumed that bloodstream trypanosomes can survive
anaerobically. By combining reverse genetics with the
use of inhibitors, we have found that trypanosomes
cannot survive anaerobically. This opens up new possibilities
for chemotherapy.
The compartmentation of glycolysis in a microbody
is unique in evolution. We would like to know which
(if any) advantages this compartmentation affords.
56
We have therefore been attempting to create conditional
mutants in glycosome membrane biogenesis.
The overall strategy is to identify genes encoding
glycosomal membrane proteins, then to use tetracycline-
inducible gene expression to make a conditional
knockout. The first candidates for this approach were
the two most abundant proteins of the glycosomal
membrane. The first to be studied, a 24kD protein, is
the trypanosome homologue of yeast Pex11p, which
is required for peroxisome division. The trypanosome
PEX11 gene complements the yeast pex11∆
deletion mutant, and parasites with reduced levels of
PEX11 have fewer, larger glycosomes than normal.
The second protein, GIM5, has a mass of 26kD and
shows no significant similarity to any other proteins
in the databases (apart from two predicted proteins
from the closely-related parasite Leishmania). Experiments
with conditional knockouts have demonstrated
that GIM5 is essential for parasite survival, but have
not yet given any indication of its precise function.
The protein spans the membrane twice, with both termini
exposed on the cytosolic side, and forms dimers
in vivo.
Because glycosomes are related to peroxisomes and
the mechanism of organelle biogenesis is conserved,
we can also find genes involved in glycosome biogenesis
by homology. One candidate for this approach is
the trypanosome PEX2 gene. PEX2 is essential for
peroxisome membrane biogenesis in yeast. The generation
of conditional knockouts is in progress.
The secretory pathway
Iris Ansorge, Michael Herberger, Alexander Maier
The secretory pathway of kinetoplastids is similar to
that of other eucaryotes. The predominance of GPIanchored
surface molecules is however a specialisa-
tion that appears to be unique to primitive unicellular
organisms. Although a few transmembrane proteins
have been identified, the vast majority of trypanosome
surface proteins are GPI-anchored. Even the transferrin
receptor is a heterodimer that is completely unrelated
to mammalian transferrin receptors; one of the
subunits, ESAG6, is GPI-anchored, while its partner,
ESAG7, lacks any direct membrane attachment. The
anchors of bloodstream trypanosomes contain 14-carbon
lipids so are shorter than the lipids on mammalian
anchors. In mammalian cells, GPI-anchored proteins
partition into lipid microdomains („rafts“) that
are poorly soluble in Triton X-100 and may be important
in selective trafficking of GPI-anchored proteins
to particular parts of the plasma membrane. The trypanosome
plasma membrane has three specialised
domains: the flagellar membrane, the rest of the surface,
and an inwardly-directed pocket at the base of
the flagellum which is the only site of membrane trafficking
(the flagellar pocket). Some proteins (such
as VSG) cover the entire surface while others are
restricted to one or two domains. Collaborative experiments
demonstrated that VSG, which forms homodimers,
partitions poorly (50%) into the detergent-insoluble
fraction, whether it is integrated into the trypanosome
membrane or in artificial lipid bilayers. The
heterodimeric transferrin receptor, which is normally
restricted to the membrane and matrix of the flagellar
pocket, is not retained at all in detergent-insoluble
fractions, perhaps because only a single GPI anchor
per dimer is available.
Coatomer is a complex of seven proteins (α, β, γ, δ,
ε and ζ –COP) that is involved in the formation of vesicles
that move between the endoplasmatic reticulum
and the Golgi apparatus. Work in Heidelberg and elsewhere
has yielded results suggesting that the endoplasmatic
reticulum and coatomer are involved in peroxisome
biogenesis. The C-terminus of mammalian
PEX11 includes a coatomer-binding sequence. To find
out whther coatomer-binding by PEX11 was conserved
throughout evolution, we tested the interaction
of the relevant PEX11 sequences with coatomer from
trypanosomes, yeast and mammals. A necessary preliminary
was the partial characterisation of trypanosome
coatomer. We found that that the specificity of
coatomer binding is conserved throughout evolution.
Trypanosome PEX11 binds coatomer, but mutants
that lack binding are still functional in vivo. Yeast
PEX11 does not have a C-terminal coatomer-binding
sequence. Thus binding of coatomer by the C-terminus
of PEX11 is not functionally essential.
Redox metabolism and drug resistance
Stephan Krieger, Sanjay Shahi, Claudia Hartmann
Like all other cells, trypanosomes require a reducing
environment in their cytosol, and contain a number of
cofactors and proteins that act to maintain the correct
redox balance. In nearly all cells from bacteria to man,
one of these cofactors is glutathione. The thiol group
of glutathione is maintained in the reduced state by
glutathione reductase. In trypanosomes, most of the
glutathione is found in the form of trypanothione (two
glutathione moieties conjugated to spermidine). Trypanothione
is maintained in the dithiol state by a
highly specific enzyme, trypanothione reductase. The
uniqueness of this system makes it a strong candidate
for targeted drug design. To test the precise role of
trypanothione reductase in vivo, we created trypanosomes
with an inducible knockout. Trypanothione
reductase was found to be essential for trypanosome
growth both in vitro and in a mammalian host. This
was the first time that an inducible knockout parasite
had been tested in an animal model system. We found
that at least 90% inhibition of trypanothione reductase
will be required for effective chemotherapy. Parasites
57

with low levels of the enzyme were highly susceptible
to oxidative stress.
A variety of drugs is excreted from cancer cells in the
form of glutathione-drug conjugates by ABC transporters
of the MRP (multidrug resistance protein)
family. In some Leishmania species, arsenite and antimonial
drugs can be sequestered as conjugates with
trypanothione. The only drugs available against latestage
trypanosomiasis in east Africa are arsenical
derivatives, but resistance to these drugs is emerging.
To find out if MRP-like trypanothione-arsenical conjugate
pumps might play a role in resistance, we have
cloned and sequenced trypanosome homologues of
two Leishmania MRP-like genes. Cells over-expressing
the transporters will be characterised with respect
to drug sensitivity and other characteristics.
Metabolic compartmentation in Toxoplasma
Martina Ding, Frank Voncken
D. Soldati (ZMBH) and I became intrigued by the
complete absence of reports of peroxisomes in malaria
parasites and related organisms such as Toxoplasma. A
partial sequence for catalase (a peroxisomal marker)
was identified in the Toxoplasma sequence database.
The predicted protein has all concensus amino acids
for enzyme activity, and a C-terminal peroxisomal
targeting signal. To our surprise, immunofluorescent
labelling of Toxoplasma with antibodies to two different
anti-peptide antibodies gave predominantly cytosolic
labelling, and the addition of the targeting signal
to the green flourescent protein also resulted in cytosolic
flourescence. Cell fractionations gave similar
results. Thus so far we have no evidence that Toxoplasma
contains peroxisomes.
58
Collaborations
Dr. Luise Krauth-Siegel (Biochemie-Zentrum, Heidelberg)
– trypanothione reductase.
Dr. Dietmar Steverding (Parasitologie, Heidelberg) –
transferrin receptor.
Dr. Martina Bremser and Prof. F. Wieland (Biochemie-Zentrum,
Heidelberg) - coatomer.
Dr. Dominique Soldati (ZMBH) – Toxoplasma peroxisomes
Drs. Jürgen Benting and Kai Simons, EMBL (rafts)
Dr. Paul Michels (Institute for Cellular Pathology,
Brussels, Belgium) - glycosomes and glycolysis.
Prof. Kenneth Stuart (Seattle Biomedical Research
Institute, Washington, Seattle) and Prof. Ullrich
Goeringer (Universität Darmstadt) – inducible knockouts
of genes involved in RNA editing.
Dr. Roland Kaminsky and Dr. Pascal Maser (Tropeninstitut,
Basel) - drug resistance.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 352 “Molekulare Mechanismen intrazellulärer
Transportprozesse”, SFB 544 “Kontrolle tropischer
Infektionskrankheiten”, Graduiertenkolleg “Kontrolle
der Genexpression in pathogenen Mikroorganismen”
and Projekt-’Sachbeihilfen’) from the Human Frontier
Science Programme Organization, the EMBO, the
DAAD, the BMBF (Forschungsschwerpunkt Tropenmedizin)
and from the Fonds der Chemischen Industrie.
PUBLICATIONS
Original articles
Ansorge, I., Steverding, D., Melville, S., Hartmann,
C., and Clayton, C. (1999). Transcription of ‘inactive’
expression sites in African trypanosomes leads
to expression of multiple transferrin receptor RNAs
in bloodstream forms. Mol. Biochem. Parasitol. 101,
81-94.
Benting, J., Rietveld, A., Ansorge, I., and Simons, K.
(1999). Acyl and alkyl chain length of GPI anchors is
critical for raft association in vitro. FEBS Lett. 462,
47-50.
Blattner, J., Helfert, S., Michels, P., and Clayton,
C. E. (1998). Compartmentation of phosphoglycerate
kinase in Trypanosoma brucei plays a critical role
in parasite energy metabolism. Proc. Natl. Acad. Sci.
USA 95, 11596-11600.
Clayton, C.E., Ha, S., Rusche, L., Hartmann, C.,
Beverley, S. M. (2000). Tests of heterologous promoters
and intergenic regions in Leishmania major. Mol.
Biochem. Parasit. 105, 163-167.
Ding, M., Clayton, C.E. and Soldati, D. (2000). Toxoplasma
gondii catalase: are there peroxisomes in Toxoplasma?
. J. Cell Sci. 105, (in press).
Drozdz, M., and Clayton, C. E. (1999). Confirmation
of a regulatory 3‘-untranslated region from Trypanosoma
brucei. RNA 5, 1632-1644.
Hartmann, C., Hotz, H.-R., McAndrew, M., and Clayton,
C. (1998). Effect of multiple downstream splice
sites on polyadenylation in Trypanosoma brucei. Mol.
Biochem. Parasitol. 93, 149-152.
Hotz, H.-R., Biebinger, S., Flaspohler, J., and Clayton,
C. E. (1998). PARP gene expression: regulation at
many levels. Mol. Biochem. Parasitol. 91, 131-143.
Krieger, S., Schwarz, W., Ariyanagam, M.R., Fairlamb,
A., Krauth-Siegel, L., and Clayton, C. E. (2000).
Trypanosomes lacking trypanothione reductase are
avirulent and show increased sensitivity to oxidative
stress. Mol. Microbiol. 35, 542-552.
Lorenz, P., Meier, A., Erdmann, R., Baumgart, E., and
Clayton, C. (1998). Elongation and clustering of glycosomes
in Trypanosoma brucei overexpressing the
glycosomal Pex11p. EMBO J. 17, 3542-3555.
McAndrew, M., Graham, S. and Clayton, C.E. (1998).
Testing promoter activity in the trypanosome genome:
isolation of a metacyclic-type VSG promoter, and
unexpected insights into RNA polymerase II transcription.
Exp. Parasitol. 90, 65-76.
Reviews
Hotz, H.-R., Biebinger, S., Flaspohler, J., and Clayton,C.E.
(1998). PARP Gene expression: regulation at
many levels. Francqui Symposium Proceedings and
Mol. Biochem. Parasitol. 91, 131-143.
Clayton, C. E., et al. (1998). Genetic nomenclature for
Trypanosoma and Leishmania. Mol. Biochem. Parasitol.
97, 221-224.
Clayton, C. E. (1999). Genetic manipulation of Kinetoplastida.
Parasitol. Today 15, 372-378.
Roditi, I., and Clayton, C. E. (1999). An unambiguous
nomenclature for the major surface glycoprotein
59

genes of the procyclic form of Trypanosoma brucei.
Mol. Biochem. Parasit. 103, 99-100.
Clayton, C.E., Maier, A., Lorenz, P., Blattner, J., Helfert,
S., Krieger, S. (2000). Strange characteristics of
kinetoplastid protists: targets for antiparasitic chemotherapy?
Nova Acta Leopoldina 80, 15-25.
Clayton, C.E. (1999). Why do we need standard
genetic nomenclature for parasites? Acta Tropica (in
press)
THESES
Diploma
Ding, M. (1998). Charakterisierung von Organellen in
Toxoplasma gondii.
Herberger, M. (1999). Klonierung und Expression
eines Cysteine Rich Acidic Integral Membrane
(CRAM)-GFP-Fusionsgens in Trypanosoma brucei
brucei.
Dissertations
Krieger, S. (1998). Die Trypanothionreduktase bei
Trypanosoma brucei:Etablierung und Chatakterisie–
rung einer TR-defizienten Zelllinie.
Maier, A. (1999). Funktionelle Charakterisierung
glykosomaler Membranproteine aus Trypanosoma
brucei.
60
STRUCTURE OF THE GROUP
E-mail: clayton@zmbh.uni-heidelberg.de
Group leader: Clayton, Christine, Prof. Dr.
Postdoctoral fellows
Ansorge, Iris, Dr.
Estévez, Antonio, PhD*
Quilada, Luis, PhD*
Voncken, Frank, PhD*
Ph.D. students Ding, Martine, Dipl. Biol.*
Drodzd, Maciej, Dipl. Biol.
Guerra-Giraldez, Christina, MSc.
Helfert, Sandra, Dipl. Biol.
Irmer, Henriette, Dipl. Biol.
Krieger, Stephan, Dipl. Biol.
Maier, Alexander, Dipl. Biol.
Diploma students Ding, Martina *
Schulreich, Sebastian *
Herberger, Michael *
Techn. assistant Hartmann, Claudia
*part of the time reported
Bernhard Dobberstein
Protein Targeting and Intracellular
Sorting
Protein translocation across the membrane of the endoplasmic
reticulum (ER) involves cytosolic chaperones,
docking receptors, a translocation channel (translocon)
and in some cases a “translocation motor” which
drives the actual translocation (for review see Schatz
and Dobberstein, 1996). Once in the ER, proteins are
folded, modified and - after a quality control - packed
into vesicles and transported to the Golgi complex and
the trans-Golgi network. From there they can either
be further transported to the plasma membrane or to
organelles of the endosomal system. A major focus of
the work of our group is the analysis of mechanisms
involved in targeting proteins to the ER membrane
and in their translocation across or insertion into this
membrane. Special emphasis is on the control and
regulation of these processes.
Cotranslational targeting of nascent secretory and
membrane proteins to the ER membrane involves
the signal recognition particle (SRP) and its receptor
(SRP-receptor or docking protein). SRP interacts with
the signal sequence of the nascent proteins and mediates
their transfer to the ER membrane. The SRP
receptor catalyzes the release of the nascent chain
from SRP and its insertion into the translocon. SRP
comprizes a 7S RNA and six polypeptides of 9, 14, 19,
54, 68 and 72 kDa. The SRP54 subunit is a GTPase
to which the signal sequence of a nascent polypeptide
chain binds. The SRP receptor consists of an α and β
subunit both of which are GTPases. The translocation
channel is formed by the Sec61p complex and several
accessory proteins.
Functional analysis of the three translocation-
GTPases, SRP54, SRP receptor α and β
G. Bacher and M. Pool
The GTPase cycle of SRP54 was studied in its functional
context, a ribosome nascent chain complex
(RNC) and membranes containing different components
of the translocation complex (Sec61p, TRAMp,
SRP receptor). It was found that the ribosome stimulates
GTP binding to SRP54 and that the GTP-bound
state of SRP54 is important for high affinity binding
of SRP to the SRP receptor α (Bacher et al. 1996).
Two ribosomal proteins were identified that contact
the GTPase domain and M-domain of SRP54 respectively
(Pool and Dobberstein, in preparation). It is proposed
that one of these proteins allows SRP to scan
the RNC for the presence of a signal sequence and
that the second protein functions in stimulating GDP /
GTP exchange on SRP 54.
The SRP receptor β subunit is anchored in the membrane
and attaches SRP receptor α to the membrane.
We have investigated GTP binding and hydrolysis of
SRP receptor β. We found that SRP receptor β binds
GTP with high affinity and interacts with ribosomes
in the GTP-bound state. Subsequently, the ribosome
increases the GTPase activity of SRP receptor β and
thus functions as a GTPase activating component for
SRP receptor β. We propose that SRP receptor β regulates
the interaction of SRP receptor with the ribosome
and thereby allows SRP receptor α to scan membrane
bound ribosomes for the presence of SRP (Bacher,
Pool and Dobberstein, 1999). Thus the two subunits
of the SRP receptor regulate ribosome binding to the
ER membrane and signal sequence insertion into the
translocon.
61

Protein Kinase C (PKC) phosphorylates essential
translocon components
O. Gruss with P. Feick, Homburg and R. Frank,
ZMBH and S. Owen
Secretion of proteins can occur in a constitutive or
regulated manner. In the exocrine pancreas Ca ++ activated
PKCs play a central role in the regulated secretion.
In order to identify possible targets for PKC
at the ER membrane we have characterized PKCphosphorylated
proteins of rough microsomes and the
rough ER of intact cells. We found that essential components
of the translocation machinery become phosphorylated
in a Ca ++ -dependent manner. Among the
proteins were the α subunit of the SRP receptor, the
β subunit of the Sec61p complex and the TRAM protein.
Isoform -specific antibodies revealed the presence
of PKC α and β on rough ER membranes. Purified
PKCs from rat brain phosphorylated the same
translocon components as the endogenous Ca ++ -
dependent kinases. Phosphorylation of translocon proteins
was also observed in vivo and could be stimulated
by phorbol esters. Phosphorylation of microsomal
proteins by PKCs increased protein translocation
efficiency in vitro. This suggests phosphorylation
as a further level of regulation of protein translocation
across the ER membrane (Gruss et al., 1999).
Protein complexes of the translocation site
L. Wang
Several proteins or protein complexes function at
different stages of the targeting or translocation process.
Among them are the targeting components (SRP
and SRP-receptor) the translocon (Sec61p complex),
translocon associated components (TRAMp, RAMP4)
oligosaccharyl transferase (OST) and signal peptidase
complex. Depending on their functional state they
62
assemble transiently into supercomplexes. This is particularly
evident for the translocon which can be unengaged,
involved in cotranslational or posttranslational
translocation or function in retrotranslocation. To analyse
protein complexes of the rough ER we used mild
solubilisation of ER membrane proteins, fractionation
and blue native PAGE. Consistent with their multiple
engagements we find targeting and translocation components
in distinct oligomeric complexes (Wang and
Dobberstein, 1999).
Translocation-pausing mediated by RAMP4
K. Schröder, B. Martoglio, M. Hofmann in collaboration
with E. Hartman, Göttingen, S. Prehn, Berlin and
T. Rapoport, Boston
Passage of nascent polypeptides across the membrane
of the ER proceeds through a proteinaceous, aqueous
channel formed by the Sec61p complex. To identify
components that may regulate translocation by interacting
with nascent polypeptides in the translocon,
we used site-specific photo-crosslinking. Crosslinkers
were placed around a consensus site for Asn-linked
glycosylation. As a model protein we used the MHC
class II-associated Invariant chain, which contains two
closely spaced N-glycosylation sites. We found that a
region C-terminal of the two consensus glycosylation
sites in the Invariant chain binds in a hydrophobic
interaction to RAMP4, a previously identified Ribosome
Associated Membrane Protein (Görlich and
Rapoport (1993) Cell, 75, 615-630). RAMP4 is a
small, tail anchored protein of 66 amino acid residues.
The interaction of RAMP4 with Ii occurred when
nascent Ii chains reached a length of 170 amino
acid residues and persisted until Ii chain completion,
suggesting translocational pausing (Nakahara et al.
(1994) J. Biol. Chem., 269, 7617-7622). Site-directed
mutagenesis revealed that the region of Ii interacting
with RAMP4 contains essential hydrophobic amino
acid residues. Exchange of these residues for serines
led to a reduced interaction with RAMP4 and inefficient
N-glycosylation. We propose that RAMP4 controls
modification of Ii and possibly also of other
secretory and membrane proteins containing specific
RAMP4-interacting sequences. Efficient or variable
glycosylation of Ii may contribute to its capacity to
modulate antigen presentation by MHC class II molecules
(Schröder et al., 1999).
Figure 1: Hypothetical model of control of Ii chain glycosylation
by RAMP4. Three stages of Ii insertion into the membrane
are depicted. (I) Nascent Ii inserts into the translocon
in a loop-like fashion. (II) When ~170 amino acid residues
have been polymerized Ii interacts via its RIS (white box) with
RAMP4. (III) Translocation is arrested and OST brought into
position to efficiently glycosylate Ii. Upon chain completion, Ii
detaches from RAMP4. Oligosaccharides are shown as forked
structures.
Signal sequences - more than just greasy peptides
B. Martoglio, ZMBH / Zürich and M. Fröschke
Signal sequences that target newly synthesized proteins
to the ER contain a hydrophobic core region but
otherwise show a great variation in both, overall length
and amino acid sequence. Recently, it has become
clear that this variation allows signal sequences to
specify different modes of targeting and membrane
insertion and even to perform functions after being
cleaved from their parent protein. Signal sequences
are therefore not simply greasy peptides but sophisticated,
multipurpose peptides containing a wealth of
functional information (Martoglio and Dobberstein,
1998). Further work is directed to the functional analysis
of unusually long signal sequences during targeting,
proteolytic processing and after signal sequence
cleavage.
Signal sequences of the prion protein (PrP)
C. Hölscher and U. C. Bach
Prion protein (PrP) is synthesised at the membrane of
the ER in three different topological forms, a secreted
one (secPrP) and two that span the membrane in opposite
orientations ( Ntm PrP and Ctm PrP). To identify signal
sequences in PrP that determine the generation of the
multiple forms of PrP we performed a deletion analysis.
We found that PrP has – besides its N-terminal
signal sequence- a C-terminal signal sequence that
functions posttranslationally. The C-terminal signal
sequence mainly mediates synthesis of Ctm PrP. Ctm PrP
has been causally connected to certain inherited forms
of neurodegeneration (Hegde et al. Nature, 402, 822 –
826, 1999). We suggest that multiple forms of PrP are
generated by the differential use of a N-terminal and
C-terminal signal sequence (Hölscher, Bach and Dobberstein,
in preparation).
mRNA transport and protein localisation
S. Frey and M. Seedorf in collaboration with R.
Jansen, ZMBH
mRNA always exits the nucleus in a complex with
RNA-binding proteins. Prior to translation complexes
63

containing a subset of mRNAs are transported to
specific regions inside the cell. This mechanism of
local translation of transported mRNAs might play
an important role to support protein targeting. The
mRNA-binding protein vigilin displays unique features
making it a good candidate to be a player in this
process. Vigilin cofractionates with free and membrane
bound ribosomes and shows an intracellular distribution
similar as the endoplasmatic reticulum (ER).
The protein consists almost exclusivly of RNA-binding
domains. These domains provide multiple binding
sites for messenger and ribosomal RNAs. To study the
function of this unique RNA-binding protein we use
budding yeast as a model system. Yeast expresses the
structurally closely related protein Scp160p, which
shows a similar intracellular distribution as vigilin.
The SCP160 gene is not required for cell growth but
a mutant without SCP160 fail to localize one specific
mRNA. The majority of Scp160 protein associates
with translating ribosomes, suggesting a function
in the control of translation. Strong expression of
Scp160p abolishes growth and reduces translation
rates of a subset of so far unknown proteins. Cytoplasmic
steady state localisation of Scp160p is independent
of bulk mRNA export from the nucleus supporting
the idea that Scp160p function is primary cytoplasmic
and that the protein is not exported from the
nucleus as constituent of a mRNA complex. Scp160p
bears a masked nuclear import signal indicating that
the function of Scp160p might be regulated by relocation
of the protein to the nucleus.
Microsomal ATP-Transport in yeast
G. Konrad, T. Schlecker and P. Mayinger in collaboration
with V. Bankaitis, Birmingham, Alabama
ATP Transport into the ER is essential for ATP con-
64
suming reactions inside the ER lumen, including protein
translocation and protein folding. We have characterized
this nucleotide transport reaction as a specific
ADP/ATP antiport and we have identified the
Sac1 protein as an important regulator for this process.
Sac1p is an ER and Golgi membrane protein with
homology to a number of yeast and mammalian inositol
phosphatases. It was shown recently that the N-terminal
portion of Sac1p displays polyphosphoinositide
phosphatase activity with unique enzymatic specificity.
Consistent with these findings we obtained strong
evidence in vitro that microsomal ATP transport is
regulated via specific phosphoinositides.
Cellular role of Sac1p in phosphoinositide
signaling
A. Then
Sac1p plays additional roles in the regulation of
intracellular membrane trafficking, phospholipid metabolism
and the actin cytoskeleton. To elucidate the cellular
function of Sac1p in more detail, we performed
a synthetic lethal screen. In this screen we identified a
novel allele of the SLT2 gene, which results in a severe
growth defect when combined with a disruption of the
SAC1 gene. The SLT2 gene codes for a PKC1-dependent
MAP kinase, which plays an important role in
cell wall integrity and in organization of cortical actin
and performs an essential function in the context of
a sac1D background. This genetic interaction with
SLT2 defines a novel important function of the SAC1
gene in regulating cell wall biogenesis and polarized
secretion. Epistatic analysis places the SAC1 gene parallel
to the PKC1 dependent SLT2 MAP kinase, linking
this kinase cascade to phosphoinositide signaling.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 352 “Molekulare Mechanismen intrazellulärer
Transportprozesse”, Graduiertenkolleg “Molekulare
Zellbiologie”), from the Landesforschungsschwerpunkt
“Protein-Faltung und -Transport: Mechanismen
und Pathobiochemie”, from the EU (“Signal Recognition
Particle-Network”) and from the Fonds der Chemischen
Industrie.
PUBLICATIONS
Martoglio, B., Hauser, S. and Dobberstein, B. (1998).
Cotranslational translocation of proteins into microsomes
derived from the rough endoplasmic reticulum
of mammalian cells. In Cell Biology, A Laboratory
Handbook. Academic Press, 2nd ed. Vol. 2, pp.
265-273.
Graf, R., Brunner, J., Dobberstein, B. and Martoglio,
B. (1998). Probing the molecular environment of proteins
by site-specific photocrosslinking. In Cell Biology,
A Laboratory Handbook. Academic Press 2nd ed.
Vol 4, pp. 495-501.
Martoglio, B. & Dobberstein, B. (1998). Signal
sequences – more than just greasy peptides. Trends in
Cell Biology 8, 410-415.
Dube, P., Bacher, G., Stark, H., Müller, F., Zemlin, F.,
van Heel, M. and Brimacombe, R. (1998). Correlation
of the expansion segments in mammalian rRNA
with the fine structure of the 80S ribosome; a cryoelectron
microscopic reconstruction of the rabbit reticulocyte
ribosome at 21A resolution. J. Mol. Biol. 279,
403-421.
Otter-Nilsson, M., Hendriks, R., Pecheur-Huet, E.I.,
Hoekstra, D. and Nilsson, T. (1999). Cytosolic
ATPases, p97 and NSF, are sufficient to mediate rapid
membrane fusion. EMBO J. 18, 2074-2083.
Gruss, O.J., Feick, P., Frank, R. and Dobberstein, B.
(1999). Phosphorylation of components of the ER
translocation site. Eur. J. Biochem. 260, 785-793.
Schröder, K., Martoglio, B., Hofmann, M., Hölscher,
C., Hartmann, E., Prehn, S., Rapoport, T.A. and Dobberstein,
B. (1999). Control of glycosylation of MHC
class II-associated invariant chain by translocon-associated
RAMP4. EMBO J. 18, 4804-4815.
Bacher, G., Pool, M. and Dobberstein, B. (1999). The
ribosome regulates the GTPase of the β-subunit of the
signal recognition particle receptor. J. Cell Biol. 146,
723-730.
Wang, L. and Dobberstein, B. (1999). Oligomeric
complexes involved in translocation of proteins across
the membrane of the endoplasmic reticulum. FEBS
Lett. 457, 316-22.
Hofmann , M.W., Höning, S., Rodinov, D., Dobberstein,
B., von Figura, K. and Bakke, O. (1999).
The leucine-based sorting motifs in the cytoplasmic
domain of the invariant chain are recognized by the
medium chains of the clathrin adaptors AP1 and AP2.
J. Biol. Chem. 274, 36153-36158.
Kochendörfer, K.-U., Then, A. R., Kearns, B. G.,
Bankaitis, V. A., and Mayinger, P. (1999). Sac1p plays
a crucial role in microsomal ATP transport, which
is distinct from its function in Golgi phospholipid
metabolism. EMBO J. 18, 1506-1515.
65

Then, A. R., Berger, J., Schwarz, H., and Mayinger, P.
(2000). Sac1p regulates yeast cell wall organization in
a signaling pathway that is functionally related to the
Slt2p MAP kinase cascade. (in press).
THESES
Diploma
Grimm, O. (1999). Isolation and partial characterization
of proteins interacting with SRP-receptor.
Schlecker, T. (1999). Einfluß der intrazellulären Lokalisation
von Sac1p auf die Regulation des mikro–
somalen ATP-Transports.
Dissertations
Gruß, O. (1998). Regulation der Translokation neusyn–
thetisierter Polypeptide über die Membran des rauhen
endoplasmatischen Retikulums.
Then, A. (1999). Identifizierung von Faktoren die
funktionell mit dem Sac1p Protein in Saccharomyces
cerevisiae interagieren.
STRUCTURE OF THE GROUP
E-mail: dobberstein@zmbh.uni-heidelberg.de
Group leader Dobberstein, Bernhard, Prof. Dr.
Research
associates Mayinger, Peter, Dr.
Seedorf, Matthias, Dr.
Postdoctoral
fellows Barrett, Jeannie, Dr.*
Hendriks, Robertus, Dr.*
66
Hölscher, Christina, Dr.
Kipp, Bettina, Dr.*
Martoglio, Bruno, Dr.*
Owens, Sue, Dr.*
Pool, Martin, Dr.
Wang, Lin, Dr.*
Ph.D. students Frey, Steffen,*
Fröschke, Marc
Gruß, Oliver*
Konrad, Gerlinde*
Then, Angela
Diploma students Grimm, Oliver*
Schlecker, Tanja*
Techn. assistants Bach, Ute
Meese, Klaus
*part of the time reported
Dirk Görlich
Nucleocytoplasmic Transport
The need for nuclear transport
The nuclear envelope (NE) divides eukaryotic cells
into a nuclear and a cytoplasmic compartment and
thereby necessitates exchange between nucleus and
cytoplasm. Not only must all nuclear proteins, such as
histones and transcription factors, be imported from
the cytoplasm, but also, tRNA, rRNA and mRNA are
synthesised by transcription in the nucleus and need to
be exported to the cytoplasm where they function in
translation. Nuclear-cytoplasmic transport constitutes
a tremendous activity that requires considerable cellular
resources and involves probably > 100 different
and often very abundant proteins. However, these
expenses pay off as indicated by the fact that only
eukaryotes evolved into complex multicellular organisms.
One can think of several reasons why a cell
nucleus is required for such cellular complexity. First,
the containment of the genome in a specialised organelle
certainly improves genetic stability and allows
eukaryotes to handle more genetic information than
prokaryotes. Second, this compartmentation permits
key cellular events to be regulated at a level that
is unavailable to prokaryotes, e.g. by controlling the
access of transcriptional regulators to chromatin. A
further reason is the composition of typical eukaryotic
genes from exons and introns, which requires primary
transcripts to be spliced before translation may occur.
Translation of unspliced pre-mRNAs would produce
proteins that are not only non-functional but which
may potentially act as dominant-negative inhibitors.
The confinement of transcription and translation to
distinct compartments can therefore be considered a
good solution to avoid this problem.
Importin beta-like transport receptors
P. Schwarzmaier, U. Kutay, G. Lipowsky, F. Paraskeva,
S. Jäckel, K. Ribbeck, K. Dean, in collaboration
with E. Hartmann (Univ. Göttingen), R. Kraft and
S. Kostka (MDC, Berlin)
Nuclear-cytoplasmic transport proceeds through
nuclear pore complexes (NPCs) which allow passive
diffusion of small molecules up to 20-40kD and also
active, i.e., carrier-mediated transport of macromolecules.
This active transport can occur along a great
variety of distinct pathways, many of which are mediated
by transport receptors that are at least distantly
related to importin β. Transport receptors that confer
import into the nucleus are referred to as importins,
while export mediators are called exportins.
A major objective of our laboratory has been to obtain
a detailed and comprehensive overview of the various
nuclear transport pathways. To identify novel transport
receptors, we have employed a biochemical approach
combined with data base searches and have so far
arrived at 22 mammalian members of the importin β
superfamily. In the following, we will briefly summarise
some of our recent insights in the mechanistic
principles of transport receptor function and elaborate
some nuclear transport pathways in more detail.
Mechanistic principles of transport receptor
function
Importins or exportins are capable of mediating multiple
rounds of transport, which requires them to shuttle
continuously between nucleus and cytoplasm. A given
transport cycle, however, can only be productive if
the corresponding transport receptor carries cargo in
one direction only and returns „empty“ to the original
compartment. This asymmetry in the transport cycles
can be explained by the RanGTP-gradient model. Ran
67

switches between a GDP- and a GTP-bound form. Its
nucleotide-bound state is controlled by a number of
activities whose asymmetric nucleocytoplasmic distribution
is thought to result in a high nuclear RanGTP
concentration and very low RanGTP levels in the cytoplasm.
Importin-β like transport receptors are RanGTP
binding proteins that respond to this gradient by
loading and unloading their cargo in the appropriate
compartment. Importins bind their substrates in the
absence of RanGTP, i.e. in the cytoplasm and release
them upon encountering RanGTP in the nucleus. The
importins then return to the cytoplasm where RanGTP
is released (involving GTP hydrolysis), thereby allowing
the importin to bind and import the next cargo molecule.
Exportins respond to the RanGTP gradient in
exactly the opposite way; they bind their cargo preferentially
in the presence of RanGTP in the nucleus,
where they form a trimeric substrate-exportin-RanGTP
complex. The trimeric export complex is disassembled
after export to the cytoplasm and the „empty“ exportin
can re-enter the nucleus to participate in another round
of export.
Transport receptors bind their transport substrates in
many cases directly, an example being nuclear export
of tRNA by exportin-t (see below). In other cases, substrate
recognition is more complicated and involves an
adapter molecule. The best studied example for that is
the classical NLS import pathway, where the NLS is
recognised by the adapter importin α, which in turn
binds the actual transport receptor importin beta. This
complicates the corresponding transport cycle considerably
in that not only importin β, but also importin
α needs to be return to the cytoplasm. Interestingly,
importin α employs a specialised exportin, namely
CAS, for this purpose.
68
Figure 1: Transport cycles of Importins (Imp) and exportins
(Exp). For details, please see main text.
Recycling of snurportin 1 back to the cytoplasm
F. Paraskeva, U. Kutay, in collaboration with R. Lührmann
(Univ. Marburg), F.R. Bischoff (DKFZ, Heidelberg),
E. Izaurralde (EMBL, Heidelberg)
Snurportin 1 functions as an import adapter for U
snRNPs. It recognises the m 3 G-cap import signal of
U snRNPs and also binds Importin β which in turn
is the actual mediator of import. Just as importin α,
also snurportin 1 needs to be returned to the cytoplasm
after each round of import in order to accomplish further
import cycles. We found that this recycling is
mediated by CRM1. CRM1 was originally identified
as the exportin specific for leucine-rich nuclear export
signals (NESs). However, the CRM1/ snurportin 1
interaction differs significantly from that with other
export substrates: Firstly, snurportin 1 binds CRM1
not through a short, leucine-rich signal, but instead
through a large domain that comprises more than 200
residues. Secondly, snurportin 1 binds CRM1 100
times more tightly than an NES from the HIV Rev
protein. In addition, snurportin 1 can bind either the
m 3 G-cap import signal or CRM1, but not both at the
same time. This property ensures that CRM1 exports
only those snurportin 1 molecules which have already
released their cargo and thereby allows snurportin 1 to
mediate productive import cycles.
tRNA export
U. Kutay, G. Liposwsky, P. Schwarzmaier, in collaboration
with E. Izaurralde (EMBL, Heidelberg) and
F.R. Bischoff (DKFZ, Heidelberg)
tRNAs are synthesised as precursor molecules (pretRNAs),
mature to functional tRNA and finally get
exported to the cytoplasm. There, they participate in
cycles of aminoacylation, binding to the elongation
factor eE1A and function in translation. We have identified
exportin-t as the tRNA-specific exportin and
showed that it functions according to the exportinparadigm
described above. The maturation of pretRNAs
to functional tRNAs involves trimming of the
3´and 5´ends, post-transcriptional addition of the 3´
CCA end to which the amino acid is later attached,
and the modification of a number of nucleosides. It
is quite remarkable that exportin-t preferentially binds
and exports mature tRNA which contain correctly processed
3´and 5´ends and the appropriate nucleoside
modifications. Exportin-t mediated export thus constitutes
a proof-reading or quality-control mechanism
that co-ordinates RNA processing with export and
thereby helps to ensure that only functional tRNA
arrives in the cytoplasm.
Nuclear import of ribosomal proteins and
histone H1
S. Jäckel
The biogenesis of ribosomes is a very complex process
that also involves nuclear import and export events:
Ribosomal proteins are first imported from the cytoplasm,
assemble with rRNA in the nucleolus to form
ribosomal subunits which are then finally re-exported
to the cytoplasm.
We have studied nuclear import of ribosomal proteins
in higher eukaryotes, focusing on rpL23a. We found
that at least 4 distinct transport receptors, namely
importin β, transportin, importin 5 and importin 7
can directly bind and import rpL23a. This not only
assigned the first functions to importin 5 and 7,
but showed an apparently quite common principle
in nuclear import, namely that some substrates can
„choose“ between several carriers. L23a binds through
the same domain or „import signal“ to each of the 4
transport receptors.
69

Ribosomal proteins are generally very basic and have
a high tendency to precipitate and aggregate before
their assembly into ribosomal subunits. It is therefore
important to note that the importins shield rather large
domains of the ribosomal proteins, thereby preventing
undesired interactions in the cytoplasm. Thus, the
importins not only fulfil an import function but also
act as chaperonins.
Histones are also very basic and cause similar aggregation
problems as ribosomal proteins. This posed the
questions of whether histones are imported the same
way as ribosomal proteins. Surprisingly, we found that
the histone H1 cannot be imported into the nucleus
by any „single“ importin. Instead, the active species
in H1 import is an importin β/importin 7 heterodimer.
Both importins appear to contact the histone, probably
because a single import receptor is not sufficient
to completely wrap the extended and extremely basic
domain of this cargo.
Recycling of Ran back to the nucleus
K. Ribbeck, G. Lipowsky
A key element of current models for importin and
exportin function is that these factors normally enter
the nucleus Ran-free and exit as a complex with
RanGTP. Importins carry cargo in their Ran-free form,
while exportins do so when complexed with RanGTP.
Both have in common to constantly deplete Ran from
the nucleus (see Fig. 1). These transport cycles can
then only be maintained by an extremely efficient
nuclear import of Ran. The assumption that each
transport cycle of an importin β-like transport receptor
results in export of one Ran molecule would imply
that Ran crosses the nuclear envelope as frequently
as all these receptors together. For reasons of stoichiometry
it then appears impossible that Ran itself
70
is imported in a conventional way by an importin β
family transport receptor.
We have established an in vitro system to study nuclear
import of Ran and found that this process requires
some soluble nuclear transport factor. Another key
observation was that wild type Ran was efficiently
imported, but the RanQ69L mutant was not. We could
thus use immobilised wild type Ran to deplete its
import factor from the cytosol, while depletion with
immobilised RanQ69L had no effect. Examination of
the eluates revealed that a 15 kD protein had bound to
immobilised wild type Ran, but was absent from the
RanQ69-bound fraction. The 15 kD protein was identified
as nuclear transport factor 2 (NTF2) and indeed,
recombinant NTF2 could restore the Ran-import activity
of the depleted cytosol (see Fig. 2). This established
that Ran uses for its own import a unique import
pathway that is mediated by NTF2.
The Ran-Transport cycles can thus be described as
follows (see Fig. 1): RanGDP is bound by NTF2 in
the cytoplasm and translocated into the nucleus. The
Figure 2: Effect of NTF2 on nuclear import of Ran. Figure
shows import of fluorescent Ran into nuclei of permeabilised
cells, performed either in the absence or presence of
NTF2.
RanGEF (nucleotide exchange factor) is a nuclear,
chromatin-bound protein and converts RanGDP to
RanGTP. RanGTP can now bind to an importin and
displace the import cargo or it can assemble into
a trimeric cargo/ exportin/ RanGTP complex. The
RanGTP/ transport receptor complexes are then transferred
to the cytoplasm, where they become disassembled
by the concerted action of RanBP1 and
the RanGTPase activating protein RanGAP (both of
which are excluded from the nuclei). This disassembly
results in the hydrolysis of the Ran-bound GTP molecule,
cargo release from exportins and it allows importins
to bind another substrate molecule. RanGDP can
now enter into another transport cycle.
Mechanism and energetics of NPC-passage
K. Ribbeck
The nuclear transport system is capable of accumulating
cargo molecules against a gradient of chemical
activity, which is obviously an energy-consuming task.
The question of how the input of metabolic energy
is coupled to active transport across the nuclear envelope
has been a central problem in nucleocytoplasmic
transport. As indicated in the scheme of Fig. 1, each
importin- or exportin-dependent transport cycle results
in the hydrolysis of one molecule of GTP. This happens
when RanGTP/transport receptor complexes are
disassembled in the cytoplasm. A surprising recent
finding by us and others has been that this single GTPhydrolysis
event constitutes the sole input of energy
into these transport cycles and that the actual translocation
process is not directly coupled to any nucleotide
hydrolysis or any other irreversible step. This
meant a radical change in concept as the actual translocation
through NPCs had previously been assumed
to be driven by NTP-hydrolysis.
There is an interesting parallel between the energetics
of the just mentioned nucleocytoplasmic transport
cycles and membrane-bound transporters for lowmolecular
weight substances. These transporters can
often use the chemical potential of a primary gradient
(e.g. of Na + ions or protons) to accumulate another
molecule against a gradient of chemical activity. One
textbook example is uptake of amino acids by mammalian
cells which is powered by the Na + ion gradient
across the plasma membrane. The Na + ion concentration
is lower inside the cell than outside and the transporter
couples the uptake of an amino acid with an
influx of Na+ ions, i.e. it symports the two molecules.
Transport cycles of exportins and importins can be
seen in a similar way in that they also use the chemical
potential of a primary gradient, namely that of
RanGTP, to drive the directed transport of cargo molecules.
Exportins are symporters for export cargo
and RanGTP (see Fig. 1). Conversely, importins can
be considered as antiporters that accomplish nuclear
cargo accumulation against a gradient of chemical
activity by „paying“ with the export of RanGTP that
occurs down a gradient. The other constituents of the
RanGTP system function to replenish the Ran gradient
and use GTP-hydrolysis by Ran as the energysource
for this purpose.
The actual translocation through NPCs constitutes a
fully reversible process that can be described as some
kind of facilitated diffusion. The mechanistic basis for
this process, however, is still unclear and for example,
we do not yet understand why a cargo-transport receptor
complex can pass the NPC so much faster than
the cargo molecule alone. The mechanistics of NPCpassage
will therefore be a major focus of our future
research activity.
71

External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 352 “Molekulare Mechanismen intrazellulärer
Transportprozesse”, DFG-Schwerpunkt “Funktionelle
Architektur des Zellkerns”, Graduiertenkolleg
“Molekulare Zellbiologie”), from the Human Frontier
Science Programme Organization and from the Fonds
der Chemischen Industrie.
PUBLICATIONS
Kutay, U., Lipowski, G., Izaurralde, E., Bischoff,
F.R., Schwarzmaier, P, Hartmann, E., and Görlich,
D. (1998). Identification of a tRNA-specific nuclear
export receptor. Mol. Cell. 1, 359-369.
Görlich, D. (1998). Transport into and out of the cell
nucleus (review). EMBO J. 17, 2721-2727.
Jäkel, S., and Görlich, D. (1998). Importin β, transportin,
RanBP5 and RanBP7 mediate nuclear import of
ribosomal proteins in mammalian cells. EMBO J. 17,
4491-4502.
Ribbeck, K., Lipowsky, G., Kent, H.M., Stewart, M.,
and Görlich, D. (1998). NTF2 mediates nuclear import
of Ran. EMBO J. 17, 6587-6598.
Ribbeck, K., Kutay, U., Paraskeva, E. and Görlich, D.
(1999). The translocation of transportin-cargo complexes
through nuclear pores is independent of Ran
and energy. Curr. Biol. 9, 47-50.
Lipowsky, G., Bischoff, F.R., Izaurralde, E., Kutay,
U., Schäfer, S. Gross, H.J., Hildburg Beier, H. and
Görlich, D. (1999). Coordination of tRNA nuclear
72
export with processing of tRNA. RNA 5, 539-549.
Ziemienowicz, A., Görlich, D., Lanka, E., Hohn, B.,
and Rossi, L. (1999). Import of DNA into mammalian
nuclei by proteins originating from a plant pathogenic
bacterium. Proc. Natl. Acad. Sci. USA 96, 3729-33.
Jäkel, S., Albig, W., Schwamborn, K., Doenecke, D.,
and Görlich, D. (1999). The importinβ/ importin7 heterodimer
is a functional nuclear import receptor for
histone H1. EMBO J. 18, 2411-2423.
Paraskeva, E., Izaurralde, E., Bischoff, F.R., Huber, J.,
Kutay, U., Hartmann, E., Lührmann, R., and Görlich,
D. (1999). Crm1p-mediated recycling of snurportin 1
to the cytoplasm. J. Cell Biol. 145, 255-264.
Görlich, D. and Kutay, U. (1999). Transport between
the cell nucleus and the cytoplasm. Annu. Rev. Cell
Dev. Biol. 15, 607-660.
Vetter, I.R., Arndt, A. Kutay, U., Görlich, D. and
Wittinghofer, A (1999). Structural view of the Ran-
Importin β interaction at 2.3 Å resolution. Cell 97,
635-646.
Jullien, D., Görlich, D., Laemmli, U.K., and Adachi,
Y (1999). Nuclear import of RPA in Xenopus egg
extracts requires a novel protein XRIPα but not importin
α. EMBO J. 18, 4348-4358.
Bayliss, R., Ribbeck, K., Akin, D., Kent, H.M., Feldherr,
C.M., Görlich, D., and Stewart, M. (1999).
Interaction between NTF2 and xFxFG-containing
nucleoporins is required to mediate nuclear import of
RanGDP. J. Mol. Biol. 293, 579-93.
Köhler, M., Speck, C., Christiansen, M., Bischoff,
F.R., Prehn, S., Haller, H., Görlich, D., and Hartmann,
E. (1999). Evidence for distinct substrate specificities
of importin alpha family members in nuclear protein
import. Mol. Cell. Biol. 19, 7782-91.
Bachi, A, Braun, I.C., Rodrigues, J.P., Pante, N.,
Ribbeck, K., von Kobbe, C., Kutay, U., Wilm, M.,
Görlich, D., Carmo-Fonseca, M., and Izaurralde, E.
(2000). The C-terminal domain of TAP interacts with
the nuclear pore complex and promotes export of
specific CTE-bearing RNA substrates. RNA J., (in
press).
Bannister, A.J., Miska, E.A., Görlich, D. and Kouzarides,
T. (2000). Acetylation of importin α nuclear
import factors by CBP/p300. Curr. Biol. 10, 467-470.
Lipowsky, G., Bischoff, F.R., Schwarzmaier, P., Kraft,
R., Hartmann, E., Kutay, U., and Görlich, D. (2000).
Exportin 4: a mediator of a novel nuclear export pathway
in higher eukaryotes. EMBO J., (in press).
STRUCTURE OF THE GROUP
E-mail: dg@zmbh.uni-heidelberg.de (Dirk Görlich)
Group leader Görlich, Dirk, Dr.
Postdoctoral
fellows Kutay, Ulrike, Dr. *
Paraskeva, Froso, Dr.
Dean, Kelly, Dr.*
Mingot, José-Manuel, Dr. *
Ph.D. students Lipowsky, Gerd, Dipl. Biol.
Jäckel, Stefan, Dipl. Biol.
Ribbeck, Katharina, Dipl. Biol.
Technical assistant Schwarzmaier, Petra
* part of the time reported
73

Richard Herrmann
Molecular Biology of the Bacterium
Mycoplasma pneumoniae
So far, the genomes of 26 different prokaryotes have
been completely sequenced and published. Among
those are the prototype of the gram-positive bacteria,
Bacillus subtilis, and of the gram-negative bacteria,
Escherichia coli, and several archaea from extreme
habitats. In addition, the first genomes of two eukaryotes,
baker‘s yeast (Saccharomyces cerevisiae) and
the nematode Caenorhabditis elegans, are available.
We expect, by the year 2000 the genome of the first
plant, Arabidopsis thaliana, and of the fruit fly Drosophila
melanogaster will be also sequenced. The annotation
of these genome sequences has made it possible
to predict functions for at least half of the proposed
genes and permits an understanding of the metabolic
pathways and the synthesis of macromolecules. But
the sequence and annotation are not sufficient to
understand the two key processes characterizing all
living systems that are metabolism, defined as the sum
of all chemical reactions taking place in a cell and
reproduction. Nevertheless, a genome sequence and
annotation lead us directly to several experimental
approaches that can bring us closer to an understanding
of the total biology of a living cell.
Such experiments will include:
• Identification of all genes and their functional
assignment,
• Classification of essential and non-essential genes
(C. Hutchison et al., 1999),
• Transcription and translation profiles,
• Analysis of quantitative changes at the level of
gene expression depending on growth conditions,
e.g. growth with or without a host cell,
• Analysis of the mechanisms of gene regulation,
• Studies on the interaction between gene products.
74
Despite the progress in methodology, such analyses
are still very complex and, therefore, we profess they
can be done most readily in a simple cell with a small
number of gene products. We have chosen the human
pathogenic bacterium Mycoplasma pneumoniae as a
model organism to study the biochemistry and molecular
biology of a minimal cell.
Mycoplasma pneumoniae is one of the smallest bacteria
known with a genome size of 816 kbp and a cell
diameter of about 0.5µ. It colonizes as a human pathogen
the respiratory tract causing an atypical pneumonia.
M. pneumoniae is host dependent in nature, but it
can be grown in the laboratory without a host cell in
a rich medium containing 5-20% serum. The cholesterol
in the serum is essential for growth and is incorporated
into the cell membrane. Lacking a cell wall
the membrane of M. pneumoniae is the only outer
layer that separates it from the environment.
I. Gene expression and functional analysis
Most of the functional assignments of proposed genes
from genome sequencing projects are based on significant
sequence similarities to genes/proteins with
known function from other organisms. Besides the
search for sequence similarities other more general
approaches for functional assignments are possible.
Our approach for a functional analysis combines gene
expression studies with functional characterization.
The assumption is that under changing growth conditions
individual genes might be differently expressed
and that specific response to a certain stimulus like
change in temperature, ionic strength or oxygen concentration,
etc., will provide indications for function.
Our gene expression studies of M. pneumoniae include
all the proposed genes and they are being done at the
level of transcription and translation. The first step in
both analyses will be the establishment of a transcription
(transcriptome) and translation (proteome) profile
of M. pneumoniae grown under standard conditions.
These standard profiles will serve as references for
profiles, which were generated from M. pneumoniae
grown under modified conditions.
II. Transcription analysis
J. Weiner, H. Göhlmann, C.U. Zimmermann,
S. Schulz, E. Pirkl
Our approach to transcription analyses was two-fold.
First, the structure of mycoplasma promoters was
studied and second, transcription of all proposed open
reading frames (transcriptome) was monitored.
Presently, little is understood of the structure of mycoplasma
promoters. Since we are interested in understanding
regulation of gene expression of an organism
possessing only one recognized sigma factor, promoter
structures of M. pneumoniae had to be defined.
For this reason the transcriptional start points of 22
genes, which were transcribed at different rates (see
below), were identified by primer extension analysis
and the regions upstream and downstream were compared.
Several possible –10 region promoter sequences
were found, but there were no obvious conserved –35
regions. Another unusual feature was the lack of an
untranslated leader region in front of the first start
codon which could contain a ribosomal binding site.
Search for other translation signals like a downstream
box was also unsuccessful. Inspection of the region
about 50 bases upstream of the proposed start codon
for all other open reading frames revealed that in the
majority of cases promoters with a –10 region but not
with a –35 region were conserved.
The first step of the transcriptome analysis is the
establishment of a reference transcription profile for
all proposed ORFs of M. pneumoniae. For this purpose,
total RNA from bacteria grown under standard
laboratory conditions was isolated, reverse transcribed
into single stranded complementary DNA and labeled
with 33 P. Further, specific probes for all proposed
ORFs were synthesized by polymerase chain reaction,
immobilized on nylon membranes and tested
for cross-hybridization with the labeled cDNA. About
500 transcripts from different ORFs were detected.
They could be divided into several classes according
to signal strength that reflects the number of individual
RNA species. We have no experimental evidence
describing how transcription is regulated. However,
we expect some information about the expression of
individual genes, operons or regulatory networks from
comparative analyses of transcription profiles from
cells grown under modified conditions, e.g., under different
temperatures (32°C, 37°C, 43°C), osmotic or
oxygen stress etc. We are extending the transcriptome
analysis to the intergenic regions. Among others, we
found a highly abundant RNA species that has the
potential to code for a 29 amino acid long cysteine
rich protein. Experiments are in progress to identify
this peptide in bacterial extracts.
III. Proteome analysis
J. Regula, B. Ueberle
Proteome analysis involves the two-dimensional (2-D)
gel electrophoresis for the separation of proteins
according to isoelectric point (pI) and molecular mass,
the knowledge of the sequences of all proteins of a
cell and mass spectrometry for the characterization
75

of individual proteins. The combination of peptide
mass finger-printing and peptide fragmentation which
matches the masses of in gel proteolytically generated
peptides against theoretically digested proteins from
the M. pneumoniae database, has proved to be very
effective and reliable. Proteome analysis is limited by
the sensitivity of mass spectrometry and the pH range
of the first dimension of the two-dimensional gel electrophoresis.
Presently proteins with isoelectric points
Figure 1: 2-D-gel of a total protein extract from M. pneumoniae.
First dimension (1-D) immobilized pH gradient,
second dimension (2-D) 12,5% SDS-polyacrylamide gel.
All stained protein spots (≈ 200) were assigned to the corresponding
genes, but only the strongest spots are marked
with a gene number.
76
Figure 2: Mock 2-D-gel of all proposed proteins of M.
pneumoniae. Based on the predicted molecular mass and
isoelectric point, each protein could be localized in this
mock 2-D-gel. The yellow and blue dots represent the sum
of all proposed proteins; the „blue“ proteins have been
assigned to a gene.
between 3-10 can only be separated routinely by
immobilized pH gradients (Fig. 1), but the annotation
of the genome sequence of M. pneumoniae predicts
236 proteins with pIs greater than 10 (Fig. 2). When
we analyze the protein extract of entire M. pneumoniae
cells by two-dimensional gel electrophoresis
and visualize the proteins by silver staining, about 450
proteins can be detected and about 200 proteins by
staining with the less sensitive Coomassie blue (Fig.
1). So far, 200 proteins have been identified by mass
spectrometry and assigned to the corresponding genes.
We found in most cases a good correlation between
DNA sequence based prediction of pI and mass of a
protein and its final position within a two-dimensional
gel. However, exceptions have been found where the
experimentally derived values were different. We suggest
that in many of these exceptions posttranslational
modification might have occurred. Furthermore,
a comparison of transcriptome and proteome analysis
shows that a relatively high copy number of a given
transcript does not always correlate with a strong pro-
tein signal and vice versa. Those genes could be interesting
examples for regulation of gene expression at
different levels. The proteome analysis is being done
in cooperation with R. Frank, Heidelberg and A. Görg,
München.
IV. Is there a cytoskeleton-like structure in M.
pneumoniae?
J. Regula, A. Boonmee, W. Schaller
The M. pneumoniae genome does not encode for a
single gene known to be involved in the synthesis
of a bacterial cell wall. M. pneumoniae is only surrounded
by a cytoplasmic membrane that, exceptionally
among bacteria, contains cholesterol as an essential
component. Over the past twenty years evidence
has accumulated that M. pneumoniae possesses a cytoskeleton-like
structure, probably as a substitute for the
missing cell wall. In analogy to the cytoskeleton of
eukaryotic cells, such a structure could provide the
necessary framework for maintaining and stabilizing
the shape of M. pneumoniae, for motility and for the
formation of an asymmetric cell.
The first experimental evidence for a cytoskeleton-like
structure in M. pneumoniae was provided by Meng
and Pfister (1980) who detected fibrous structures
by electron microscopy after treating M. pneumoniae
cells with the nonionic detergent Triton X-100, which
removed the membrane and the cytoplasm. These
observations were confirmed and extended by several
other researchers. These experiments and studies on
the architecture and composition of eucaryotic cytoskeletons
from cells which have been treated with the
detergent Triton X-100 suggested that a cytoskeletonlike
structure would also be enriched in the Triton
X-100 insoluble fraction of M. pneumoniae.
Therefore, we decided to determine the protein com-
position of the Triton X-100 insoluble fraction of
M. pneumoniae by 2-D-gel electrophoresis and mass
spectrometry.
Silver staining of 2-D gels of the Triton X-100 insoluble
fraction revealed about 100 protein spots. By
staining with colloidal Coomassie blue about 50 protein
spots were visualized of which 41 were identified
by determining the mass and the partial sequence of
their tryptic peptides following enzymatic digestion.
The identified proteins belonged to several functional
categories, mainly energy metabolism, translation and
heat shock response. In addition, we found lipoproteins
and most of the proteins involved in cytadherence
which were known to be components of the
Triton X-100 insoluble fraction based on evidence
from previous experiments. There were also 11 functionally
unassigned proteins. The quantitatively most
prevalent proteins were the heat shock protein DnaK,
the elongation factor Tu and the subunits α and ß
of the pyruvate dehydrogenase (PdhA, PdhB). To
prove whether a cytoskeleton-like structure exists
in Mycoplasma pneumoniae, further experiments are
required.
One of the promising newer methods to identify in
vivo interacting proteins is the two-hybrid system in
Saccharomyces cerevisiae. We started in cooperation
with M. Kögel (LION Bioscience, Heidelberg) a twohybrid
analysis with the aim of finding which of the
proposed proteins of the cytoskeleton-like structure
interact directly. We expect that this approach will
also reveal new candidates for structural components.
As starting material for our analysis we used the gene
hmw2. Our selection is based on data from various
laboratories indicating that the gene hmw2 codes for
one of the key components in the formation of the
cytoskeleton-like structures. So far, we have identified
several interacting proteins. These results are now
being verified by a second independent method.
77

V. Genetic stability of infectious M. pneumoniae
isolates
W. Reiser, I. Catrein, M. Götzmann, W. Schaller,
E. Pirkl
M. pneumoniae causes sporadic endemic disease but
epidemics have been reported to appear in certain
time intervals. Infectious M. pneumoniae isolates from
patients showed that either one of the two subtypes
(I and II) was the causative agent in an outbreak.
The prototypes of these subtypes are M. pneumoniae
M129(I) and M. pneumonia FH(II). The basis for their
classification is the P1 adhesin, which is essential for
adherence of the bacterium to its host. To obtain more
information about the differences between the two
prototypes and various isolates collected over about
30 years, we examined M. pneumoniae M129 and
FH for the following features: genome organization,
sequence similarities of selected genes, protein patterns
of two-dimensional gels and transcription profiles.
Comparing the complete nucleotide sequence of the
genome of M.p. M129 with about 150.000 bp of the
genome of M. pneumoniae FH showed that the isolates
are very conserved and so far the main differences
are located in the P1 operon
The P1 operon consists of the following three genes
named ORF4, ORF5 (=P1 gene) and ORF6. The gene
product of ORF6 are the two proteins P40 and P90,
which are involved in the correct localization of P1 in
the membrane. While the ORF4 gene is highly conserved
in both prototypes, the genes P1 and ORF6 are
different. Experiments are in progress to extend the
comparative analyses to other isolates from patients to
examine whether the P1 operon is indeed the decisive
difference between subtypes.
78
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(Graduiertenkolleg “Kontrolle der Genexpression
in pathogenen Mikroorganismen”, Projekt-
’Sachbeihilfen’) from the BioRegion Rhein Neckar
(BMBF - Fa. Genzyme Virotech and BMBF - Fa.
EML) and by the Fonds der Chemischen Industrie.
PUBLICATIONS
Pyrowolakis, G., Hofmann, D. and Herrmann, R.
(1998). The subunit b of the F 0 F 1 type ATPase of the
bacterium Mycoplasma pneumoniae is a lipoprotein.
J. Biol. Chem 273, 24792-24796.
Herrmann, R. and Reiner, B. (1998). Mycoplasma
pneumoniae – Mycoplasma genitalium, a comparison
of two closely related bacterial species. Curr. Opinion
in Microbiol. 1, 572-579.
Herrmann, R., Göhlmann, H.W.H., Regula, J.T.,
Weiner III, J., Pirkl, E., Ueberle, B. and Frank, R.
(1999). Mycoplasmas, the Smallest Known Bacteria.
In Microbial Evolution and Infection. (U. B. Göbel,
B.R. Ruf, eds.), Einhorn-Presse Verlag, 71-79.
Fisseha, M., Göhlmann, H.W.H., Herrmann, R. and
Krause, D.C. (1999) Identification and Complementation
of Frameshift mutations associated with loss of
cytadherence in Mycoplasma pneumoniae. J. Bacteriol.
181, 4404-4410.
Göhlmann, H.W.H, Weiner, J., Schön, A. and Herr–
mann, R. (2000). Identification of a small RNA
within the pdh gene cluster of Mycoplasma pneumoniae
and Mycoplasma genitalium. J. Bacteriol.,
182, 3281-3284.
Dandekar, T., Huynen, M., Regula, J.T., Zimmermann,
C.U., Ueberle, B., Andrade, M., Doerks, T., Sanchez,
L., Snel, B., Suyama, M., Yuan, Y.P., Herrmann, R.,
and Bork, P. (2000). Re-annotating the Mycoplasma
pneumoniae genome sequence: Adding value, function
and reading frames. Nucleic Acids Res. (in press).
Regula, J.T., Ueberle, B., Boguth, G., Görg, A.,
Schnölzer, M., Herrmann, R., and Frank, R. (2000).
Towards a two-dimensional proteome map of Mycoplasma
pneumoniae. Electrophoresis (in press).
Weiner, J., Herrmann, R., and Browning, G.F. (2000).
Transcription in Mycoplasma pneumoniae. Nucleic
Acids Res. (in press).
THESES
Dissertations
Göhlmann, Hinrich (1999). Transcriptionsanalyse
einer gesamten Zelle am Beispiel von Mycoplasma
pneumoniae.
Regula, Jörg (1999). Auf dem Weg zum Proteom von
Mycoplasma pneumoniae.
STRUCTURE OF THE GROUP
E-mail: r.herrmann@zmbh.uni-heidelberg.de
Group leader Herrmann, Richard, Prof. Dr.
Research associates Reiser, Walter, Dr.*
Hofmann, Diana, Dipl. Biol.*
Regula, Jörg, Dipl. Chem.
PhD. students Göhlmann, Hinrich, Dipl. Biol.*
Weiner, January, Dipl. Biol.
Catrein, Ina, Dipl. Biol.*
Ueberle, Barbara, Dipl. Biol.
Diploma students C.U. Zimmermann*
Stefan Schulz*
Atcha Boonmee*
Techn. assistants Pirkl, Elsbeth
Götzmann, Martina*
Schaller, Werner*
*part of the time reported
79

Ralf-Peter Jansen
Asymmetric Cell Division and RNA
Transport in Yeast
During embryogenesis, cell type diversity is generated
by different mechanisms, one of which is asymmetric
cell division. Such divisions produce progeny differing
in morphology, size, or gene expression pattern.
However, asymmetric divisions are not only found in
multicellular organisms. A typical unicellular eukaryote
that divides asymmetrically is the yeast Saccharomyces
cerevisiae. A „mother cell“ generates a
„daughter cell“ by polarized growth, a process known
as „budding“. After separation, the two cells show
a differential gene expression pattern. Whereas the
mother cell can express the HO gene and eventually
undergoes a process called „mating type switching“,
the daughter cell is unable to do so.
HO expression depends on multiple positively and
negatively acting transcription factors but the differential
expression pattern of HO is regulated by the
asymmetric distribution of a transcriptional repressor
known as Ash1p. Ash1p can be detected in post-anaphase
cells predominantly in the nucleus of daughter
cells where it persists to the end of the daughter cell‘s
following G1 phase. In the daughter cell it is both sufficient
and essential to shut off HO expression in G1.
The asymmetric distribution of Ash1p is the result of
an asymmetric localization of its mRNA during anaphase.
ASH1 mRNA is transported from the nuclei of
both mother and daughter cell to the cell cortex of
the daughter. The localization depends on a functional
microfilament system and three cis-acting localization
signals. These signals appear to be independent of each
other since each signal is sufficient to target a reporter
RNA to the daughter cell. In addition to the cis signals,
5 so called She proteins are essential for ASH1 mRNA
localization. Among these proteins is a type V myosin
80
(She1p/Myo4p) and She5p/Bni1p, a member of the
FH („formin homology“) protein family required for
actin cytoskeleton organization in different organisms.
Homologs of another She protein, She4p have recently
been predicted to be involved in myosin assembly or
function. The last two She proteins, She2p and She3p
do not show any sufficient homology to other proteins.
Myo4p, a motor protein essential for mRNA
transport
D. Djandji, A. Frank, C. Kruse, S. Münchow, and C.
Sauter
To date, Myo4p is the only motor protein with an essential
role in mRNA localization. In order to understand
its role we initially wanted to test if the myosin
is directly involved in ASH1 mRNA transport.
Using a combination of in-situ hybridization and indirect
immunofluorescence, we could show a colocalization
of the myosin and ASH1 mRNA not only at the
final targeting site (the tip of the daughter cell) but
also on filamentous structures that are most likely microfilaments
running from the mother to the daughter
cell (Fig. 1). In addition, ASH1 mRNA was shown to
specifically coprecipitate with an epitope-tagged version
of Myo4p. Both sets of data strongly suggest that
ASH1 mRNA is in fact a cargo of the Myo4p myosin.
What other proteins are required for the association of
ASH1 mRNA with Myo4p? One candidate was She3p
since it‘s intracellular localization was very similar
to that of Myo4p. We were able to demonstrate that
She3p is essential for Myo4p-ASH1 association. Furthermore,
She3p coprecipitated both with Myo4p and
Figure 1
ASH1 mRNA. In addition, She3p‘s aminoterminus
that is predicted to form a coiled coil can bind to
the carboxyterminal tail of Myo4p in a two hybrid assay
whereas She3p‘s carboxyterminus interacts with
She2p. She3p and She2p might be adapters between
the mRNA and the motor protein.
The role of Myo4p‘s tail domain was further investigated
since the tail domains of other myosins are
involved in cargo binding. Expression of the tail in an
otherwise wildtype cell had the effect of a dominant
negative mutant. It abolished the localization of ASH1
mRNA, She3p and endogenous Myo4p as well as the
association of endogenous Myo4p with She3p, most
likely by sequestering essential factors. The effects are
specific since the expression of another type V myosin
tail did not interfere with mRNA localization.
The myosin tail domain should be useful to biochemically
identify and copurify components of the putative
mRNA transport complex, especially the RNAbinding
factor(s).
Ash1p as a regulator of pseudohyphal differentiation
K. Kahlina and S. Münchow
Besides it‘s role in regulating HO expression and
mating type switching, Ash1p is required for another
differentiation process in Saccharomyces cerevisiae,
pseudohyphal growth. During this differentiation,
yeast cells elongate and grow unipolar to form chains
of connected cells that allow a yeast colony to penetrate
it‘s medium. Several signalling pathways converge
during induction of pseudohyphal growth and
at least four transcription factors (including Ash1p)
are required for this differentiation process. However,
nothing is known about the signalling pathway that
regulates Ash1p or about the target genes that are regulated
by Ash1p.
We have recently shown that mislocalization of ASH1
mRNA and Ash1 protein in a myo4 mutant dramatically
increases pseudohyphal growth suggesting that
RNA targeting plays a crucial role in proper pseudohyphal
differentiation. In order to identify ASH1-dependent
genes, two independent screens based on dif-
81

ferential display methods and gene arrays have been
initiated and putative candidates are currently analyzed.
Pseudohyphal and hyphal growth plays an important
role during pathogenesis of the yeast Candida albicans.
In the course of the C. albicans sequencing project,
a DNA fragment of the putative C. albicans ASH1
gene has been identified. Using this fragment, we have
cloned the full length C. albicans ASH1 gene and are
currently characterizing its function.
Novel localized mRNAs in Saccharomyces
cerevisiae
F. Böhl and D. Ferring
In a number of cell types, several mRNAs are sorted
differentially in a single cell. In order to identify novel
localized RNAs in yeast, we devised two different
screens based on in-situ hybridization. The first one
was based on the rationale that asymmetrically sorted
mRNAs should be expressed in the same cell cycle
window as ASH1, the M-/G1-phase boundary. Therefore,
we fused the 3‘ parts of 40 mRNAs that are expressed
at this boundary to a reporter mRNA (GFP,
green fluorescent protein) and expressed these hybrids
from a regulatable promoter. In the case of ASH1 such
an RNA hybrid is clearly localized to the daughter cell.
However, none of the other mRNA hybrids showed a
localized hybridization signal, indicating that ASH1 is
the only M-/G1-phase mRNA that is asymmetrically
distributed.
In a second approach, we tempted to screen the complete
yeast genome for localized mRNAs. A plasmidbased
DNA library was generated that allowed random
fusions of genomic DNA fragments to the GFP coding
sequence. The resulting hybrid genes can be expressed
from a regulatable GAL1 promoter. Localized RNAs
82
can be detected by in-situ hybridization against the
GFP part of the resulting hybrid mRNAs. The constructed
library covers approximately 95% of the complete
yeast genome. After transformation into yeast,
pools of 25 clones are currently analyzed for the presence
of novel localized mRNAs. Such a screen should
be useful to assess the complete number of localized
mRNAs in a polarized cell.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (Graduiertenkolleg “Molekulare Zellbiologie”,
Projekt-’Sachbeihilfen’).
PUBLICATIONS
Münchow, S., Sauter, C., and Jansen, R.-P. (1999). Association
of the class V myosin Myo4p with a localised
messenger RNA in budding yeast depends on She proteins.
J. Cell Sci. 112, 1511-1518.
Jansen, R.-P. (1999). RNA-cytoskeletal associations.
FASEB J. 13, 455-466.
Hurt, E., Segref, A., Sträßer, K., Bailer, S., Schlaich,
N., Presutti, C., Tollervey, D., and Jansen, R.-P. (2000).
Mex67p mediates nuclear export of a variety of Pol II
transcripts. J.Biol.Chem. 275, 8361-8368.
THESES
Diploma
Münchow, S. (1998). Isolierung und Charakterisierung
des Myo4-Protein/ASH1 mRNA Komplexes aus Sac-
charomyces cerevisiae.
Sauter, C. (1998). Detektion lokalisierter mRNAs in
Saccharomyces cerevisiae mittels Fluoreszenz-in situ-
Hybridisierung.
Theurer, J. (1998). Versuch der Identifikation neuer
Bestandteile der ASH1 mRNA Lokalisationsmaschi–
nerie von Saccharomyces cerevisiae durch Überexpression
einer cDNA-Bibliothek.
Djandji, D. (1999). Die Assoziation des She3 Proteins
mit ASH1 mRNA in Saccharomyces cerevisiae.
STRUCTURE OF THE GROUP
e-mail: r.jansen@mail.zmbh.uni-heidelberg.de
Group leader Jansen, Ralf-Peter, Dr.
Ph.D. students Böhl, Florian, Dipl. Biol. *
Jaedicke, Andreas, Dipl. Biol. *
Kruse, Claudia, Dipl. Biol. *
Münchow, Sonja, Dipl. Biol.
Diploma students Djandji, Dominic *
Sauter, Claus *
Theurer, Jörg *
Kahlina, Kornelia *
Techn. assistants Ferring, Dunja *
Frank, Andrea *
* part of the time reported
83

Stefan Jentsch
Ubiquitin-Dependent Proteolysis
The central importance of selective proteolytic systems
in regulating cellular events has been recognized
recently. Progression through the eukaryotic cell
cycle, for example, is substantially regulated through
a timed and coordinated degradation of cyclins and
inhibitors of cyclin-dependent protein kinases. Similarly,
the shift from one transcriptional or developmental
program to another is often achieved through
a timed destruction of regulatory proteins. Proteolysis
is irreversible and therefore proteolytic enzymes are
usually employed for controlling unidirectional cellular
pathways. Selective degradation in eukaryotes is
primarily mediated by the ubiquitin/proteasome pathway.
Substrates of this pathway are covalently modified
by conjugation to ubiquitin, a small and highly
conserved protein. In most cases several ubiquitin
molecules are added to the substrate as a multiubi–
quitin chain in which ubiquitin molecules are arranged
like beads on a string. Multiubiquitinated proteins are
targeted for degradation by a large protease complex,
known as the 26S proteasome. Our research focuses
primarily on functional aspects of this pathway. We
identify from yeast and mammalian cells the enzymatic
components of this system, clone the corresponding
genes, and study their functions in vitro and
in vivo.
A novel ubiquitination factor, E4, involved in
multiubiquitin chain assembly
M. Kögl, T. Hoppe, S. Schlenker, H. D. Ulrich, T. U.
Mayer
Proteins modified by multiubiquitin chains are the
preferred substrates of the proteasome. Previous work
has suggested that E1, E2, and E3 enzymes are both
84
required and sufficient for the formation of multi–
ubiquitinated substrates. Recently, however, we could
show that efficient multiubiquitination needed for proteasomal
targeting of a model substrate (ubiquitin
fusions) requires an additional conjugation factor,
which we termed E4. This protein, previously known
as UFD2 in yeast, binds to the ubiquitin moieties of
preformed conjugates and catalyzes ubiquitin chain
assembly in conjunction with E1, E2, and E3. Intriguingly,
E4 defines a new protein family, which includes
two human members and the regulatory protein NOSA
from Dictyostelium required for fruiting body development.
In yeast E4 activity is linked to cell survival
under stress conditions, indicating that eukaryotes utilize
E4-dependent proteolysis pathways for multiple
cellular functions. Interestingly, UFD2 (E4) interacts
with CDC48, a member of the large family of AAAtype
ATPases. CDC48 is not involved in the ubiquitinconjugation
reaction but is needed for proteolysis of
some substrates of the ubiquitin/proteasome pathway.
Ubiquitin-like proteins from mammals and
yeast
D. Liakopoulos, G. Doenges
Ubiquitin is one of the most highly conserved eukaryotic
proteins known to date. Human ubiquitin differs
from its yeast homolog by only three amino acid residues
(out of 76 residues) and the proteins are functionally
equivalent. In addition to ubiquitin, two classes
of ubiquitin-related proteins have been identified. Proteins
of the first class carry ubiquitin-like domains
linked to unrelated sequences and are not conjugated
to other cellular proteins. One example is the mammalian
ubiquitin-related protein BAG-1 for which we
showed that it functions as a regulatory cofactor of
the Hsc70 chaperone. Proteins of the second class of
ubiquitin-like proteins are distinguished by their property
to become posttranslationally attached to other
cellular proteins analogously to ubiquitin. Known
members are SUMO-1 from higher eukaryotes and
its yeast ortholog SMT3. Recently, we have identified
a novel ubiquitin-like protein from yeast, which
we termed RUB1. RUB1 displays 53% amino acid
sequence identity to ubiquitin. We found that RUB1
conjugation to other cellular proteins requires at least
three proteins in vivo. ULA1 and UBA3 are related
to the amino- and carboxyl-terminal domains of the
E1 ubiquitin-activating enzyme, respectively, and together
fulfill E1-like functions for RUB1 activation.
RUB1 conjugation also requires UBC12, a protein
related to E2 ubiquitin-conjugating enzymes, which
functions analogously to E2 enzymes in RUB1-protein
conjugate formation. Conjugation of RUB1 is not
essential for normal cell growth and appears to be
selective for a small set of substrates. Remarkably, we
found that CDC53/cullin, a common subunit of the
multifunctional SCF ubiquitin ligase, is a major substrate
for RUB1 conjugation. This suggests that the
RUB1-conjugation pathway is functionally affiliated
with the ubiquitin/proteasome system and may play a
regulatory role.
Conjugation of the ubiquitin-like protein RUB1/
NEDD8 to cullin-2 is linked to von Hippel-Lindau
(VHL) tumor suppressor function
D. Liakopoulos, G. Doenges; in collaboration with T.
Büsgen, A. Brychzy, and A. Pause (Max Planck Institute
for Biochemistry, Martinsried)
Yeast RUB1 has homologs in higher eukaryotic cells.
The human RUB1 homolog, NEDD8, when expressed
in a yeast rub1 mutant, can complement its RUB1
deficiency. Recently we found that both hCUL-1
and hCUL-2 (homologs of yeast CDC53, see above)
are modified by the conserved ubiquitin-like protein
RUB1/NEDD8. Whereas hCUL-1 is part of a human
SCF complex, hCUL-2 assembles with elongin B/C
and the von Hippel-Lindau tumor suppressor protein
pVHL, forming a protein complex, CBC VHL , that
resembles SCF ubiquitin ligases. We could show that
NEDD8-hCUL-2 conjugates are part of CBC VHL complexes
in vivo. Remarkably, the formation of these
conjugates is stimulated by the pVHL tumor suppressor.
A tumorigenic pVHL variant, however, is
essentially deficient in this activity. Thus, ligation of
NEDD8 to hCUL-2 is linked to pVHL activity and
may be important for pVHL tumor suppressor function.
Moreover, our data indicate that modification of
cullins by NEDD8 requires the existence of a preassembled
ubiquitin ligase complex.
External Funding
During the period reported our research was supported
by grants from Deutsche Forschungsgemeinschaft
(project grants, SFB 352, Schwerpunktprogramm
Ubiquitin/Proteasomsystem, Leibniz Program,
Graduiertenkolleg), European TMR Ubiquitin
Research Network, American Cancer Society, and
Fonds der Chemischen Industrie.
PUBLICATIONS
Finley et al., (1998). Unified nomenclature for subunits
of the Saccharomyces cerevisiae proteasome regulatory
particle. Trends Biochem. Sci. 23, 244-245.
Hauser, H.-P., Bardroff, M., Pyrowolakis, G., and
85

Jentsch, S. (1998). A giant ubiquitin-conjugating
enzyme related to IAP apoptosis inhibitors. J. Cell
Biol. 141, 1415-1422.
Jentsch, S. and Ulrich, H.D. (1998). Ubiquitous déjà
vu. Nature 393, 321-323.
Liakopoulos, D., Doenges, G., Matuschewski, K., and
Jentsch, S. (1998). A novel protein modification pathway
related to the ubiquitin system. EMBO J. 17,
2208-2214.
Mayer, T.U., Braun, T., and Jentsch, S. (1998). Role of
the proteasome in membrane extraction of a short-lived
ER-transmembrane protein. EMBO J. 17, 3251-3257.
Scheffner, M., Smith, S., and Jentsch, S. (1998).
The ubiquitin-conjugation system. In: Ubiquitin. J.M.
Peters, J.R. Harris , and D. Finley, eds., Plenum Press,
New York, pp 65-98.
Schwarz, S.E., Matuschewski, K., Liakopoulos, D.,
Scheffner, M., and Jentsch, S. (1998). The ubiquitinlike
proteins SMT3 and SUMO-1 are conjugated by
the UBC9 E2 enzyme. Proc. Natl. Acad. Sci. USA 95,
560-564.
Koegl, M., Hoppe, T., Schlenker, S., Ulrich, H.D.,
Mayer, T.U., and Jentsch, S. (1999). A novel ubiquitination
factor, E4, is involved in multiubiquitin chain
assembly. Cell 96, 635-644.
Liakopoulos, D., Büsgen, T., Brychzy, A., Jentsch, S.,
and Pause, A. (1999). Conjugation of the ubiquitinlike
protein NEDD8 to cullin-2 is linked to von Hippel-Lindau
(VHL) tumor suppressor function. Proc.
Natl. Acad. Sci. USA 96, 5510-5515.
86
Honors/Awards
Elected Member of the German Academy of Science,
Leopoldina, 1998.
Member of the Advisory Editorial Board of EMBO
Journal, 1998.
Member of the Advisory Editorial Board of EMBO
Reports, 1999.
THESES
Dissertations
Braun, T. (1998). Analyse des Abbaus von Membranproteinen
am Endoplasmatischen Retikulum.
Liakopoulos, D. (1998). Identifizierung eines neuen
Protein-Modifikations-Systems mit Verwandtschaft
zum Ubiquitin-System.
Matuschewski, K. (1998). Genetische Charakterisie–
rung der Ubiquitin-Protein-Ligase RSP5.
Mayer, T. (1998). Rolle des Proteasoms bei der
Extraktion eines kurzlebigen Proteins aus der ER-
Membran.
STRUCTURE OF THE GROUP
Group leader: Jentsch, Stefan, Prof. Dr.
Postdoctoral
fellows: Ulrich, Helle, Ph.D.
Kögl, Manfred, Dr.
Ph.D. students:
Bardroff, Michael, Dipl. Biochem.
Braun, Thorsten, Dipl. Chem.
Doenges, Georg, Dipl. Biol.
Hoege, Carsten, Dipl. Biol., M. Phil.
Hoppe, Thorsten, Dipl. Biol.
Liakopoulos, Dimitris, Dipl. Chem.
Matuschewski, Kai, Dipl. Biochem.
Mayer, Thomas, Dipl. Biol.
Pyrowolakis, George, Dipl. Biol.
Thoms, Sven, Dipl. Chem.
Schlenker, Stephan, Dipl. Biochem.
Technical
assistants: Hubbe, Petra
Jepsen, Kathrin
Löser, Eva
87

Project Group Jörg Höhfeld
Function and Regulation of the Mammalian
Chaperone Hsc70
We are focusing on the characterization of the Hsc70
chaperone system in the mammalian cytosol and
nucleus. Hsc70 is not only involved in the folding of
newly-synthesized polypeptides but also in the sorting
of proteins to different cellular compartments and
appears to present proteins to the degradative system
of the cell. Thus, elucidating the regulation of Hsc70
will provide insight into central events during protein
biogenesis. In recent years we have been able to
identify and characterize several chaperone cofactors
that modulate the chaperone activity of Hsc70. Thus,
essential tools have become available to decipher the
molecular mechanisms that determine the functional
specificity of Hsc70 and that enable Hsc70 to efficiently
cooperate with other chaperone systems and
with the proteolytic machinery.
Structural requirements for the interaction of
Hsc70 with distinct cofactors
J. Demand
The modulation of the chaperone activity of the heat
shock cognate Hsc70 protein in mammalian cells
involves a cooperation with chaperone cofactors such
as Hsp40, BAG-1, the Hsc70-interacting protein Hip,
and the Hsc70/Hsp90-organizing protein Hop. By
employing the yeast two-hybrid system and in vitro
interaction assays we were able to determine structural
requirements for Hsc70‘s cooperation with different
cofactors. The carboxy-terminal domain of Hsc70,
previously shown to form a lid over the peptide bind-
88
ing pocket of the chaperone protein, mediates interaction
of Hsc70 with Hsp40 and Hop. Remarkably, the
two cofactors bind to the carboxy terminus of Hsc70
in a non-competitive manner, revealing the existence
of distinct binding sites for Hsp40 and Hop within this
domain. In contrast, Hip exclusively interacts with
the amino-terminal ATPase domain of Hsc70. Hence,
Hsc70 possesses separate non-overlapping binding
sites for Hsp40, Hip, and Hop. This appears to enable
the chaperone protein to cooperate simultaneously
with multiple cofactors. On the other hand, BAG-1
and Hip have recently be shown to compete in bind-
Figure 1. Cofactor-binding sites of Hsc70
ing to the ATPase domain. Our data thus establish the
existence of a network of cooperating and competing
cofactors regulating the chaperone activity of Hsc70
in the mammalian cell.
Cofactor-induced modulation of the functional
specificity of the molecular chaperone Hsc70
J. Lüders
Molecular chaperones differ in their ability to stabilize
nonnative polypeptides and to mediate protein
folding, defining ‚holding‘ and ‚folding‘ systems. We
analyzed how the chaperone activity of Hsc70 is modulated
by different cofactors after in vitro reconstitu-
tion of the chaperone system. It was observed that
Hsc70 in conjunction with the cofactor Hsp40 stabilizes
heat-denatured firefly luciferase, acting as a
‚holding‘ system. The stabilizing activity turns into
a folding activity in the additional presence of the
Hsc70-interacting protein Hip. In contrast, the cofactor
BAG-1 abrogates the ‚holding‘ function of the
Hsc70/Hsp40 system and blocks the action of Hip on
Hsc70. The competing cofactors Hip and BAG-1 thus
directly determine the fate of a nonnative polypeptide
stabilized by Hsc70 and Hsp40. The findings reveal
on a molecular level how functional specificity of
Hsc70 can be induced through the action of chaperone
cofactors.
Figure 2. Cofactors determine the function of Hsc70
Defining a pathway that links molecular chaperones
to the proteolytic machinery
J. Höhfeld
It is intriguing that Hsc70 participates in protein folding
but can also present non-native proteins to the
ubiquitin/proteasome system, which mediates protein
breakdown. Regulatory mechanisms might exist to
modulate the action of Hsc70 at the interface between
protein folding and protein degradation.
Remarkably, the Hsc70 cofactor BAG-1 possesses a
ubiquitin-like domain at its amino-terminus, suggesting
a link to the proteolytic system. To verify this
notion we analyzed whether BAG-1 stably associates
with the proteasome. An association of BAG-1 with
the proteolytic complex was indeed observed in
human HeLa cells. Binding of the chaperone cofactor
to the proteolytic complex is regulated by ATP-hydrolysis
and is not mediated by Hsc70. Intriguingly, targeting
of BAG-1 to the proteasome promotes an association
of Hsc70 with the proteaseome in vitro and
in vivo. BAG-1 apparently acts as a coupling factor
between the chaperone system and the proteolytic
complex. BAG-1 may thus fulfill a key regulatory
function at the interface of protein folding and protein
degradation.
Figure 3. BAG-1 acts as a coupling factor between
Hsc70 and the proteasome
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (Sachbeihilfen).
89

PUBLICATIONS
Heyrovska, N., Frydman, J., Höhfeld, J., and Hartl,
F.-U. (1998). Directionality of polypeptide transfer
in the mitochondrial pathway of chaperone-mediated
protein folding. Biol. Chem. 379, 301-309.
Höhfeld, J. (1998). Regulation of the heat shock cognate
Hsc70 in the mammalian cell: the characterization
of the anti-apoptotic protein BAG-1 provides
novel insights. Biol. Chem. 379, 269-274.
Demand, J., Lüders, J., and Höhfeld, J. (1998). The
carboxy-terminal domain of Hsc70 provides binding
sites for a distinct set of chaperone cofactors. Mol.
Cell. Biol. 18, 2023-2028.
Lüders, J., Demand, J., Schönfelder, S., Frien, M.,
Zimmermann, R., and Höhfeld, J. (1998). Cofactorinduced
modulation of the functional specificity of
the molecular chaperone Hsc70. Biol. Chem. 379,
1217-1226.
Lüders, J., Demand, J., and Höhfeld, J. (1999). The
ubiquitin-related BAG-1 provides a link between the
molecular chaperones Hsc70/Hsp70 and the proteasome.
J. Biol. Chem, 275, 4613-4617.
Habilitation
Höhfeld, J. (1998). Regulation of molecular chaperones
in the eukaryotic cell.
STRUCTURE OF THE GROUP
Group leader Höhfeld, Jörg, PD Dr.
Ph.D. students Demand, Jens, Dipl.Chem.
Lüders, Jens, Dipl.Biol.
90
Klaus-Armin Nave
Myelin Genetics and Developmental
Neurobiology
The assembly of myelin in the nervous system of
higher vertebrates is the highly specialized function of
oligodendrocytes and Schwann cells. Myelin-forming
glial cells enwrap axons with multiple layers of
membranes and provide the electrical insulation that
is necessary for a rapid impulse propagation. We
are interested in the principles of these neuron-glia
interactions and are studying genes that are required
for normal myelin assembly and maintenance. Transgenic
and natural mouse mutants are useful tools
which also serve as models for corresponding myelin
diseases in human. One gene of interest encodes
PMP22, a myelin membrane protein of Schwann
cells, and is frequently duplicated in human patients
with Charcot-Marie-Tooth disease. Mutations of the
proteolipid protein (PLP) gene underlie Pelizaeus-
Merzbacher disease, a lethal white matter disease.
Proteolipids are not restricted to glial cells: PLPhomologous
genes (M6A, M6B) are expressed in
nearly all CNS neurons but their function is still
unknown. Mouse mutants of these genes and the overexpression
of proteolipids in heterologous cells shed
some light onto their cellular function.
In an attempt to identify regulatory genes that control
the terminal differentiation of neuronal and glial cells,
we have cloned several basic helix-loop-helix (bHLH)
transcription factors from the postnatal rodent brain.
One bHLH factor that we termed NEX defines a
new subfamily of neuronal bHLH genes expressed
in the mammalian CNS. This familiy of Drosophila
atonal-related genes also includes BETA2/NeuroD
and NDRF. The targeted inactivation of NeuroD and
NEX in transgenic mice reveals a critical function of
these genes in the differentiation program of hippocampal
neurons.
I. Myelin protein PMP22 in a rat model of
Charcot-Marie-Tooth disease
S. Niemann, M. Sereda
Tetraspan myelin proteins play an important role in
CNS and PNS myelination. To address the role of
PMP22 in development and in disease, we have generated
a transgenic rat model of the most frequent
human neuropathy, Charcot-Marie-Tooth disease type
1A. CMT1A is associated with a partial duplication
of chromosome 17 and we have proven experimentally
that the underlying cause of CMT1A is overexpression
of the PMP22 gene. Transgenic rats have
typical clinical features of the human disease, including
muscle weakness, reduced nerve conduction
velocities, and marked Schwann cell hypertrophy. In
the phenotypical analysis of the CMT rat we can now
adress questions which could previously not be studied
in the corresponding human disease. Of specific
interest is the analysis of homozygous PMP22-transgenic
rats which completely fail to elaborate a myelin
sheath. The severalfold increased PMP22 gene dosage
results in a remarkable „uncoupling“ of normal molecular
parameters of Schwann cell differentiation which
coincide with a premature myelination arrest. The
abnormal „gain of function“ effect places the role of
PMP22 upstream in the myelin assembly process.
II. Proteolipid protein in myelin assembly
and dysmyelinating disease
M. Jung, E. Krämer, M. Klugmann
PLP is a highly conserved, myelin-specific protein
with four transmembrane domains. We have previously
identified mutations in the mouse PLP gene
which differ in phenotype depending on the nature of
the specific molecular defect. There is good evidence
that mutations of the PLP gene are lethal because the
91

expression of misfolded PLP is „toxic“ to myelinforming
oligodendrocytes. Jimpy mice can not be
„rescued“ by transgenic complementation with a wildtype
PLP transgene. Moreover, mice which completely
lack expression of PLP or any truncated
polypeptide (a null allele created by gene targeting)
develop normal motor functions and show no signs
of abnormal glial cell death. In fact, CNS myelin is
assembled as a multilamellar and compacted structure
in the absence of PLP. Although the overall stability
of such PLP-deficient myelin appears reduced, these
observations question the view that the major mem-
Figure 1. Mutations of the gene for myelin proteolipid
protein (PLP) cause Pelizaeus-Merzbacher Disease and
severe dysmyelination in PLP-transgenic mice. The molecular
pathomechanism, which is associated with abnormal
protein trafficking, is poorly understood. We are using confocal
microscopy to study the intracellular localisation and
export of myelin proteins into the processes of cultured oligodendrocytes.
The folding-sensitive O10-epitope of PLP
(in green) which is lacking in all mutant PLP isoforms
emerges after the C-terminal epitope (in red) of newly synthezised
PLP (provided by E. Krämer).
92
brane protein of CNS myelin is essential to obtain
myelin membrane adhesion and compaction. Recent
evidence suggest that PLP deficiency has profound
effects on axonal integrity which becomes clinically
manifest in aged mice with signs of „neuroaxonal
dystrophy“. The basis of the underlying axon-glia
interaction is currently under investigation.
We have expressed PLP cDNAs from natural mouse
mutants in cultured non-glial cells. These studies show
that fibroblasts are much less sensitive to the overexpression
effect and mutant isoforms of PLP than
oligodendrocytes in vivo. However, also transfected
fibroblastoid cells recognize subtle alterations in the
PLP primary structure, because these translation products
are retained inside the cell and do not reach the
cell surface. A direct demonstration of protein misfolding
is also possible with a conformation-sensitive
monoclonal antibody (O10) which distinguishes
wildtype PLP from the known mutant isoforms.
III. Neuronal members of the proteolipid protein
family: M6A and M6B
L. Dimou, M. Klugmann, H. Werner
PLP is the prototype of a small family of sequencerelated
tetraspan membrane proteins which includes
M6A and M6B proteins, abundantly expressed on
neuronal processes in the central nervous system.
M6B is also detectable in oligodendrocytes. There are
multiple M6B-mRNAs, encoding at least eight protein
isoforms. Smaller, presumably soluble polypeptides
result from alternatively spliced M6B mRNAs
with a stop codon upstream of the exon for the first
transmembrane domain. In cells of the oligodendroglial
lineage, M6B localizes to the cell surface and transiently
also on cellular processes. By in situ-hybrid-
ization, oligodendrocytes express one of two alternative
C-termini, whereas cortical neurons express
both. Regulated expression of the various intracellular
domains of M6B, termed α, β, γ, ψ, and ω (some of
which are structurally conserved in M6A and PLP),
suggests that proteolipids can interact with cytoplasmic
proteins present in a variety of neural cell types.
The N-terminal domains of M6B affect transport features
and the subcellular distribution of this protein
when expressed in fibroblasts. The analysis of M6A
and M6B mouse mutants is ongoing and suggests a
role in neuronal process outgrowth and CNS myelination,
respectively.
IV. NEX and NeuroD regulate hippocampal
granule cell differentition
A. Bartholomä, S. Göbbels, M. Rossner, M.
Schwab
Parts of our laboratory‘s activity was devoted to the
function of basic helix-loop-helix (bHLH) proteins in
the nervous system. The neuronal transcription factors
NEX (neuronal helix-loop-helix protein-1), NeuroD
(neurogenic differentiation factor), and NDRF comprise
a family of Drosophila atonal-related bHLH
proteins with highly overlapping expression in the
developing forebrain. A role for NeuroD and NEX
in terminal neuronal differentiation is demonstrated
by mutations in mice. For example, in the hippocampus,
presumptive granule cells of the dentate gyrus
are generated but fail to mature, lack normal sodium
currents, and show little dendritic arborization. Longterm
hippocampal slice cultures reveal secondary
alterations, such as abnormal projections from the
entorhinal cortex. Ongoing experiments aim at restricting
the mutations of the NeuroD gene to subsets of
cortical neurons, utilizing homologous recombination
of the loxP/Cre system. This will allow to circumvent
perinatal lethality and study the role of bHLH proteins
in adult neuronal functions.
Figure 2. Cre-mediated recombination of genes that are
flanked by loxP sites allows the generation of cell type-specific
somatic mutations in transgenic mice. We have utilized
the spatio-temporal expression pattern of the NEX gene
to drive expression of Cre specifically into CNS neurons. A
„knock in“ strategy was chosen, to insert Cre into exon 2 of
the endogenous NEX gene. To demonstrate the specificity of
this system, a lacZ reporter function was specifically activated
by Cre recombination in neurons of the hippocampus
and neocortex of transgenic mice. This mouse is used to
circumvent the perinatal lethality of a null mutation in the
neuroD gene (provided by S. Goebbels).
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(SFB 317 “Molekulare Biologie neuraler Mechanismen
und Interaktionen”, Graduiertenkolleg “Molekulare
und zelluläre Neurobiologie”) from the BMBF
and from the EU.
93

PUBLICATIONS
Anderson, T.J., Klugmann, M., Thomson, C.,
Schneider, A., Readhead, C., Nave, K.-A., and
Griffiths, I.R. (1999). Distinct phenotypes associated
with increasing dosage of the PLP gene: implications
for gene duplications in CMT1A. Annals N.Y. Acad.
Sci. 883, 234-236.
Anderson, T.J., Schneider, A., Barrie, J.A., Klugmann,
M., McCulloch, M.C., Kirkham, D., Kyriakides, E.,
Nave, K.-A. and Griffiths, I.R. (1998). Increased
dosage of the proteolipid protein gene causes late-onset
neurodegeneration. J. Comp. Neurol. 394, 506-519.
Bradl, M., Bauer, J., Inomata, T., Zielasek, J., Nave,
K.-A., Toyka, K., Lassmann, H., and Wekerle, H.
(1999). Transgenic Lewis rats overexpressing the proteolipid
protein gene: myelin degeneration and its
effect on T cell-mediated experimental autoimmune
encephalomyelitis. Acta Neuropathol. 97, 595-606.
Coetzee, T., Suzuki, K., Nave, K.-A., and Popko,
B. (1999). Myelination in the absence of galactolipids
and proteolipid proteins. Mol. Cell. Neurosci. 14,
41-51.
Dimou, L., Klugmann, M., Werner, H., Jung, M.,
Griffiths, I.R., and Nave, K.-A. (1999). Dysmyelination
in mice and the proteolipid protein gene family.
Adv. Exp. Med. Biol. 468, 261-271.
Griffiths, I., Klugmann, M., Anderson, T. and Nave,
K.-A. (1998). Current concepts of PLP and its role
in the nervous system. Microscop. Res. Techn. 41,
344-358.
94
Griffiths, I., Klugmann, M., Anderson, T., Yool, D.,
Thomson, C., Schwab, M.H., Schneider, A., Zimmermann,
F., McCulloch, M., Nadon, N., and Nave,
K.-A. (1998). Axonal swellings and degeneration in
mice lacking myelin proteolipid protein. Science 280,
1610-1613.
Klein, L., Klugmann, M., Nave, K.-A., and Kyewski,
B. (1999). Shaping of the autoreactive T-cell repertoire
by a splice variant of self protein expressed in
thymic epithelial cells. Nature Medicine 6, 56-61.
Montague, P., Kirkham, D., McCallion, A.S., Davies,
R.W., Kennedy, P.G., Klugmann, M., Nave, K.-A.,
and Griffiths, I.R. (1999). Reduced levels of a specific
myelin-associated oligodendrocytic basic protein isoform
in shiverer myelin. Dev. Neurosci. 21, 36-42.
Nave, K.-A. (1999) X-linked dysmyelination: mouse
models of Pelizaeus-Merzbacher disease. In Mouse
models in the study of genetic neurological disorders.
B. Popko, ed. (Plenum Press, New York), pp 25-41.
Nave, K.-A. (1999). Zentrale Myelinisierungsstörungen:
Biologische Grundlagen, transgene Modelle und
molekulare Pathologie. In Handbuch der Molekularen
Medizin, Band V. D. Ganten and K. Ruckpaul, eds.
(Springer Verlag Berlin), pp 370-394.
Nave, K.-A. and Trapp, B. (2000). Myelin-forming
glial cells. Glia 29, 103.
Nave, K.-A. (2000). Myelin-specific genes and their
mutations in the mouse. In Glial Cell Development,
2 nd edition. K.R. Jessen and W.D. Richardson, eds.
(Oxford University Press), (in press).
Niemann, S., Sidman, R.L. and Nave, K.-A. (1998).
Evidence against altered forms of MAG in the dysmyelinated
mouse mutant claw paw. Mammalian
Genome 9, 903-904.
Niemann, S., Sereda, M., Rossner, M., Stewart, H.,
Suter, U., Meinck, H.-M., Griffiths, I.R., and Nave,
K.-A. (1999). The ‚CMT rat‘: Peripheral neuropathy
and dysmyelination caused by transgenic overexpression
of PMP22. Annals N.Y. Acad. Sci. 883,
254-261.
Niemann, S., Sereda, M.W., Suter, U., Griffiths,
I.R., and Nave, K.-A. (2000). Uncoupling of myelin
assembly and Schwann cell differentiation by transgenic
overexpression of PMP22. J. Neuroscience, 20,
4120-4128.
Schwab, M., Druffel-Augustin, S., Gass, P., Jung,
M., Klugmann, M., Bartholomae, A., Rossner, M.,
and Nave, K.-A. (1998). Neuronal basic helix-loophelix
proteins (NEX, neuroD, NDRF): spatio-temporal
expression and targeted disruption of the NEX
gene in transgenic mice. J. Neurosci. 18, 1408-1418.
Schwab, M.H., Bartholomä, A., Heimrich, B., Feldmeyer,
D., Druffel-Augustin, S., Goebbels, S., Naya,
F.J., Frotscher, M., Tsai, M.-J., and Nave, K.-A.
(2000). Neuronal bHLH proteins (NEX and BETA/
2NeuroD) regulate terminal granule cell differentiation
in the hippocampus. J. Neurosci. 20, 3714-3724.
Suter, U. and Nave, K.-A. (1999). Transgenic mouse
models of CMT1A and HNPP. Annals N.Y. Acad. Sci.
883, 247-253.
Vouyiouklis, D.A., Werner, H., Griffiths, I.R., Stew-
art, G.J., Nave, K.-A., and Thomson, C.E. (1998).
Molecular cloning and transfection studies of M6b-2, a
novel splice variant of a member of the PLP-DM20/M6
gene family. J. Neurosci. Res. 52, 633-640.
Werner, H., Jung, M., Klugmann, M., Sereda, M.,
Griffiths, I., and Nave, K.-A. (1998). Mouse models
of myelin diseases. Brain Pathol. 8, 771-793.
THESES
Staatsexamen
Kassmann, C. (1999). Funktionelle Charakterisierung
von basischen Helix-Loop-Helix Proteinen.
Dissertations
Rossner, M. (1998). Molekulare unf funktionale Charakterisierung
zweier neuer Transkriptionsfaktoren,
SHARP-1 und -2: Eine Rolle für bHLH Proteine in
neuronaler Plastizität.
Schwab, M. (1998). Herstellung und Analyse einer
Mausmutante mit einer Nullmutation des NEX Gens.
Klugmann, M. (1999). Funktionsanalyse des Myelin
Proteolipid Proteins (PLP) und seines neuronalen
Homologs M6A in vivo.
Sereda, M. (1999). Ein transgenes Rattenmodell für
die Charcot-Marie-Tooth Erkrankung.
95

STRUCTURE OF THE GROUP
E-mail: nave@em.mpg.de
Group leader Nave, Klaus-Armin, Ph.D., Prof.
Postdoctoral
fellows Bartholomä, Angelika, Dr. rer. nat.
Klugmann, Matthias, Dr. rer. nat.*
Krämer, Evi, Dr. rer. nat.
Niemann, Stefan, Dr. med.*
Rossner, Moritz, Dr. rer. nat.*
Schwab, Markus, Dr. rer. nat.*
Sereda, Michael, Dr. med.*
Yonemasu, Tomoko, Ph.D.*
Ph.D. students Dimou. Leda, Dipl.-Biol.
Goebbels, Sandra, Dipl.-Biol.
Jung, Martin, Dipl.-Biol.
Lappe, Corinna, Dipl. Biol.*
Schardt, Anke, Dipl. Biol.*
Werner, Hauke, Dipl. Biol.
Technical
assistants Druffel-Augustin, Silke
Krischke, Hartmut
* part of the time reported
96
Renato Paro
Chromatin-Controlled Epigenetic Regulation
of Transcription
During the development of an organism, mechanisms
of pattern formation program cells for particular functions
and structures. The program code is based on
a cell-specific differential repression or activation of
master control genes. Once established such codes
need to be maintained over many cell divisions during
the growth phases and eventually also into adulthood,
a process termed “cellular memory”. In this context,
the stable and mitotically heritable inactivation of
transcription (Silencing) becomes an important developmental
function, necessary to generate and to maintain
defined gene expression patterns in determined
cells. The proteins of the Polycomb group (PcG) control
the permanent inactivation of program coding factors
like the HOX genes. PcG proteins appear to inactivate
their target genes by generating heterochromatin-like
structures. Conversely, the proteins of the trithorax
group (trxG) counteract the silencing role of
the PcG and maintain the active expression state of the
target genes. Thus, the maintenance of gene expression
patterns is controlled at the levels of higher order
chromatin structures. Chromatin components such as
histones, through their modifications, and other chromatin
proteins seem to be able to generate an “epigenetic
code” that influences the processing of the underlying
genetic information. We are studying and trying
to decipher this epigenetic code in the fruit fly Drosophila
melanogaster by assessing the molecular functions
of the PcG and trxG proteins in the chromatinbased
control of HOX gene expression.
Chromatin-based silencing mechanisms appear to have
many evolutionary conserved features. Thus, their
analysis in a genetically well tractable system like
Drosophila serves as a paradigm for other epigenetic
phenomena like the mammalian X-chromosome inactivation
or genomic imprinting. In addition, there is
increasing evidence that the missregulation of elements
of cellular memory in adulthood can result
in tumorigenesis in mammals. We would like to
understand and to identify the common denominator
involved in these apparently diverse processes. What
creates and forms particular chromatin structures that
maintain defined gene expression patterns? Is the
DNA-encoded genetic information overlaid by an epigenetic
code, and how is this code transmitted from
one cell generation to the next?
I. Chromosomal elements conferring epigenetic
inheritance
G. Cavalli; S. Ehrhardt; F. Lyko; M. Prestel; DFG,
HFSP (in collaboration with A. Surani (Wellcome,
Cambridge) and R. Ohlson (Univ. of Uppsala))
Using chromatin-IP (X-CHIP) methodology developed
in the lab, we have identified several chromosomal
elements of the bithorax complex (BX-C)
through which the PcG and trxG proteins exert their
functions. We have shown in a particular transgenic
set-up that chromosomal elements, such as Fab-7 controlling
the Abdominal-B gene in the BX-C, are able
to maintain a decision made early during embryogenesis
through many mitotic cell divisions during development.
Fab-7 is switched from a silenced (PcGcontrolled)
to an activated (trxG-controlled) chromatin
state by a pulse of embryonic transcription. Once
activated the expression of a nearby reporter gene
becomes independent of the primary transcription
factor. Surprisingly, in the transgenic system, an active
97

Fab-7 state can also be transmitted meiotically in a
substantial portion of the progeny (Figure 1).
While we initially established the system using Fab-7,
we have now demonstrated a similar maintenance
function for two other elements of the BX-C, MCP
and BXD. Thus, our results indicate that in genetic
complexes containing master control genes like the
Figure 1. In a transgene construct an activated CMM is
stable during mitotic cell divisions and to a certain degree
also stable through meiosis. This result shows that in principle
it is possible to transmit a chromatin-based epigenetic
state through the germ line into the next generation.
98
homeotic genes, defined chromosomal elements act
as “cellular memory modules” (CMMs) capable of
memorizing active or repressed gene expression patterns
over developmental time.
In the previous period, we have used Drosophila to
identify chromosomal elements controlling mammalian
epigenetic mechanisms like genomic imprinting.
In collaboration with the Surani lab we have identified
an upstream element of the imprinted H19 gene acting
as a silencer in Drosophila. The subsequent deletion
of this element in the endogenous murine H19 locus
by the Surani lab resulted in the loss of the imprint. In
parallel, we could demonstrate in collaboration with
the Ohlson lab that the nucleosomal patterning of the
upstream region containing the imprinting element of
H19 in transgenic flies highly resembles the endogenous
mouse H19 pattern. Thus, conserved features
seem to be involved in maintaining the epigenetically
controlled imprinted states of H19. Indeed, in another
approach we were able to demonstrate that a different
chromosomal element, necessary for the maintenance
of H19 expression, in transgenic Drosophila is bound
and controlled by a member of the PcG (PSC). These
results witness the usefulness of Drosophila in studying
basic conserved mechanisms of chromatin regulation
in epigenetic processes.
II. Epigenetic marking of CMMs
B. Breiling, G. Cavalli, C. Maurange, G. Rank,
DFG, HFSP (in collaboration with P. Becker (Univ.
of Munich))
PcG proteins show an extended binding profile at
CMMs. At the molecular level we could demonstrate
a highly specific binding of PC protein through its
C-terminal part with in vitro reconstituted nucleosomes.
Surprisingly, we found that also in the acti-
vated state PcG proteins are bound to the CMM. Conversely,
trxG proteins are still bound to a repressed
CMM. Thus, the interplay between PcG and trxG
members appears to be vital for the proper function
of CMMs. What defines a CMM, like Fab-7, to be in
an active or in an inactive mode and how this tag
is maintained over many cell generations, are questions
we are currently addressing. We could demonstrate
that hyperacetylated histone H4 tags an activated
CMM, and this epigenetic mark is transmissible
mitotically (Figure 2).
We are now assessing how CMMs become switched
from an apparent silenced default state to an activated
state. After embryogenesis the epigenetic state
of CMMs become fixed and cannot be changed anymore.
Thus, a specific event occurring during this
early stage appears to switch a CMM. After passing a
specific developmental threshold, the epigenetic state
is fixed and clonally inherited in subsequent cell generations.
We have evidence that early transcription
through the element might be important to change
the overall chromatin structure and thus the epigenetic
state of the CMM. Additionally, we are testing
the extent histone modifications at CMMs potentially
have as primary epigenetic tags and how such modifications
can be inherited at DNA replication and at
mitosis.
III. The TRITHORAX protein in cancer formation
I. Chen-Muyrers, S. Schönfelder, DKFZ-MOS,
DFG (in collaboration with E. Canaani (Weizmann,
Rehovot) and V. Orlando (DIBIT, Milano))
The gene trithorax (trx), the name giving member of
the trxG, was identified by its requirement for normal
expression of multiple homeotic genes of the bithorax
and Antennapedia complex. Different homeotic
genes and their promoters are affected differently in
trx mutants, suggesting a coordinated spatial and tem-
Figure 2. Immunostaining of larval
polytene chromosomes at the
integration site of a Fab-7 (CMM)
transgene construct. After embryonic
activation the Fab-7 construct
remains marked by histone
H4 hyperacetylation also at larval
stages (pannel D; arrow). Control
constructs or non-activated Fab-7
do not show a H4 hyperactylation
signal (pannels A-C) nor is H3
hyperacetylation observed in any
of the cases (pannels F-H).
99

poral requirement for trx in order to achieve normal
expression patterns. We have shown with X-CHIP a
dynamic association of TRX with CMMs and promoters
of the BX-C and found a close cooperation of TRX
with PcG members at CMMs.
Several members of the Drosophila trxG have counterparts
in humans and mice. The mixed-lineage leukaemia
(MLL) gene (also known as ALL-1, HRX), a
TRX homologue, was found through its implication in
the majority of infantile acute lymphocytic and mixed
lineage leukemias. Disruption of the murine Mll gene
by gene targeting causes homeotic transformations of
the vertebrae in heterozygotes and loss of homeotic
gene expression, supporting the notion that Mll is a
functional equivalent of trx.
To date, not much is known at the molecular level
how translocation of MLL with various other chromosomal
locations generate leukemia. At least 23 fusion
MLL proteins involved in leukemia have been identified.
As part of our collaborative efforts with
the Canaani lab, we have generated transgenic flies
expressing full length MLL, and fusion proteins
MLL-AF9, MLL-AF4, and MLL-AF17 under GAL4
control. Expression of these proteins in Drosophila
may reveal their effects by determining with which
pathway in Drosophila development they influence.
Crosses between our transgenic flies and various
GAL4 drivers revealed that only MLL-AF9 and
MLL-AF17 constructs had a slight effect on Drosophila
development. When we took a closer look at the
molecular level, we could not detect the proteins on
Western, but at the RNA level, we see transcripts from
all four constructs. Interestingly, this same effect is
also observed with MLL-AF9 in mouse. We are currently
testing whether overexpression of the murine
proteins results in cell death as recent published observations
link MLL with apoptosis.
100
IV. Drosophila as a model system to identify
components involved in the processing
and the function of the human Amyloid Precursor
Protein
G. Merdes, B. Brückner, DFG (in collaboration
with K. Beyreuther (ZMBH))
The human amyloid precursor protein (APP) is an
integral membrane protein with two homologues in
invertebrates: the amyloid protein-like protein 1 (C.
elegans, APL-1) and the amyloid precursor proteinlike
protein (D. melanogaster, APPL). Flies deficient
for expression of APPL show a phototaxis impairment
that can be rescued by the expression of human APP
suggesting an evolutionary conservation of APP function.
The importance of APP in the pathogenesis of
Alzheimer’s disease (AD) became apparent through
the identification of distinct mutations in the gene
causing early onset familial AD. The major component
of the plaques and amyloid found in senile Alzheimer
patients is a proteolytic product of APP, the
βA4 peptide. APP is cleaved in vivo by several proteases
termed α−, β−, and γ−secretase. Cleavage of
APP by the α−secretase prevents the production of
βA4. Despite the availability of APP-null mutants and
transgenic mice expressing human APP, the physiological
role of APP remains unknown and many factors
involved in the processing of APP have not yet
been clearly identified. Therefore we have established
Drosophila melanogaster as a model system to study
the function and processing of APP.
Various forms of human APP were expressed in
Drosophila tissue culture cells as well as in transgenic
fly lines. Studies in both systems revealed that also in
flies APP is processed in a similar way as in mammalian
cells in regard to the α- and γ-cleavages. In addition,
transgenic flies expressing full-length forms of
APP in the wing imaginal discs are characterized by a
blistered-wing phenotype, suggesting that the expression
of human APP interferes with the cell adhesion
between the dorsal and ventral epithelial cell monolayers
of the wing (Figure 3). To define the involvement
of APP in the blistered wing phenotype in more
detail as well as to identify factors involved in the processing
of APP, we perform biochemical and genetic
studies. Since the observed wing phenotype depends
on full-length APP, an increased processing of APP,
e.g. by the simultaneous overexpression of an APPspecific
protease, results in a suppression of the phenotype.
This observation enables us to identify proteins
involved in the processing of APP by the use of
genetic screens and to subsequently identify the corresponding
mammalian homologues. In a similar way,
gene products required for the effect of APP on cell
adhesion and therefore for APP function, can also be
identified by genetic screens. Mutations in these genes
result in suppression or an enhancement of the blis-
tered wing phenotype, dependent on their function.
Drosophila can be mutagenized by the use of chemicals
or by the use of the transposable P-element.
The advantage of P-element mutagenesis is the easier
identification of the mutagenized genes by isolating
and sequencing the flanking genomic DNA. Therefore
we have recently performed a P-element based genetic
screen as well as screened collections of already existing
Drosophila mutants. The analysis of 150 000 flies
with potential new P-element insertion sites resulted
in the isolation of several enhancer- and suppressormutations,
together with several genes interfering with
wing patterning. The corresponding genes, which we
are currently characterizing further, encode factors
involved in signal transduction, maintenance of cell
shape and cell adhesion, and protein processing.
We utilize the fruit fly Drosophila melanogaster as
a genetic model system. Since Thomas Hunt Morgan
introduced Drosophila as a model organism into genetics
at the beginning of this century and isolated the
Figure 3. Expression of different
isoforms of human APP in transgenic
Drosophila. Only full-length
APP result in a blistered wing
phenotype.
101

first mutation, thousands of genes and mutations have
been isolated and characterized, making Drosophila
one of the best-studied multicellular organisms. In
Drosophila many factors and basic principles of
development and differentiation have been uncovered,
which were subsequently found to also play important
roles during the development of mammals, e.g. morphogens
and the homeodomain. Surprisingly, despite
the difference in morphology, even gene products
required for the development of specific organs have
been evolutionary conserved in structure and function.
This conservation of fundamental developmental and
cellular principles as well as the vast assortment of
genetic, cell and molecular biological tools available,
allows us to study biological problems of medical relevance
in Drosophila and to identify conserved factors
and mechanisms required for epigenetics.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(Sachbeihilfe and Graduiertenkolleg “Signalsysteme
und Genexpression in entwicklungsbiologischen
Modellsystemen”, from the Human Frontier
Science Program, from the Deutsche-Israelische
Zusammenarbeit in der Krebsforschung (DKFZ/
MOS), from the BMBF (Humangenomprojekt) and
the Fonds der Chemischen Industrie.
PUBLICATIONS
Brenton, J.D., Ainscough, J.F., Lyko, F., Paro, R., and
Surani, M.A. (1998). Imprinting and gene silencing
in mice and Drosophila. Novartis Found Symp. 214,
233-442.
Cavalli, G. and Paro, R. (1998). Chromo-domain proteins:
linking chromatin structure to epigenetic regula-
102
tion. Curr. Opin. Cell. Biol. 10, 354-360.
Cavalli, G. and Paro, R. (1998). The Drosophila Fab-7
chromosomal element conveys epigenetic inheritance
during mitosis and meiosis. Cell 93, 505-518.
Fossgreen, A., Brückner, B., Czech, C., Masters,
C.L., Beyreuther, K., and Paro, R. (1998). Transgenic
Drosophila expressing human amyloid precursor protein
show gamma-secretase activity and a blisteredwing
phenotype. Proc. Natl. Acad. Sci. U.S.A. 95,
13703-13708.
Gasser, S.M., Paro, R., Stewart, F., and Aasland, R.
(1998). The genetics of epigenetics. Cell. Mol. Life
Sci. 54, 1-5.
Lyko, F., Buiting, K., Horsthemke, B., and Paro, R.
(1998). Identification of a silencing element in the
human 15q11-q13 imprinting center by using transgenic
Drosophila. Proc. Natl. Acad. Sci. U.S.A. 95,
1698-1702.
Orlando, V., Jane, E.P., Chinwalla, V., Harte, P.J., and
Paro, R. (1998). Binding of trithorax and Polycomb
proteins to the bithorax complex: dynamic changes
during early Drosophila embryogenesis. EMBO J.
17, 5141-5150.
Paro, R., Strutt, H., and Cavalli, G. (1998). Heritable
chromatin states induced by the Polycomb and trithorax
group genes. Novartis Found. Symp. 214, 51-61.
Paro, R. (1998). Das Zellgedächtnis. Biospektrum 6,
21-24.
Breiling, A., Bonte, E., Ferrari, S., Becker, P.B., and
Paro, R. (1999). The Drosophila Polycomb protein
interacts with nucleosomal core particles in vitro via its
repression domain. Mol. Cell. Biol. 19, 8451-8460.
Cavalli, G. and Paro, R. (1999). Epigenetic inher–
itance of active chromatin after removal of the main
transactivator. Science 286, 955-958.
Dietzel, S., Niemann, H., Brückner, B., Maurange, C.,
and Paro, R. (1999). The nuclear distribution of Polycomb
during Drosophila melanogaster development
shown with a GFP fusion protein. Chromosoma 108,
83-94.
Lyko, F. and Paro, R. (1999). Chromosomal elements
conferring epigenetic inheritance. Bioessays 21,
824-832.
Paro, R. (1999) Trithorax Group Genes. In Encyclopedia
of Molecular Biology (ed. Creighton, T.E.) Wiley,
New York, pp 2670-2672.
Paro, R. (2000). Mapping Protein Distributions on
Polytene Chromosomes by Immunostaining. In Drosophila
Protocols Manual (ed. Ashburner, M.) CSHL
Press, CHS, (in press)
THESES
Diploma
Erhardt, Sylvia (1998). Etablierung und Charakterisierung
transgener Drosophila melanogaster mit
einem cis-regulatorischen Element geprägter Gene der
Maus und die Analyse der am Gen-Silencing beteiligten
Faktoren.
Dissertations
Breiling, Achim (1998). Charakterisierung der funk-
tionellen Aufgaben der beiden evolutionär konser–
vierten Bereiche des Polycomb-Proteins aus Drosophila
melanogaster: die Chromodomäne und der
C-Terminus.
Lyko, Frank (1998). Identifikation und Charakterisie–
rung von Silencing-Elementen in cis-regulatorischen
Regionen geprägter Säugergene mit Hilfe transgener
Drosophila melanogaster.
(GFM-Preis für beste Doktorarbeit)
STRUCTURE OF THE GROUP
E-mail: paro@sun0.urz.uni-heidelberg.de
Group leader Paro, Renato, Prof. Dr.
Research associates Cavalli, Giacomo, Dr.*
Merdes, Gunter, Dr.
Ph.D. students Breiling, Achim, Dipl. Biol.*
Brückner, Bodo, Dipl. Biol.
Chen-Muyrers, Inhua
Lyko, Frank, Dipl. Biol.*
Maurange, Cédric*
Prestel, Matthias, Dipl. Biol.*
Rank, Gerhard, Dipl. Biol.
Diploma students Erhardt, Sylvia*
Schönfelder, Stefan*
Techn. assistants Albrecht, Steffen *
Bas-Orth, Carlos *
Friedrich, Daniela *
Ehret, Heidi
Eysel, Georg *
Mund, Cora *
Schwarz, Gabriele
*part of the time reported
103

Frank Sauer
Mechanisms of Transcriptional Regulation
Our research focuses on the molecular mechanisms
governing regulation of eukaryotic gene expression.
The capability of living organisms to control gene
expression is essential for the execution of complex
biological events, such as body pattern formation or
cell differentiation, as well as the response to extra-
and intra-organismal stimuli. Eukaryots have evolved
refined mechanisms that regulate the precise temporal
and spatial expression of genes. At the molecular
level, eukaryotic gene expression is predominantly
regulated at the level of transcriptional initiation. A
widely accepted model proposes that initiation of
transcription requires the precisely regulated assembly
of distinct multi-protein complexes on eukaryotic
genes. Major efforts have led to the identification of
the key players that initiate m-RNA synthesis. The
first player is the eukaryotic gene, that in general
is comprised of a protein coding region, which
is flanked by two essential cis-regulatory regions:
core-promoter and enhancer/silencer. The core-promoter
defines the start-site of transcription while the
enhancer/silencer mediate the precise temporal and
spatial expression of the coding region. The core promoter
is the target for the general RNA polymerase II
(pol II) transcription machinery (GTM). This multiprotein
complex contains general transcription factors
(TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH), RNA
polymerase II, the catalytic motor of m-RNA synthesis
and associated protein-complexes. Enhancer/
silencer-regions are characterized by their repertoire
of DNA-sequences, which serve as targets for transcription
factors, a diverse family of DNA-binding
Figure 1. Dorsal-dependent transcriptional activation is alleviated in TAF250-mutant embryos. In situ hybridization of
whole mount Drosophila embryos (left and middle panel) showing twist and snail expression. Note that the expression of
both twist and snail is significantly reduced in TAF250 mutant embryos. (Right panel) Cuticula preparations of Drosophila
embryos. Note that the Drosophila body pattern is significantly altered in TAF250-mutant embryos.
104
proteins. It is thought, that transcription factors contact
components within the general transcription machinery
to either activate or repress the process of transcriptional
initiation. This communication between
transcription factors and GTM requires in many cases
so called co-factors, which function as receivers of the
regulatory signals provided by transcription factors.
Although co-factors are a diverse group of proteins, a
common feature of these proteins is their direct interaction
with transcription factors and recruitment of
co-factors has been established as an essential step
of transcriptional regulation. It is thought that co-factors
function as molecular bridge between transcription
factors and GTM. The finding that the same cofactors,
which function as molecular bridge between
transcription factors and GTM, can mediate posttranslational
modification of histones has added an unexpected
level of complexity to the model of transcriptional
regulation. Histones (H1, H2A, H2B, H3,
H4) are the basic building block of nucleosomes, the
most fundamental structural entity within chromatin.
Nucleosomes inhibit the most fundamental step of
transcriptional regulation, namely the interaction of
transcription factors and GTM with DNA. To overcome
this “nucleosome-barrier” it is thought that transcription
factors recruit co-factors, which by posttranslational
modification of histones establish transcriptional
active or inactive chromatin structures.
Abundant evidence has been provided that co-factor
mediated acetylation of core-histones is correlated
with transcriptional activation while deacetylation is
concomitant with repression. Fuelled by the functional
diversity of co-factors deciphering the molecular
mechanisms for how co-factors convert regulatory
signals into activated or repressed states of transcription
lies at the heart of the transcription field.
TAFs mediate transcriptional activation in
Drosophila
Anh-Dung Pham
Transcription factors contact one or multiple components
of the GTM to activate transcription. One target
of activators is the general transcription factor TFIID
a multi-protein complex comprised of the TATA-box
binding protein (TBP) and at least eight TBP-associated
factors (TAF II s). Distinct TAF II s serve as targets
for different activation domains. As TAF II -activator
interactions mediate transcriptional activation in
vitro, TAF II s are thought to function as co-activators,
receivers, of activation signals. However, the role and
function of TAF II s for transcriptional activation in
vivo remains unclear. We have used Drosophila as a
model system to investigate the functional importance
of TAF II s for activation of transcription. To identify
transcription factors, which activate transcription in a
TAF II -dependent manner, we made use of TAF II 250
mutant flies. As TAF II 250 is the central subunit in the
TBP-TAF-complex, a disruption of this TFIID-subunit
is thought to abrogate the structure and function
of TFIID and, hence TFIID-dependent transcriptional
activation. We could show that a mutation within
TAF II 250 alleviates Dorsal-dependent transcriptional
activation (Figure 1). The Rel transcription factor
Dorsal establishes dorso-ventral polarity in the early
Drosophila embryo by activating the expression of
different tissue-determining genes in ventral and lateral
cells. We could show that Dorsal interacts with
two TAF II -subunits, TAF II 110 and TAF II 60. By using a
reconstituted Drosophila in vitro transcription system
and in vitro assembled partial TBP-TAF II -complexes
we could show that interactions between Dorsal and
TAF II 110 and/or TAF II 60 mediate transcriptional activation
in vitro. To provide evidence for the biological
relevance of these Dorsal: TAF II -interactions in vivo,
105

we made use of TAF mutant flies. The expression
of Dorsal target genes was significantly reduced in
TAF110 and/or TAF60 mutant embryos indicating
that these co-activators contribute to Dorsal-dependent
transcriptional activation in Drosophila. Our
results provide evidence that TAF II s play an important
role for transcriptional activation in Drosophila.
Figure 2. Model for Dorsal-dependent transcriptional activation.
For details see ”TAFs mediate transcriptional activation
in Drosophila”.
Mechanisms underlying STAT-dependent transcriptional
regulation in Drosophila
Simona Kwocynski
STATs (signal transducers and activators of transcription)
are phylogenetic highly conserved latent cytoplasmic
transcription factors that have been identified
in Dictyostelium, Drosophila, mouse and human.
STATs activate the expression of specific target genes,
which encode proteins that are involved in complex
biological processes such as immune response or
embryogenesis. Although the biological function and
importance of STATs has well established, the molec-
106
ular mechanisms underlying STAT-dependent transcriptional
activation remain poorly understood. To
address this question we have established a STATdependent
in vitro transcription assay that enables us
to characterize the co-factor requirements for STATmediated
transcriptional activation. The functional
relevance of these co-factors has been tested using
reconstituted in vitro transcription systems supplemented
with mutant co-factors. We use the Drosophila-embryo
as model system to determine the biological
relevance of the in vitro identified co-factors for
STAT-dependent transcriptional activation.
Mechanisms of transcriptional repression in
Drosophila
Thorsten Belz
The molecular mechanisms underlying repression of
transcription in eukaryotic cells remain opaque. We
use the Drosophila proteins Snail and Krüppel as
models to decipher modes of transcriptional repression
in Drosophila. The gap gene krüppel (Kr) is
expressed in the central region of the blastoderm
Drosophila embryo and encodes a zinc-finger type
transcription factor (Krüppel). Genetic analyses have
established that Krüppel represses the expression of
the gap-gene giant and the second expression domain
of the pair-rule gene even-skipped (eve). Snail (sna)
is expressed in 18 ventral-most cells of the embryo.
Sna encodes a zinc-finger type transcription factor
that represses the expression of neuroectoderm-specific
genes, including rhomboid (rho) and singleminded
(sim). We use a combination of biochemistry
and genetics to decipher the molecular mechanisms
underlying Snail- and Krüppel-dependent transcriptional
repression.
Identification of histone modifying enzymes
Christian Beisel
Various posttranslational modifications of histones
have been described. Although these modifications
have been correlated with transcriptional activation,
their precise role for transcriptional regulation remains
unknown. Two of the least understood histone modifications
are methylation and ubiquitination of histones.
The predominant histones, which are methylated,
are H3 and H4. Different studies have linked
histone methylation with regulation of gene expression.
In Drosophila changes in the gene expression
pattern in response to heat shock are concomitant
with changes in the methylation pattern of histones.
In Tetrahymena, transcriptionally active macronuclei
contain histone methyltransferase (HMT)-activity
whereas transcriptional inactive micronuclei do not.
It, therefore, has been proposed that histone methylation
plays a role in the reconfiguration of chromatin
under stress conditions. A functional link between histone
methylation and transcriptional regulation came
with the finding that the co-activator CARM-1 possesses
a histone-specific HMT-activity. Like methylation,
ubiquitination of histones has been correlated
with the execution of various DNA-dependent processes.
In Tetrahymena, ubiquitinated histones are
present in transcriptional active chromatin structures
implying that ubiquitination of histones correlates
with activation of transcription.
However, until now the role and function(s) of histone-methylation
and -ubiquitination for transcriptional
activation and the responsible enzymes remain
unknown. It is our aim to identify the enzyme(s)
responsible for methylation and ubiquitination of
histones.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (Graduiertenkolleg “Signalsysteme und Genexpression
in entwicklungsbiologischen Modellsystemen”,
Projekt-’Sachbeihilfen’) and the BMBF.
PUBLICATIONS
Schock, F., Sauer, F., Jäckle., H and Purnell, BA.
(1999). Drosophila head segmentation factor buttonhead
interacts with the same TATA box-binding protein-associated
factors and in vivo DNA targets as
human Sp1 but executes a different biological program.
Proc. Natl. Acad. Sci. USA 96, 5061-5065.
Pham, A.D., Müller, S. and Sauer F. (1999). Mesoderm-determining
transcription in Drosophila is alleviated
by mutations in TAF II 60 and TAF II 110. Mech.
Dev. 84, 3-16.
La Rosee-Borggreve, A., Hader,T., Wainwright, D.,
Sauer F. and Jäckle H. (1999). Hairy stripe 7 element
mediates activation and repression in response to different
domains and levels of Krüppel in the Drosophila
embryo. Mech. Dev. 89, 133-140.
Pham, A.-D., and Sauer, F. (2000). Ubiquitinactivating/conjugating
activity of TAF II 250 mediates
activation of gene expression in Drosophila. Science
(in press).
107

THESES
Diploma
Belz, Thorsten (1998). Untersuchungen zur Assoziation
von Repressoren der Transkription aus Drosophila
melanogaster mit Histon-Deacetylase-Aktivität.
Kwoczynski, Simona (1998). Genetische Untersu–
chung zum Mechanismus der STAT-abhängigen Transkriptionsaktivierung
in Drosophila melanogaster.
Bierlein, Nicole (1999). Untersuchungen zum Einfluß
von Mutationen des TBP-assoziierten Faktors
TAF II 250 auf die differentielle Genexpression in
Drosophila melanogaster.
STRUCTURE OF THE GROUP
E-mail: f. sauer@mail.zmbh.uni-heidelberg.de
Group leader Sauer, Frank, Dr.
Ph.D. students Beisel, Christian
Belz, Thorsten
Kwoczynski, Simona
Dung-Pham, Anh
Diploma students Egle, Markus*
Bogin, Jochen*
Tech. assistants Leibfried, Ute*
Müller, Sandra
* part of the time reported
108
Hans Ulrich Schairer
I. Stigmatella aurantiaca, a Prokaryotic
Organism for Studying Intercellular Signalling
and Morphogenesis
The organisms
Stigmatella aurantiaca belongs to the myxobacteria
that are Gram-negative soil bacteria. Myxobacteria
show both, features of unicellular and multicellular
organisms. As the eukaryotic organism Dictyostelium
dicoideum they are thought to lie on the boundary
between unicellular and multicellular organisms.
Myxobacteria grow and divide as separate cells. But
they may be regarded as a multicellular organisms
whose cells feed in swarms and which under conditions
of starvation, assemble to well defined regular
three dimensional structures called fruiting bodies
which enclose about 10 5 dormant cells, the myxospores.
The shape of the fruiting bodies is species specific
and is genetically determined. The fruiting body
of S. aurantiaca consists of a stalk bearing several
sporangioles on branches at its top.
Myxobacteria secrete hydrolytic enzymes together
with slime with which they degrade particulate organic
matter of the soil. It has been shown that the growth
rate increases with cell density if myxobacteria were
grown on a macromolecular substrate as sole nutrient,
such as casein. This suggests that cells feed co-operatively
and the association in a swarm allows them to
feed more efficiently. The advantage of cooperative
feeding may have driven the evolution of fruiting body
formation. When nutrients are again available after a
period of starvation, myxospores germinate and form
vegetative cells. The multicellular nature of the fruiting
body ensures that a swarm of cells is formed for a
new growth cycle.
Myxobacteria move by gliding on solid surfaces. This
facilitates the stabilisation of a swarm and of fruiting
body formation. Gliding permits tight cell-cell contact
and efficient signal exchange between the cells
by diffusible molecules. Both features are a prerequisite
for the transmission of positional information of
the single cell necessary for the coordination of the
metabolism and movement of the cell in the course
of fruiting body formation. One of the developmental
signals – Stigmolone – that is involved in early cell
aggregation has been recently isolated and characterised
by Wulf Plaga et al.
Apart from their ability to form fruiting bodies, myxobacteria
form a broad range of secondary metabolites.
All these unique features are reflected in the size of the
genome and its organisation. The size of the myxobacterial
genome has been shown to be about 9.5 Mbp.
A gene cluster involved in S. aurantiaca fruiting
body formation
Susanne Müller, Barbara Silakowski and Diana Hofmann
To investigate the genes involved in S. aurantiaca
fruiting body formation and the co-ordination of their
expression, Tn5 transposon insertion mutagenesis was
performed. Three different mutant types impaired in
fruiting body formation were detected by screening
the insertional mutants. They include mutants that
form neither fruiting bodies nor aggregates. mutants
that aggregate to unstructured clumps, and mutants
that undergo only part of the differentiation process.
One of the mutants (AP182) that formed clumps
during starvation was analysed further. Mixing of the
109

cells of this strain with those of a mutant (AP191),
which was unable to form aggregates prior to starvation,
lead to a partial phenotypic complementation.
Instead of clumps, a mushroom-like structure, similar
to a champignon was obtained. Sequencing of the
mutant gene of strain AP182 and of the adjacent
genomic segments resulted in four open reading
frames that were involved in fruiting body formation.
One of the genes, fbfB, and the other three genes,
fbfA, fbfC, and fbfD are arranged in a divergent orientation.
FbfB shows significant homology to the
secreted copper enzyme galactose oxidase from the
fungus Dactylium dendroides. fbfA encodes a polypeptide
that is homologous to chitin synthases. The
start codon of fbfC overlaps with the stop codon of
fbfA. FbfC has no significant homology to any of the
known proteins. FbfD has similarities to the human
phosphoprotein synapsin I. Inactivation of either of
these genes by insertion of the neo gene cassette
resulted in mutants that formed only unstructured
clumps during starvation. This indicates the gene products
of the fbf-genes to be involved in fruiting body
formation. No additional open reading frame involved
in fruiting was detected downstream of fbfD. Mixing
of cells from either the fbfA or the fbfB mutant with
those of the non-aggregating strain AP191 before starvation
resulted in mushroom-like fruiting bodies with
the form of a morel or a champignon, respectively.
These forms are observed during wild-type fruiting
body formation 12 or 15 hours after the beginning of
starvation.
The partial phenotypic complementation suggests that
factors involved in fruiting and which are lacking in
one mutant may be obtained from the other. A reason
for the incomplete phenotypic complementation may
be that not all substances involved in fruiting (e.g.,
intracellular macromolecules) which are lacking in
110
one of the strains can be supplemented by the other
mutant and vice versa.
For the analysis of the time dependence of the expression
of the genes fbfA, fbfB, fbfC, and fbfD during
fruiting body formation or indole induced sporulation
merodiploid strains were constructed. They harboured
the wild type genes and in addition a 3’ truncated fbfgene
with 5’ regions of variable length. The truncated
genes were fused to the promotorless hybrid indicator
gene ∆trp-lacZ and the neo gene for selection.
β-Galactosidase activity increased 8 or 14 hours
after the beginning of starvation in the merodiploid
strains but not during indole induced sporulation. This
unequivocally proves the four fbf-genes to be involved
in the morphogenic process of fruiting. RT-PCR analyses
of fbf-gene transcription revealed these genes to
be induced during starvation. Low levels of fbf-gene
transcript are found in vegetative cell and in the case
of fbfC or fbfD during indole induced sporulation.
Analysis of the protein patterns of the wild-type and
the mutant strains by 2D electrophoresis is in progress.
Alternative sigma factors
Barbara Silakowski, Susanne Müller and Chi-Hyuk
Chang
The genes of two alternative sigma factors, sigB and
sigC have been cloned. These sigma factors harbour
two domains that were shown for σ 32 of E. coli to
be necessary for DnaK binding and thus for its proteolytic
clearance. Merodiploid strains containing the
wild type gene and the corresponding 3’ truncated
gene fused to an indicator gene were analysed for the
expression of the sigma factor genes during development
or heat shock. sigB was shown to be expressed
early during indole-induced sporulation and fruiting
body formation as well during heat shock. These
results agree with those of the RT-PCR analysis of
sigB transcription. Inactivation of either sigB or sigC
by insertional mutagenesis did not impair fruiting
body formation, indole-induced sporulation or the
heat shock response. No changes in either the spores’
ultrastructure (H. Lünsdorf, GBF, Braunschweig) or
in spore germination have been detected.
A gene cluster of S. aurantiaca DW4/3-1 for
Myxothiazol biosynthesis
H. Ehret and B. Silakowski in collaboration with H.
Reichenbach, GBF, Braunschweig
Sequence analyses downstream of the developmental
fbfB gene resulted in the detection of the mta (myxothiazol)
gene cluster. The first ORF, mtaA, (formerly designated
hesA), encodes a phosphopantetheinyl transferase.
P-pant transferases activate polyketide synthases
(PKS) by the transfer of the P-pant moiety from
coenzyme A to a conserved serine residue of the PKS.
Downstream of mtaA a second ORF, mtaB, (formerly
pksA) was found, of which 7 kbp were sequenced. It
encodes a PKS. Inactivation of mtaA by insertional
mutagenesis or deletion of part of the gene or insertional
inactivation of mtaB gene impairs myxothiazol
synthesis. In addition mutants defective in mtaA fail to
form a sofar unknown metabolite. This suggests MtaA
to be able to activate at least two different polyketide
synthases.
For studying the expression of the mtaB gene a merodiploid
strain BS64 was constructed that harboured
a functional wildtype and a 3‘ truncated mtaB gene
to which an indicator gene was fused. Measurement
of indicator gene expression showed, mtaB to be
expressed under all conditions tested, such as vegetative
growth, fruiting, indole induced sporulation and
heat shock. The project was stopped at this stage at the
ZMBH. Further investigations of the gene cluster are
performed at the GBF in Braunschweig.
CspA, a prominent cold-shock(-like) protein
I. Stamm and W. Plaga
Several related proteins of about 7 kDa constitute a
prominent fraction of the S. aurantiaca cell extract.
One of the genes encoding such a protein was cloned
and named cspA. The delineated protein sequence of
68 amino acid residues displays a high sequence identity
with bacterial cold-shock(-like) proteins. A RNA
chaperon-function was proposed for these proteins
in E. coli. Using a cspA::(trp-lacZ) fusion gene that
was introduced into Stigmatella by electroporation the
transcription was analyzed during development and
at lowered temperature. These experiments indicated
cspA to be constitutively transcribed at a high level.
The cspA promoter was used to express the gene for
the green fluorescent protein (GFP). GFP fluorescence
was found to be detectable in whole fruiting bodies
as well as in single cells. The GFP-labelled cells are
easily distinguished from wild type cells using a fluorescence-activated
cell sorter (FACS). Fruiting body
formation is not impaired in gfp expressing wild type
strains. Thus the gfp gene under the control of the
cspA promoter seems to be suitable to label Stigmatella
strains. With this kind of labelling it should be
possible to analyze the fate of mutant strain cells
in phenotypic complementation experiments during
fruiting body formation; in such experiments fruiting
bodies are formed by the combined action of two
mutant strains which are unable to develop properly
on their own.
111

Fluorescence based analysis of gene expression:
Identification of pheromone target
genes
The pheromone stigmolone (2,5,8-trimethyl-8-hydroxy
-nonan-4-one) is instrumental in early steps of fruiting
body formation. To identify stigmolone-responsive
genes a promoter trap vector (pTRAP1) was constructed
which allows the creation of random promoter
fusions to gfp in S. aurantiaca. With the aid
of a flow cytometer a selection strategy exploiting the
differential fluorescence induction (DFI) of these promoter
fusions by stigmolone is feasible. First analyses
of the random insertion mutants by flow cytometry
have shown, that about 2.5% of the mutants express
the gfp during vegetative growth. Screening for genes
affected by the addition of stigmolone is in progress.
HspA (SP21): Transcriptional regulation and
biological function
Hui Shen
HspA (formerly SP21) of S. aurantiaca is synthesised
during development or under stress such as heat shock,
oxygen limitation or indole treatment. Sequence alignment
revealed HspA to belong to the α-crystallin
family of the low molecular weight heat shock proteins.
HspA is located mainly at the cell periphery in
heat shocked cells and in fruiting body derived myxospores
and either at the cell periphery or within the
cytoplasma of indole treated cells as shown by immunoelectron
microscopy (H. Lünsdorf, GBF, Braun–
schweig).
Two transcripts of the hspA gene were detected after
heat shock and only one after indole treatment. A
unique transcription initiation site of the monocistronic
hspA gene was detected by primer extension
either after heat shock or indole treatment. Analysis
112
of the promoter proved various upstream regions to
be required for maximum expression of hspA under
stress conditions. For maximum hspA transcription
225 bp and 587 bp upstream of the ATG start codon
are required in the case of heat shock or indole treatment,
respectively.
To delimit the regulatory elements involved in hspA
transcription that depends on heat shock, deletion
/ insertion mutagenesis as well as gel shift assays
were performed. Three regulatory promoter regions
involved in heat shock response were defined. The first
region spans from bp -56 to bp -85 upstream of the
hspA ORF and harbours the RNA polymerase binding
sites. Deletion of this region completely blocks hspA
transcription. The second domain ranges from bp -141
to bp -223 upstream of the hspA ORF and carries putative
regulator-binding sites. Heat shock and phosphorylation
enhance binding of the regulator(s) to the hspA
promoter. Deletion of this region reduces hspA transcription
by more than half. Deleting a sequence ranging
from bp -86 to bp -140 upstream of the hspA ORF
abolishes hspA transcription suggesting a cis-acting
element to exist just upstream of the -35, -10 regions
of the hspA promoter. These results suggest the transcription
of hspA to be mainly positively regulated
under heat shock conditions.
A His-tagged fusion protein of Hspa (HspA His ) was
produced in E. coli. This polypeptide tends to assemble
into a large complex that consists of 26 subunits
with a molecular mass of 560 kDa as judged by size
exclusion chromatography. This oligomer of HspA His
interacts with unfolded cytrate synthetase (CS) and
prevents the enzyme‘s precipitation. The unfolded
B-chain of insulin is not protected from precipitation.
A stable complex is formed between HspA His and
unfolded CS because the unfolded enzyme does not
dissociate from the complex. Though thermotoler-
ance and differentiation of S. aurantiaca cells are not
affected in the absence of this protein, one may suggest
HspA to play a protective role in vivo.
II. Molecular Biology of the Infection Process
by the Entomopathogenic Fungus Beauveria
bassiana
E. Duperchy, H. Wan, G. Mitina and A. Leclerque in
cooperation with P. Zink and G. Zimmermann (Bioassays),
Biologische Bundesanstalt f. Land- und Forstwirtschaft,
Darmstadt
The organism
The filamentous fungus Beauveria bassiana (Balsamo)
Vuillemin belongs to a class of insect pathogenic
hyphomycetes (fungi imperfecti). The different
Beauveria strains are highly adapted to particular host
insects. A broad range of B. bassiana species is found
world wide under very different climatic conditions.
The different Beauveria strains cover a rather wide
spectrum of insect hosts that are of medical or agricultural
significance. Hosts of medical importance
include vectors for agents of tropical infectious diseases
such as the tsetse fly Glossina morsitans morsitans,
the sand fly Phlebotomus that transmits Leishmania,
and bugs of the genera Triatoma and Rhodnius,
the vectors of Chagas’ disease. Hosts of agricultural
significance include the Colorado potato beetle
Leptinotarsa decemlineata, the codling moth Carpocapsa
pomonella and several genera of termites.
In the absence of the specific insect host, the facultative
insect pathogen passes through an asexual vegetative
life cycle that includes germination, filamentous
growth, and the formation of sympoduloconidia from
poorly differentiated conidiophores. In the presence of
its host insect, Beauveria switches to the pathogenic
life cycle. The conidiospores germinate on the surface
of the cuticle; the newly generated hyphae penetrate
the insect’s integument and liberate single cells, socalled
blastospores, or hyphal bodies, when reaching
the hemocoel. The hemolymph distributes the blastospores
through the whole body cavity, that thus form
points of origin of new hyphae that invade all host tissues.
When the nutrients of the carcass are used up,
a thick layer of aerial hyphae is formed on the surface
of the insect’s cadaver, of which conidiospores
are released.
During the infectious process fungal structures are
subject to several defense response mechanisms of the
host-insect. These include humoral as well as hematocytic
encapsulation reactions, hemolymph clotting
reactions with the aim to hinder blastospore propagation,
and the action of defensin-like small antifungal
peptides.
To overcome the host’s defense mechanisms and physical
barriers it is suggested the parasite to have two
classes of virulence factors. One class comprises the
more general virulence factors that are independent
of the host insect. They include hydrophobin like proteins
that mediate spore adhesion to cuticular structures,
lytic enzymes such as proteases or chitinases for
cuticule penetration and hemocoel invasion and diffusible
cyclopeptide- or depsipeptide-toxins that suppress
the defense reactions of the host. The second
class comprises factors that are needed for the switch
from saprophytic growth to the growth on a specific
host-insect. These factors include a specific receptor-
and signal-transduction-system that allows the fungus
to differentiate between its correct host and other
insects.
113

Characterisation of genes involved in B. bassiana
virulence
For the identification of virulence factors of a B. bassiana
strain that is highly adapted to the Colorado potato
beetle, two complementary strategies are employed.
Firstly, in a REMI (“restriction enzyme mediated integration”)
transformation approach mutants are generated.
Mutants that result from a monolocal recombination
event are tested for their ability to grow on their
host-insect. In the case of a significant reduction of
their virulence the disrupted genomic region will be
cloned and sequenced. Secondly, a mRNA differential
display analysis is performed to identify fungal genes
that are transcriptionally up regulated by the interaction
with the host using cultures grown in the absence
or presence of host structures These genes are cloned
from a wild type genomic DNA library. The significance
of the thus found potential virulence factor
genes for pathogenesis are tested by site-specific inactivation
of the single cloned genes or of groups of
functionally related genes and the determination of
the pathogenicity phenotype of the corresponding null
mutants.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (Graduiertenkolleg “Biotechnologie”, Gradu–
iertenkolleg “Kontrolle der Genexpression in pathogenen
Mikroorganismen” and Projekt-’Sachbeihilfen’)
and from the DAAD.
PUBLICATIONS
Silakowski, B., Ehret, H. and Schairer, H.U. (1998).
FbfB, a gene encoding a putative galactose oxidase, is
114
involved in S. aurantiaca fruiting body formation. J.
Bacteriol. 180, 1241-1247.
Zhang, J., Schairer, H.U., Schnetter, W., Lereclus, D.
and Agaisse, H. (1998). Bacillus popilliae cry18Aa
operon is transcribed by σ E and σ K forms of RNA
polymerase from a single initiation site. Nucleic Acids
Res. 26, 1288-1293.
Börcsök, I., Schairer, H.U., Sommer, U., Wakley, G.K.,
Schneider, U., Geiger, F., Niethard, F.U., Ziegler, R.,
and Kasperk, C.H. (1998). Glucocorticoids regulate
the expression of the human osteoblastic Endothelin A
receptor gene. J. Exp. Med. 188, 1563-1573.
Plaga, W., Stamm, I., and Schairer, H.U. (1998). Intercellular
signaling in Stigmatella aurantiaca: Purification
and characterization of stigmolone, a myxobacterial
pheromone. Proc. Natl. Acad. Sci. USA 95,
11263-11267.
Hull, W.E., Berkessel, A., and Plaga, W. (1998). Structure
elucidation and chemical synthesis of stigmolone,
a novel type of prokaryotic pheromone. Proc. Natl.
Acad. Sci. USA 95, 11268-11273.
Stamm, I., Leclerque, A., and Plaga, W. (1999). Purification
of cold-shock-like proteins from Stigmatella
aurantiaca – molecular cloning and characterization
of the cspA gene. Arch. Microbiol. 172, 175-181.
Plaga, W., and Schairer, H.U. (1999). Intercellular signalling
in Stigmatella aurantiaca. Curr. Opin. Microbiol.
2, 593-597.
Plaga, W., Vielhaber, G., Wallach, J., and Knappe, J.
(2000). Modification of Cys-418 of pyruvate formate-
lyase by methacrylic acid, based on its radical mechanism,
FEBS Lett. 466, 45-48.
Silakowski, B., Schairer, H.U., Ehret, H., Kunze, B.,
Weinig, S., Nordsiek, G., Brandt, P., Blöcker, H.,
Höfle, G., Beyer, S. and Müller, R. (1999). New lessons
for combinatorial biosynthesis from myxobacteria:
the myxothiazol biosynthetic gene cluster of
Stigmatella aurantiaca DW4/3-1. J. Biol. Chem. 274,
37391-37399.
White, D. and H.U. Schairer (1999). Development of
Stigmatella. In Procaryotic Development. Y.V. Brun
and L. J. Shimkets edts. American Society for Microbiology,
Washington, DC pp 285-294.
THESES
Dissertation
Shen, Hui (1999). Transcriptional regulation of hspA
gene in Stigmatella aurantiaca and function analysis
of HspA protein. Universität Heidelberg.
STRUCTURE OF THE GROUP
E-mail: hus@zmbh.uni-heidelberg.de
Group leader Schairer, Hans Ulrich, Prof. Dr.
Postdoctoral
fellows Plaga, Wulf, Dr.
Silakowski, Barbara, Dr.*
Visiting scientist Mitina, Galina, Dr.*
Ph.D. students Chang, Chi-Hyuk, Dipl. Biol.*
Duperchy, Esther, Dipl. Biol.*
Hofmann, Diana, Dipl. Biol.*
Leclerque, Andreas, Dipl. Chem.*
Müller, Susanne, Dipl. Biol.
Shen, Hui, Dipl. Biol.
Wan, Hong, Dipl. Biol.*
Techn. assistant Stamm, Irmela
* part of the time reported
115

Heinz Schaller
Regulation of Hepatitis B Virus Replication
Hepatitis B viruses (also hepadnaviruses, HBVs) are
small, enveloped DNA viruses which cause acute and
chronic liver infections in mammals and birds. Their
prototype, the human HBV, is the causative agent of
a world-wide major public health problem with about
5 % of the world population being chronic HBV carriers
and at high risk of developing liver cirrhosis and
hepatocellular carcinoma. As ‘pararetroviruses’, these
viruses are related to the retroviruses by genome organization
and replication strategy, but differ in major
features, e.g. by containing a DNA genome in the
extra-cellular state, or by producing transcripts from
a non-integrated, circular DNA template. The hepadnaviruses
are characterized by a tissue tropism and
a very narrow host range restricting them to their
respective natural hosts and close relatives. These features
have been an obstacle to the establishment of an
HBV cell culture infection system, which is urgently
needed for the development of targeted therapy.
Despite these experimental difficulties with the HBV
prototype, the hepadnavirus infection cycle has been
worked out in considerable detail from molecular
analysis of HBV replication in cells transfected with
cloned viral DNA, as well as from infection studies
in the duck Hepatitis B virus (DHBV) animal system.
The resulting replication model (Fig. 1) is well estab-
Figure 1. The HBV replication cycle. Individual steps are indicated: (1,2) attachment to, and endocytosis into the host cell;
(3,4) cytosolic release of the capsid and transport to the nucleus; (5) conversion of the viral genome into ccc-DNA, the
template for transcription; (6) synthesis of genomic RNA, its maturation and transport to the cytoplasm, and (7) its translation
into the core and polymerase gene products, followed by (8), coassembly into RNA-containing nucleocapsids, ((8‘)
cyto-nucleoplasmic shuttling of core protein subunits, postulated to participate in genomic RNA maturation); (9) reverse
transcription of the RNA genome into DNA, and (10) nucleocapsid maturation and export from the cell as enveloped virion.
Alternatively, nucleocapsids import the mature DNA genome into the nucleus for replenishment of the ccc-DNA pool (11).
For simplicity, synthesis and assembly of the envelope proteins from subgenomic RNA are omitted.
116
lished with respect to the basic mechanisms leading to
the production of progeny virions (steps 7-10), but still
uncertain as to the mechanisms and cellular components
involved in determining receptor-mediated virus
uptake (steps 2-4), and also as to those that control the
establishment and persistence of productive infection
(primarily steps 4, 6, and 11). These latter processes
are difficult to study under controlled conditions
since no infectable cell lines exist even in case of
the available animal models. Nevertheless, we have
been increasingly focussing our research onto these
research areas, as their better understanding appears
to be crucial for a rational approach towards our longterm
goal, which is to establish an infection system for
studying (and to interfere with) all steps of HBV replication
cycle of the human virus in cultured cells, and
in a small test animal.
I. Parameters influencing the establishment
and maintenance of hepadnavirus replication
in primary hepatocyte cultures
U. Klöcker, B. Zachmann-Brand, B. Glass, C.
Kuhn, K. Rothmann, U. Protzer
Hepatitis B viruses use a well-balanced replication
strategy and an intimate cross talk with the host to
establish productive, but non-cytotoxic, long-term persistent
infections. As part of this strategy, virus replication
and gene expression vary greatly in response
to environmental changes of the state of the cell. For
example, transfer of preinfected duck hepatocytes out
of the tissue structure into cell culture results in an initial
reduction of virus production followed by upregulation
(Fig. 2). Furthermore, extracellular stimuli such
as cytokines, the peptide hormone glucagon, or endotoxin
from gram negative bacteria inhibit the establishment
of DHBV replication in vitro. We have now
shown that endotoxin acts indirectly by activating liver
Figure 2. Changes in virus production from DHBV-infected
primary duck hepatocytes in response to environmental
changes. After plating, preformed virus is released, followed
initially by decreased rates of production. From
day 4 on, virus production increases, reaching constant
levels around day 8. This process is inhibited by endotoxin-induced
cytokines produced from liver macrophages
(Klöcker et al., 2000).
macrophages to release cytokines which act at inhibitory
an early step of DHBV replication (Fig. 2).
Another example for cross talk with the host is our
finding that the large envelope protein, although a
structural protein, is varibly phosphorylated in cytosolic
preS domains by ERK-type MAP kinases in
response to to the state of the cell. In turn, changes in
preS-phosphorylation correlated closely with the ability
of DHBV L to activate gene expression in trans,
in vitro and in vivo. Furthermore, a pathogenic phenotype
with severe growth retardation and pathologic
liver histology was observed in ducklings infected
with a variant mimicking constitutive L phosphorylation.
The above observations from experimental DHBV
infection are complemented by data obtained with
HBV-transgenic mice, which produce HBV from a
chromosomally integrated HBV genome in titers comparable
to chronically infected humans. In this system,
117

the HBV genome is expressed only in a particular
subset of hepatocytes, again demonstrating the importance
of liver architecture and of hepatocyte microenvironment.
Analysis of virus replication in HBVtransgenic
mice lacking the large envelope protein do
not support a prominent role of the L protein for regulating
the levels of intracellular replication intermediates
in HBV, in contrast to evidence for such a mechanism
for the DHBV system.
II. Hepadnavirus replication after transfer of
hepadnaviral genomes mediated by adenoviral
vectors
M. Sprinzl, H. Oberwinkler, B. Zachmann-Brand,
J. Dumortier, U. Protzer
To bypass the species barrier that precludes animal
studies with the human HBV, we have established
recombinant adenoviruses that transfer HBV and
DHBV genomes into heterologous hepatocytes, or
into cell lines which are not infectable but capable
of supporting virus replication. In contrast to transfection,
this approach allows to introduce a defined
number of hepadnavirus genomes, thereby simulating
natural initiation of hepadnavirus replication from
extrachromosomal DNA templates; it should enable
us to study in more detail in vitro and in vivo the influence
of the state of the host hepatocyte, and the use of
immune mediators on hepadnavirus replication. Thus,
we have started to use adenoviral genome transfer into
cultured mouse hepatocytes to test for the influence of
defined immune mediators (from the mouse) on hepatitis
B virus replication in vitro. Furthermore, transduction
of hepadnavirus genomes was shown to efficiently
establish HBV and DHBV replication in the
mouse liver, yielding serum titers comparable to those
from HBV-transgenic animals. In these mice, viral
118
gene expression and replication was maintained for
up to three months, provided that an (adenovirusdirected)
immune response was suppressed.
III. A Golgi-resident protein participates as
an uptake receptor in duck hepatitis B
virus infection
K. Breiner, B. Glass, S. Urban
Major efforts through two decades have produced a
growing list of HBV receptor candidates, mostly only
defined as virus binding proteins, but have failed to
characterize any of these functionally. More progress
has been made in the DHBV animal model which
allows infection experiments with primary hepatocytes.
Including this essential complement for analysis,
we have now identified and characterized in detail
a bona fide DHBV receptor molecule, which plays
a crucial role in a conceptually new mechanism of
viral entry. This protein, a cellular carboxypeptidase
(CPD, historically termed gp180), had been initially
only characterized as a virus binding protein - and met
with scepticism since it lacked the expected tissue tropism,
species specificity, and since is predominantly
concentrated in the trans-Golgi network and not at the
plasma membrane where conceptually viruses must
interact with host cell receptor(s). Our conclusion that
gp180 is nevertheless a functional DHBV receptor
is based on a comprehensive series of experimental
observations: (i) gp180 is the only duck protein that
binds to recombinant DHBV preS, a ligand on the
virus surface previously shown to be essential for
infection, (ii) a preS subdomain, functionally defined
in infection competition experiments, coincides with
the domain determining physical gp180 binding, (iii)
anti-gp180 antibodies, as well as recombinant gp180/
CPD, efficiently block DHBV infection of cultured
duck hepatocytes, (iv) expression of gp180 in a
human hepatoma cell line mediates cellular attachment
and subsequent internalization of fluorescently
labeled DHBV particles into vesicular structures;
gp180 expression does, however, not render these
heterologous cells permissive for productive DHBV
infection. Furthermore, gp180/CPD is down-regulated
in DHBV-infected cells through intracellular interaction
with the preS ligand, a finding matching our
observation that DHBV infection reduces subsequent
superinfection about 20-fold. Taken together, these
and further data support the model that gp180/CPD
acts as the uptake receptor in a multistep process
(Fig.3). Although predominantly concentrated intracellularly,
the very limited cell surface exposition of
gp180/CPD (varied experimentally by gp180-transducing
adenoviruses) was found to be sufficient to
mediate virus particle uptake by coendocytosis, while
fusion at an endosomal membrane is predicted to
require an (yet unknown) species-specific fusion coreceptor.
Thus, gp180/CPD represents a first example
for a receptor that is virtually absent from the cell surface,
but nevertheless recognized and utilized for virus
Figure 3. Model of cellular gp180 traffic and DHBV entry.
DHBV-complexed gp180 is arrested in the endosome to
allow interaction with a second receptor postulated to trigger
membrane fusion (Breiner et al., 2000).
internalization leading to productive infection. These
new insights into hepadnaviral entry from the DHBV
model are now being used to re-design the strategies
in our search for the receptor of the medically relevant
human HBV.
IV. Signals for bidirectional cyto-nucleoplasmic
transport in the DHBV capsid protein
H. Mabit, K. Breiner, A. Knaust, B. Zachmann-
Brand
Like other viruses that replicate in the nucleus, the
hepadnaviruses rely on the the cellular cyto-nucleoplasmic
transport machinery to bring the infecting
nucleocapsid to the nucleus. To get insight into the
mechanism operating and the signals used, we have
started to study this process in the DHBV system. By
analyzing the subcellular localization of various segments
of the DHBV core protein (DHBc) fused to
the green fluorescent protein, and by further mutational
analysis, we mapped a single nuclear localization
signal (NLS) in the DHBc sequence. Mutational
inactivation of this sequence prevented DHBc nuclear
targeting (Fig. 4), and in the context of the complete
virus genome, it blocked virus production from
transfected cells. The mutant genome was still capable
of directing the early steps of DHBV infection in
duck hepatocytes, however, compared to wild type,
with delayed gene expression and without infecting
neighbouring cells, both results supporting the interpretation
that the NLS is essential for intracellular
genome amplification and virus production.
Finally, evidence from heterokaryon experiments suggests
that the DHBc sequence contains, in addition to
its NLS, also a nuclear export signal. We propose
that this NES, or a bidirectional shutling signal, counterbalances
NLS function in the productive state of
119

Figure 4. Loss of nuclear import of DHBV core protein
after mutational changes in its nuclear import signal. (A)
Schematic representation of the GPF-fusion used and the
mutation introduced (215RRRKVK220 → RGGEVK). (B)
Cellular distribution of wild type (RRK; left panel) and
mutant (GGE; right panel) in transfected HuH7 cells. GFP
fluorescence in live cells was analyzed using a confocal
microscope, at day 1 post transfection.
the infected cell, thereby preventing abortive nuclear
accumulation of capsids as observed in chronic HBV
patients and in HBV transgenic mice. On the other
hand, we have observed DHBc accumulation in distinct
nuclear dots (particularly early in infection).
These dots also accumulate pregenomic DHBV RNA,
suggesting a role in RNA maturation and nuclear
export.
V. Envelope-independent membrane-binding
preceeds budding and secretion of mature
hepadnaviral nucleocapsids
H. Mabit
Hepadnaviruses are DNA viruses, but as pararetroviruses,
their morphogenesis initiates with the encapsi-
120
dation of a RNA pregenome and these viruses have
therefore evolved mechanisms to exclude immature
nucleocapsids from participating in budding and secretion.
Using the duck virus model and a flotation assay,
we provide evidence that binding of cytoplasmic core
particles to their target membrane is a distinct step
in morphogenesis, discriminating different populations
of intracellular capsids. The membrane-associated
subpopulation contained largely mature, double
stranded DNA genomes and was devoid of detectable
core protein phosphorylation, both features characteristic
for secreted virions. Against expectation, however,
the selective membrane attachment observed did
not require the presence of the large DHBV envelope
protein, whose interaction has been implicated to be
crucial for selective nucleocapsid-membrane interaction.
Furthermore, removal of surface-exposed phosphate
residues from non-floating capsids, did by itself
not suffice to confer membrane affinity. Collectively,
these observations argue for a model in which nucleocapsid
maturation, involving the viral genome, the
phosphorylation state, and the structure of the capsid,
leads to the exposure of a membrane-binding signal as
an initial step crucial for selecting nucleocapsids destined
to be enveloped and secreted.
VI. Recombinant hepadnaviruses as vectors
for hepatocyte-directed gene transfer
U. Protzer, U. Klöcker, A. Frank, in collaboration
with M. Nassal, Med. Uniklinik Freiburg
Being hepatropic and non-cytopathic viruses, the
hepadnaviruses have been envisaged for many years
to be potential candidates for the development of livertargeted
viral vectors. However, due to the compact
organization of the viral genome (3 kb with overlapping
genes and numerous cis-acting sequence ele-
ments), realization of this potential proved to be rather
difficult. We have now demonstrated that, by appropriate
design (i.e. replacement of the S-gene), recombinant
replication-defective HBVs and DHBVs can be
generated which specifically infect hepatocytes, and
transduce and express foreign genes under control of
an endogenous viral promoter. Exclusive expression
of the transgenes in hepatocytes, but not in non-parenchymal
liver cells, confirmed the predicted tissue- and
cell-type specificity.
Being produced at rather high titers (ca.10 8 /ml),
recombinant hepadnaviruses are useful tools for experimental
gene transfer in hepatocyte cultures and for
the study of hepadnaviral infection and its therapeutic
treatment. For example, genetically marked DHBVs
were used to demonstrate superinfection of hepatocytes
with an established hepadnaviral infection. Furthermore,
expression of a type I interferon, a secreted
protein of therapeutic use, from such a vector interfered
with the replication of the resident virus (Fig.5).
These data introduce the use of local cytokine produc-
Figure 5. Therapeutic effect of a recombinant DHBV transducing
an interferon gene. Preinfected duck hepatocytes
were superinfected at various ratios (moi) with rDHBV-
IFN or with rDHBV-GFP as a negative control. The time
course of progeny virus release is shown.
tion by HBV-mediated gene transfer as a new concept
for the treatment of acquired liver diseases, including
chronic hepatitis B. Finally, by developing genetically
marked hepadnaviruses, provide a simple assay to the
identification of infectable cells, and the opportunity
for gene transfer approaches in searching for the missing
DHBV-coreceptor or other missing co-factors.
VII. A spring-loaded topology of the large
envelope protein of Duck hepatitis B
virus
In collaboration with E. Grgacic, Melbourne
The structure and fusion potential of the duck hepatitis
B virus (DHBV) envelope proteins was examined
by treating viral particles with deforming agents
known to release envelope proteins of viruses from
a metastable to a fusion-active state. Exposure of
DHBV particles to low pH triggered a major structural
change in the large envelope protein (L) resulting in
exposure of a new trypsin cleavage site within its
S domain, but without affecting the same region in
the small surface protein (S) subunits. This conformational
change was associated with increased hydrophobicity
of the particle surface most likely arising
from surface exposure of the hydrophobic first transmembrane
domain. In the hydrophobic conformation,
DHBV particles were able to bind to liposomes and
intact cells, while in their absence these particles
aggregated, resulting in viral inactivation. These results
suggest that some L molecules are in a spring-loaded
metastable state which, when released, expose a previously
hidden hydrophobic domain, a transition potentially
representing the fusion active state of the envelope.
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VIII. A novel mode of high affinity virus-receptor
interaction
S. Urban, in collaboration with G. Multhaup (ZMBH),
and C. Schwarz, U.C. Marx, P. Rösch, Lehrstuhl für
Biopolymere, Universität Bayreuth.
Entry of duck hepatitis B virus (DHBV) is initiated
by specific interaction of a subdomain of about 85
amino acids of the exterior preS domain of the large
viral envelope protein with the cellular entry receptor
gp180/carboxypeptidase D (CPD). This receptor binding
domain is highly variable in sequence, between
DHBV strains and related avian hepadnaviruses, without
significantly affecting receptor interaction as
shown by either binding-competition or infectioncompetition
experiments. These observations had suggested
that the functionality of the DHBV receptor
binding domain might be predominantly determined
by a particular 3D structure rather than by sequential
amino acid motifs.
In a further biochemically analysis, using recombinant
preS polypeptides from E.coli and soluble, truncated
forms of duck CPD from a baculovirus expression
system, the ligand binding site was mapped to an
enzymatically inactive Carboxypeptidase-like repeat
domain, adjacent to the cellular membrane. Ligand
binding to the receptor was characterized by a 1:1
stoichiometry, and shown to induce significant conformational
changes in the receptor. Kinetic analysis by
surface plasmon resonance spectroscopy determined
the complex dissociation constant KD to 1.5 nM, an
extraordinary high affinity compared to other virus
systems. Including a set of mutant preS polypeptides,
this analysis allowed us also to assess contributions of
different preS-sequence elements to complex formation
and complex stability. A conformational analysis
by 2D-NMR supplemented this approach, revealing
the overall structure of the receptor binding site to be
122
largely unstructured, with a low amount of α-helix.
In combination, these data suggest that receptor binding
proceeds in two steps: Initially, a short α-helical
element near the C-terminus of the receptor-binding
domain initiates formation of a low-affinity complex.
This complex is stabilized sequentially, involving
sixty, essentially non-structured amino acids preceding
the helix. We propose that this mechanism of high
affinity receptor interaction, preserving the potential
of the ligand to adapt structure during binding, has
evolved as an alternative strategy to escape immune
surveillance and the evolutionary pressure inherent to
the compact hepadnaviral genome organization.
External Funding
During the period reported, our research was supported
by grants from the Deutsche Forschungsgemeinschaft
(Sachbeihilfen, and Graduiertenkolleg „Kontrolle der
Genexpression in Pathogenen Mikroorganismen“), a
grant from the BMBF (Förderschwerpunkt „Therapie
mit Molekulargenetischen Methoden“ im Programm
„Gesundheitsforschung 2000“), from the Boehringer
Ingelheim Foundation, and from the Fonds der Chemischen
Industrie.
PUBLICATIONS
Hild, M., Weber, O., and Schaller, H. (1998). Glucagon
Treatment Interferes with an Early Step of Duck
Hepatitis B Virus Infection. J. Virol. 72, 2600-2606.
Urban, S., Breiner, K. M., Fehler, F, Klingmüller, U.,
and Schaller, H. (1998). Avian Hepatitis B Virus Infection
is Initiated by Interaction of a Distinct Pre-S Subdomain
with the Cellular Receptor gp180. J. Virol. 72,
8089-8097.
Breiner, K.M., Urban, S., and Schaller, H. (1998).
Carboxypeptidase D (gp180), a Golgi-Resident Protein,
Functions in the Attachment and Entry of Avian
Hepatitis B Viruses. J. Virol. 72, 8098-8104.
Rothmann, K., Schnölzer, M., Radziwill, G., Hildt, E.,
K. Mölling, and Schaller, H. (1998). Host Cell - Virus
Cross Talk: Phosphorylation of a Hepatitis B Virus
Envelope Protein Mediates Intracellular Signaling. J.
Virol. 72, 10138-10147.
Protzer, U., Nassal, M., Chiang, P-W., Kirschfink, M.,
& Schaller, H. (1999). Interferon gene transfer by a
novel hepatitis B virus vector efficiently suppresses
wild-type virus infection. Proc. Natl. Acad. Sci. USA,
96, 10818-10823.
Urban, S., Kruse, C., and Multhaup, G. (1999). A Soluble
Form of the Avian Hepatitis B Virus Receptor. J.
Biol. Chem. 274, 5707-5715.
Grgacic, E.V.L., Kuhn, C. and Schaller, H. (2000).
Hepatitis B Virus Envelope Topology: Membrane
Insertion of a Loop Region and Role of S in L Protein
Translocation. J. Virol. 74, 2455-2458.
Breiner, K.M. & Schaller, H. (2000). Cellular Receptor
Traffic is Essential for Productive Duck Hepatitis
B Virus Infection. J. Virol. 74, 2203-2209.
Urban, S., Schwarz, C., Marx, U.C., Zentgraf, H.,
Schaller, H., and Multhaup, G. (2000). Receptor recognition
by a hepatitis B virus reveals a novel mode of
high affinity virus-receptor interaction. EMBO J. 19,
21227-1227.
Hegenbarth, S., Gerolami, R., Protzer, U., Trans, P.L.,
Brechot, C., Gerken, G., and Knolle, P.A. (2000).
Liver sinusoidal endothelial cells are not permissive
for adenovirus type 5. Hum. Gene Ther. 11, 481-486.
Grgacic, E.V.L., and Schaller, H. (2000). A springloaded
topology of the large envelope protein of duck
hepatitis B virus: low pH release results in a transition
to a hydrophobic potentially fusogenic conformation.
J. Virol. 74, 5116-5122.
Klöcker, U., Schultz, U., Schaller, H., and Protzer,
U. (2000). Liver macrophages release mediators after
endotoxin stimulation that inhibit an early step in
hepadnavirus replication. J. Virol. 74, 5525-5533.
Protzer, U. and Schaller, H. (2000). Immune Escape
by Hepatitis B Viruses. in Virus Genes 21, 27-37.
Breiner, K.M., Urban, S., Glass, B., and Schaller, H.
(2000) Envelope Protein-Mediated Down-Regulation
of a Hepatitis B Virus Receptor in Infected Hepatocytes.
J. Virol. (in press)
Mabit, H., and Schaller H. (2000) Intracellular Hepadnaviral
Nucleocapsids are Selected for Secretion by
Envelope Protein-independent Membrane Binding. J.
Virol. (in press).
THESES
Breiner, Klaus M. (1998): Carboxypeptidase D
(gp180): Rezeptor, Transzeptor und Signalmolekül für
Vogelhepatitis B Viren.
Rothmann, Kirsten (1998): Kommunikation zwischen
Virus und Wirtszelle: Die Phosphorylierung des grossen
Hüllproteins des Enten Hepatitis B Virus vermit-
123

telt intrazelluläre Signaltransduktion.
Klöcker, Uta (2000): Regulation der hepadnaviralen
Replikation: Untersuchung des Effektes antiviraler
Mediatoren mit Hilfe hepadnaviraler Vektoren.
Sprinzl, Martin Franz (2000): Der adenovirale Genomtransfer
überwindet die Speziesbarriere der Hepatitis-
B-Viren.
Habilitations
Hug, Hubert (1998). Signalübertragung der CD95
(Fas/APO-1)-vermittelten Apoptose.
Knolle, Percy (2000). Die Leber als immunregulatorisches
Organ: Bedeutung der sinusoidalen Endothelzellen
und Kupffer Zellen.
Protzer, Ulrike (2000). Virus-Wirt-Interaktion bei der
Hepatitis B Virus Infektion: vom verbesserten Verständnis
zur Entwicklung neuer Therapien.
Urban, Stephan (2000). Identifizierung des aviären
Hepatitis B Virus Rezeptors und Charakterisierung
seiner Interaktion mit dem viralen Hüllprotein.
STRUCTURE OF THE GROUP
E-mail: hshd@zmbh.uni-heidelberg.de
Group leader Schaller, Heinz, Prof. Dr.
Research associates/
postdoctoral
fellows Knaust, Andreas, Dr. *
Kuhn, Christa, Dr.
Mabit, Hélène, Dr.*
124
Obert, Sabine, Dr.*
Protzer, Ulrike, Dr.
Sirma, Hüseyin, Dr.*
Urban, Stephan, Dr.
Visiting
scientists Wimmer, Eckard, Prof.*
from SUNY at Stony Brook, USA
(Humboldt Awardee)
Grgacic, Elizabeth, Dr.*
from Macfarlane Burnet Centre,
Fairfield, Australia
Ph.D. students Breiner, Klaus, Dipl.Ing.,
MA chem.
Dumortier, Jêrome*
Klöcker, Uta
Rothmann, Kirsten*
Sprinzl, Martin*
Thake, Sandra*
Techn.
assistants Glass, Bärbel, Dipl. Biol.
Götzmann, Martina*
Oberwinkler, Heike*
Zachmann-Brand, Beate
*) part of the time reported
Project Group Percy Knolle
Regulation of the Immune Response in the
Liver
The liver is a unique organ with regard to the induction
of peripheral immune tolerance. Delivery of antigen
into the liver results in tolerance induction towards this
specific antigen as is seen by the increased survival
of organ transplants following portal venous injection
of donor antigens or following portal venous drainage
of the organ transplant. A number of different hepatic
cell populations and the unique hepatic microenvironment
seem to cooperate to induce immune tolerance.
We are studying the contribution to immune tolerance
of the sinusoidal endothelial cells of the liver (LSEC),
which interact constitutively with leukocytes in the
sinusoidal lumen (see Fig. 1) as well as two other
Figure 1. Schematic drawing of the relative position and
cell sizes in the hepatic sinusoid. Leukocytes are in intimate
contact with LSEC during liver passage and constitutively
adhere to LSEC under physiologic conditions.
hepatic sinusoidal cell populations, the Kupffer cells
and the liver associated lymphocytes. Kupffer cells
and LSEC have the capacity to act as antigen-presenting
cells and activate CD4 + T cells suggesting
immune-surveillance function for the liver. However,
antigen-presentation by LSEC and Kupffer cells is
stringently controlled by physiological constituents
of portal venous blood (e.g. endotoxin) and by both,
autocrine and paracrine action of soluble mediators,
which are induced in resident liver cells by endotoxin.
Activation of naive CD4 + T cells by LSEC outside
lymphoid tissue may constitute one mechanism of
hepatic tolerance induction, because antigen-presention
by LSEC leads to the generation of CD4 + T cells
that express immunosuppressive cytokines and act as
regulatory T cells.
I. Functional inactivation of CD8+ T cells
by cross-presenting sinusoidal endothelial
cells
J. Ohl und A. Limmer
Presentation of endocytosed antigens on MHC-class
I molecules on antigen-presenting cells to cytotoxic
CD8 + T cells (cross-presentation) is important for
immunity to intracellular pathogens and was supposed
to be restricted to dendritic cells and macrophages.
Once in the blood, antigen is taken up efficiently by
LSEC, but not significantly by other cell populations
of the liver (Fig. 2). In vitro analysis of isolated
LSEC reveiled that cross-presentation of endocytosed
antigen to a antigen-specific CD8 + T cell hybridoma
occured in LSEC. Cross-presentation in LSEC compared
to other antigen-presenting cells requires only
125

small numbers of antigen-molecules. Dependence of
cross-presentation on the proteasom and TAP suggests
that LSEC possess an efficient mechanism to deliver
endocytosed antigen from the endosom to the cyto-
Figure 2. Antigen uptake by LSEC in vivo and in vitro. (A) Mice
were intravenously injected with 100 µg of Texas-red labelled
ovalbumin and antigen-uptake into liver cells was examined
by confocal microscopy of perfusion fixed liver slices. Antigen
accumulates in a sinusoidal cell population which can be identified
as LSEC by specific uptake of acetylated LDL. (B) Isolated
LSEC were incubated with 10 µg/ml of Texas-red labelled ovalbumin
and examined for antigen-uptake by confocal microscopy
1 hour later. Ovalbumin accumulates in vesicular structures in
LSEC; note the absence of a diffuse cytoplasmic distribution of
ovalbumin typically observed in dendritic cells.
126
plasm for antigen-degradation and MHC-class I peptide-loading.
Further evidence for the contribution of
LSEC to immune tolerance in the liver comes from
the observation that CD8 + T cells activated by crosspresenting
LSEC loose their capacity to express Interferon
γ and loose their cytotoxic activity towards
specific target cells. Thus, LSEC seem to contribute
to hepatic immune tolerance by inducing regulatory
CD4 + T cells and silencing CD8 + T cell mediated
immune responses. Further experiments concentrate
on the role of LSEC in tolerance induction in vivo using
a newly established method of orthotopic implantation
of LSEC into mice.
II. Sinusoidal endothelial cells are not permissive
for adenovirus type 5 infection
S. Hegenbarth and P. Knolle
Adenoviral gene therapy vectors efficiently transduce
hepatocytes in vivo and are already successfully
employed to correct hepatic metabolic disorders. Little
is known, however, on the susceptibility to adenovirus
infection of non-parenchymal liver cells, such as
LSEC. For instance, LSEC express Factor VIII and
are therefore a relevant gene therapeutic target for correction
of Factor VIII deficiency. We have shown, that
LSEC of different species (mouse, tupaia berlenghi
and human) are not permissive for infection with adenovirus
type 5 in vitro or in vivo. In cocultures of
hepatocytes and LSEC, no infection of LSEC was
observed at virus titers that infected 100% of hepatocytes
(Fig. 3). Employing extremely high virus titers,
that could never be obtained in vivo, only single LSEC
were transduced by adenovirus in vitro (Fig. 3). Macrovascular
endothelial cells, however, were efficiently
infected by adenovirus in vitro (Fig. 3). The apparent
Figure 3. Lack of infection of LSEC by Ad5.CMV-GFP.
(A) Cocultures of murine hepatocytes and LSEC were incubated
for one hour with Ad5.CMV-GFP and examined
for GFP-expression 24 hours later. LSEC were identified
by uptake of DiI-acetylated LDL. No infection of LSEC
by Ad5.CMV-GFP is observed. Hepatocytes are efficiently
infected and express GFP. (B). Isolated LSEC were incubated
with Ad5.CMV-GFP at 5.000 infectious units per cell
for one hour. Infection of single LSEC was observed after
24 hours. LSEC identification by uptake of DiI-ac-LDL. (C)
Macrovascular endothelial cells at passage 3 were incubated
with Ad5.CMV-GFP at a concentration of 5 infectious
units per cell. More than 70% of endothelial cells
express GFP after 24 hours as a marker of productive infection.
lack of LSEC infection by adenovirus may be the
consequence of the absent expression of the integrin
α v β 3 , which is important for adenovirus cell entry.
Two other viral vectors, Herpes simplex virus and lentivirus
based vectors, do not productively infect LSEC
either. We conclude that modified or new vectors are
needed for successful targeting of gene expression to
LSEC.
III. Role of sinusoidal endothelial cells for
DHBV infection of hepatocytes
In cooperation with K. Breiner from the FG of
H. Schaller
Infection of hepatocytes by hepatotropic viruses is
thought to occur via specific hepatocellular receptors.
We studied the contribution of LSEC to infection
of hepatocytes by the avian hepaDNA virus, DHBV.
Although the primary binding and uptake receptor
for DHBV-infection of hepatocytes is known (gp180),
its ubiquitous expression appears to contradict its
involvement in viral hepatocyte targeting. We have
found that blood borne DHBV-particles are endocytosed
and accumulate in LSEC but not in hepatocytes
both, in vivo and in vitro. The preferential uptake of
DHBV-particles by LSEC is not compatible with
passive diffusion of viral particles through endothelial
fenestrae as the mechanism of hepatocyte infection.
Once endocytosed, viral particles colocalize with
gp180 in LSEC suggesting a function of the receptor
not only in hepatocyte infection but also in intracellular
trafficking in LSEC. To comply with low dose infection,
we propose that endocytosed viral particles are
transported with the viral receptor through LSEC to
infect neighbouring hepatocytes.
127

External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (Graduiertenkolleg “Kontrolle der Genexpression
in pathogenen Mikroorganismen” and Projekt-
’Sachbeihilfen’) and from Bayer AG.
PUBLICATIONS
Knolle, P.A., S. Kremp, T. Hohler, F. Krummenauer,
P. Schirmacher and G. Gerken (1998). Viral and host
factors in the prediction of response to interferon-
alpha therapy in chronic hepatitis C after long-term
follow-up. J. Viral. Hepat. 5 (6): 399-406.
Knolle, P.A., A. Uhrig, S. Hegenbarth, E. Loser, E.
Schmitt, G. Gerken and A. W. Lohse (1998). IL-10
down-regulates T cell activation by antigen-presenting
liver sinusoidal endothelial cells through decreased
antigen uptake via the mannose receptor and lowered
surface expression of accessory molecules. Clin. Exp.
Immunol. 114 (3): 427-33.
Knolle, P.A., A. Uhrig, U. Protzer, M. Trippler, R.
Duchmann, K.H. Meyer zum Büschenfelde and G.
Gerken (1998). Interleukin-10 expression is autoregulated
at the transcriptional level in human and murine
Kupffer cells. Hepatology 27 (1): 93-9.
Knolle, P. A., T. Germann, U. Treichel, A. Uhrig, E.
Schmitt, S. Hegenbarth, A. W. Lohse and G. Gerken
(1999). Endotoxin Down-Regulates T Cell Activation
by Antigen-Presenting Liver Sinusoidal Endothelial
Cells. J. Immunol. 162 (3): 1401-1407.
Knolle, P. A., E. Schmitt, S. Jin, T. Germann, R. Duch-
128
mann, S. Hegenbarth, G. a. Gerken and A. Lohse
(1999). Liver sinusoidal endothelial cells can prime
naive CD4+ T cells in the absence of IL-12 and induce
IL-4 production in primed CD4+ T cells: Implications
for tolerance induction in the liver. Gastroenterology
116 (6): 1428-40.
Hegenbarth, S., R. Gerolami, L. Tran, U. Protzer, C.
Brechot, G. Gerken and P. A. Knolle (2000). Liver
sinusoidal endothelial cells are not permissive for
infection with adenovirus type 5. Human Gene Therapy
11, 481-486.
Knolle, P. A. and G. Gerken (2000). Local regulation
of the immune response in the liver. Immunological
Reviews 174, 21-34
STRUCTURE OF THE GROUP
E-mail: P.Knolle@ZMBH.Uni-Heidelberg.de
Project group leader Knolle, Percy, Priv. Doz. Dr.
Postdoctoral fellow Limmer, Andreas, Dr.
Ph.D. students Ohl, Jutta, Dipl. Biol.
Wingender, Andreas,
Dipl. Biol.*
Techn. assistant Hegenbarth, Silke
*part of the time reported
Blanche Schwappach
Quality Control of Ion Channels and
ABC Proteins
Each cell type possesses an exquisitely regulated set
of plasma membrane proteins. It is essential that these
be expressed in their properly folded and correctly
assembled form in appropriate numbers at the cell surface.
This is particularly true for ion channels since
a very small number of channel proteins can have
dramatic effects on the electrical excitability of a
cell. ATP-binding cassette (ABC) proteins constitute a
large superfamily of transporters present in both pro-
and eukaryotes. They utilize ATP to move substrates
as diverse as metal ions, peptides, and lipids across
membranes of cells and cellular organelles. Channels
and ABC transporters play an important role in human
disease - in inherited disorders where many mutations
in the corresponding genes have been identified,
as drug targets, or a cause of drug resistance. In
some diseases like cystic fibrosis or Liddle’s syndrome
intracellular trafficking of the mutated ABC or
channel protein is known to be abnormal. The same
may be true for some cases of familial hyperinsulinism,
a disorder caused by mutations in the genes for
the K ATP channel complex.
Almost all ion channels are multimeric protein complexes
and the respective composition of the complex
can greatly affect its functional properties. How is
assembly and precise stoichiometry of ion channels
controlled? What determines the subcellullar localization
of ion channel and ABC protein subunits at different
stages of assembly? How is the activity of newly
synthesized channel proteins compatible with ionic
homeostasis of the endoplasmic reticulum? How does
the cell regulate the number of channels and transporters
at the cell surface? How are the relevant quality
control and trafficking processes regulated in response
to environmental change?
To address these questions we have started to employ
new methods that expand the traditional set of tools
like site-directed mutagenesis, immunofluorescence,
co-immunoprecipitation, and analysis of glycosylation
patterns. We have developed a very sensitive, quantitative
assay for surface protein in Xenopus oocytes so
that surface trafficking and functional properties can
now be precisely correlated even for low-expressing,
multimeric channel proteins. The assay is the basis of
a novel approach to studying protein-protein interactions
between membrane proteins in a native environment.
Retroviral gene transfer methods in mammalian
cells now make it possible to introduce large cDNA
libraries permanently into diverse cell types. Combination
of this approach with the advanced possibilities
of flow cytometry greatly increases the feasibility of
expression cloning and random screening projects in
mammalian cells. We intend to probe the consensus
and context dependence for known and new trafficking
signals in efficient, unbiased random screens.
Detailed knowledge of these parameters, particularly
for common motifs, will be indispensable to identify
putative trafficking signals in the growing databases.
Background: trafficking of the K ATP channel
complex
To address issues of quality control during ion channel
assembly, we have studied the assembly-dependent
trafficking of ATP-sensitive K+ channels. K ATP channels
couple the metabolic state of the cell to membrane
excitability. They are important in many tissues
and regulate insulin secretion in the pancreas, control
129

vascular tone, protect neurons and muscles from ischemia,
and are responsive to leptin. K ATP channels have
an unusual octameric stoichiometry consisting of four
pore-lining inward rectifier a α subunits (Kir6.1/6.2;
two transmembrane segments) like other K+ channels,
but also contain four regulatory sulphonylureabinding
ß subunits (SUR1/2A/2B; probably seventeen
transmembrane segments) that belong to the ATPbinding
cassette (ABC) family of proteins.
We found that only octameric K ATP channel complexes
were capable of expressing on the cell surface, implying
that quality control mechanisms must exist to prevent
monomers and partial complexes from expressing
on the cell surface. Surprisingly, we found that
the primary quality control mechanism during K ATP
assembly did not involve ER degradation or ER chaperones,
but rather the exposure of an ER retention/
retrieval signal (RKR) present in cytosolic domains
of each subunit. Mutating the retention sequences did
not affect protein levels, but allowed surface expression
of monomers and partially assembled complexes,
including improperly gated channel combinations. We
further showed that the RKR trafficking signal was
functional in a variety of eukaryotic cells including
yeast, mammalian cells, and Xenopus oocytes. Interestingly,
this RKR motif did not require proximity
to the N- or C-terminus like all other known ER
retention/retrieval signals and may, therefore, be more
common. In conclusion, quality control during K ATP
assembly is mediated by a short trafficking signal
whose exposure reflects the assembly state of the
channel. These results provide a clear example of how
a trafficking checkpoint serves as an important quality
control mechanism during the assembly of an ion
channel complex. Furthermore, they identify a new
ER retention/retrieval motif that could explain transient
or permanent ER localization of many proteins.
130
Our current projects are designed to better define this
new class of ER retention/retrieval signals, to extend
the analysis of their role in assembly-dependent trafficking
using K ATP channels as a model system, and
to identify and characterize the molecular machinery
that recognizes this class of signals.
PUBLICATIONS
Schwappach B., Stobrawa S., Hechenberger M., Steinmeyer
K., and Jentsch T.J. (1998). Golgi Localization
and functionally important domains in the NH 2 and
COOH terminus of the yeast CLC putative chloride
channel Gef1p. J. Biol. Chem. 273, 15110–15118.
Zerangue N.*, Schwappach B.*, Jan Y.N., and Jan
L.Y. (1999). A new ER trafficking signal regulates
the subunit stoichiometry of plasma membrane K ATP
channels. Neuron 22, 537-548 (*these authors contributed
equally).
Schwappach, B.*, Zerangue, N.* Jan Y.N., and Jan
L.Y. (2000). Molecular basis for K ATP assembly: transmembrane
interactions mediate association of a K +
channel with an ABC transporter. Neuron 26, 155-167
(*these authors contributed equally).
STRUCTURE OF THE GROUP*
E-mail: b.schwappach@zmbh.uni-heidelberg.de
Group leader Schwappach, Blanche, Dr.*
Ph.D. student Yuan, Hebao, M.A.*
Techn. assistant Metz, Jutta*
* since July/August 2000
Dominique Soldati
Cell and Molecular Biology of the Obligate
Intracellular Parasite Toxoplasma
gondii
Apicomplexan parasites are of enormous medical
and veterinary significance, being responsible for a
wide variety of diseases including malaria, toxoplasmosis,
coccidiosis and cryptosporidiosis. Successful
attachment and invasion of the host cells are key to
the survival of these obligate intracellular parasites.
T. gondii, the most ubiquitous of the Apicomplexa,
has developed a remarkable ability to actively penetrate
almost any nucleated cells from virtually all
warm-blooded animals. Most invasive forms of the
Apicomplexa share a common set of apical structures
and exhibit an unusual form of substrate-dependent
gliding motility as an adaptative mechanism to
actively penetrate host cells. In absence of locomotive
organelles such as cilia or flagella, the basic
engine for gliding locomotion is the actin cytoskeleton
and involves myosin(s) to generate the mechanochemical
force along the actin filaments, allowing
parasites to move at rates from 1 to 10 micrometers
per second.
Successive exocytosis of regulated compartments
including rhoptries and micronemes is part of the
invasion process. Micronemal proteins are apparently
used for host-cell recognition, binding, and possibly
motility, while rhoptry and dense granule proteins
contribute to the parasitophorous vacuole formation
and its remodeling into a metabolically active compartment.
Release by micronemes occurs in the earliest
phase of invasion, upon contact with the host cells
and raise in intracellular Ca 2+ .
Molecular motors, adhesins and proteases are among
the distinct classes of genes coding for invasion factors.
These genes are anticipated to be essential for
the survival of these parasites and therefore the ability
to conditionally turn on or off their expression is
prerequisite for their in vivo studies.
I. Myosins: molecular motors in Apicomplexa
parasites
Five myosins of T. gondii and three myosins of P. falciparum
have been identified so far. These unconventional
myosins are the founders of a novel phylogenetic
and structural class of myosin (XIV), restricted
to the Apicomplexa. Members of this class are small
with molecular weights ranging between 90 and 125
kDa and exhibit unusual structural features.
Myosins have been implicated either genetically, biochemically
or by cytolocalization in a variety of cellular
functions. Three distinct forms of gliding characterize
T. gondii motility: circular gliding, upright
twirling, and helical rotation. Inhibitors of actin filaments
(cytochalasin D) and myosin ATPase (butanedione
monoxime) disrupted all three forms of motility.
When applied on intracellular parasites, these
inhibitors also interfere with proper parasite division.
To determine whether one of these myosins functions
as a motor in actin-based gliding motility and
plays a role in cell replication, we have examined
their stage-specific pattern of expression, subcellular
distribution and biochemical properties.
131

TgMyoA: a candidate motor to power gliding
motility
C. Hettmann, A. Geiter , F, Delbac
T. gondii myosin A (TgMyoA) is constitutively
expressed and localizes beneath the plasma membrane
of the parasite. Thus, TgMyoA is in an
ideal position to transmit mechanical energy into
forward motion propelling parasites into host cells.
We mapped the membrane localization determinant
within the short carboxy-terminal tail of TgMyoA,
and site-directed mutagenesis revealed two essential
arginine residues. The closest homologue in P. falciparum,
PfMyoA is transcribed specifically in the
invasive blood stage form and shows the same subcellular
localization in merozoites. The nature of the
receptor at the plasma membrane remains to be determined.
TgMyoD and TgMyoE are similar to TgMyoA, but
predominantly transcribed in the dormant bradyzoite
stage. We have abrogated the expression of TgMyoD
by double homologous recombination in the tachyzoites
of the virulent RH strain without any apparent
detectable phenotype. We need now to reproduce this
knockout in a persistent strain capable to differentiate
into bradyzoites, in order to analyze the phenotypic
consequences of the absence of TgMyoD in the
appropriate stage.
Biochemical characterization of TgMyoA
A. Herm, in collaboration with M. Geeves (Kent
UK), D. Manstein (MPI, Heidelberg) and E. Meyhöfer
(Hannover)
To power gliding motility, TgMyoA must be able to
track along actin filaments upon ATP hydrolysis at
a speed of 1-3 µm/s. We have determined the biochemical
and physical properties of histidine tagged-
132
TgMyoA purified from culture recombinant parasites.
Transient kinetic analysis of TgMyoA was
obtained by stopped-flow and flash photolysis methods
and revealed strong similarities to typical fast
myosins, like rabbit skeletal myosin. The sliding
velocity of fluorescently labeled actin filaments on
TgMyoA attached to nitrocellulose-coated glass was
5-6 µm/s. A step size of 5 to 6 nm was determined by
single molecule assay monitoring filament displacement
using optical tweezers. TgMyoA shows all the
characteristics of a fast myosin of the class II but
considering the significant divergence in the converter
domain and the quasi absence of neck, it is
unclear at the moment how such a motor can generate
a power stroke.
TgMyoB/C: One gene, two tails, and two localizations.
F. Delbac, C. Hettmann, A. Sänger
TgMyoB and TgMyoC are alternative-spliced variants
of the same gene, which produce two myosins
exhibiting different tails. TgMyoB distributes evenly
in the cytosol and is essentially soluble. In contrast,
TgMyoC localizes at the posterior pole of the parasites
describing a ring structure reminiscent of the
termination of the inner membrane complex (IMC).
TgMyoC protein fractionates with membranes and
partitions in the detergent-insoluble fraction. Inhibitors
of myosin ATPase and actin polymerization disrupt
the orderly turnover of mother cell organelles
during daughter cell formation. The circular distribution
of TgMyoC suggests a possible role of this
myosin in the assembly and elongation of the IMCmicrotubule
complex during parasite division. Moreover
the overexpression of TgMyoB slows the rate of
replication and leads to the formation of large resid-
ual bodies, providing an additional indication of the
involvement of TgMyoB/C in cell division.
Figure 1. Immunolocalization of Myosin C (in red) and
MIC6 (in green).
II. Micronemal proteins of several apicomplexan
parasites are sharing common
structural and functional features
The rapid and efficient nature of invasion by Apicomplexa
relies on a sequence of events that are
tightly controlled in time and space. Micronemes are
involved in the trafficking and sequestration of binding
ligands for host cell receptors. These organelles
ensure the appropriate release of ligands in high concentration,
at the tip of the parasite and upon response
to external stimuli which senses contact with the
host cells. To accommodate for the broad host range
specificity of T. gondii, we postulated that adhesion
should involve the recognition of ubiquitous surfaceexposed
host molecules or, alternatively, the presence
of various parasite attachment molecules able
to recognize different host cell receptors. We identified
and characterized a soluble protein MIC4, as
well as a novel family of transmembrane microne-
mal proteins in T. gondii. These proteins are released
at the time of invasion and share domains of homology
with proteins described in other members of the
phylum Apicomplexa, supporting the hypothesis of a
common molecular mechanism for host recognition
attachment and invasion. They contain a combination
of adhesive motifs including thrombospondin, integrin,
apple and EGF-like domains. Invasion is successfully
achieved when the newly formed parasitophorous
vacuole is sealed and at this point, the
tight interaction between host cell receptors and parasite
ligands must be disrupted. The proteolytic processing
of micronemal proteins that occurs on the
parasite surface could fulfil such a function.
TgMIC4 carries six apple domains and binds
to host cells
S. Brecht, U. Jäkle
TgMIC4 carries six apple domains, a signal peptide
at the amino terminus and no apparent membranespanning
domain. Apple domains are found in plasma
coagulation factors, kallikrein and factor XI and are
involved in highly specific protein-protein interactions.
MIC4 is expressed in all invasive stages of the
parasites, secreted at the time of invasion and proteolytically
processed twice onto the parasite surface,
after secretion by the micronemes. MIC4 binds efficiently
to host cells and the adhesive properties map
within the carboxy-terminal apple domain of the protein.
Preliminary experiments showed that galactose
inhibits specifically binding to host cells and we are
currently characterizing further the lectin properties
of MIC4 and attempting to identify the host cell
receptor(s) by expressing heterologously the apple
domain in P. pastoris.
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A novel family of secreted protein carrying
EGF-like domains.
M. Meißner and M. Reiss
We have characterized a new family of T. gondii
micronemal proteins containing various numbers of
EGF-like domains (TgMIC6, TgMIC7 TgMIC8 and
TgMIC9) and their deduced amino acids sequences
predict type I transmembrane proteins. The short
cytoplasmic tails of these proteins are conserved
across the Apicomplexa.
According to the capping model for host cell invasion
by Apicomplexa, transmembrane proteins are
anticipated to establish tight and specific interactions
with host cell receptors through their extracellular
adhesive domains. At the same time, their carboxyterminal
domain shall promote a direct or indirect
connection with actomyosin system of the parasite
to transfer the mechanical force across the plasma
membrane. The cytoplasmic domains of these MICs
failed to interact with TgMyoA tail in the yeast
“two hybrid” assay. We are pursuing a “two hybrid”
screening with a T. gondii cDNA fusion library and
biochemical approaches to identify partners interacting
with the MIC cytoplasm domains.
TgMIC6 functions as cargo receptor with two
soluble microneme proteins
M. Reiss, N. Viebig, in collaboration with J.F.
Dubremetz (Institut Pasteur, Lille)
TgMIC6 carries three EGF-like domains, a transmembrane
spanning region and a short conserved
cytoplasmic tail. The disruption of MIC6 gene by
homologous recombination revealed that this gene is
not essential for the survival of tachyzoites in culture.
Interestingly, the sorting of two soluble micronemal
134
proteins is compromised by the absence of MIC6.
MIC1 and MIC4 are mistargeted into the default
pathway, which in T. gondii transits through the
dense granules and ends in the parasitophorous vacuole.
The distribution of other micronemal proteins
such as the soluble MIC3 and the transmembrane
MIC2 are not affected in the mic6-mutant. We
have demonstrated genetically by complementing
the mic6-mutant with truncated or chimeras versions
of MIC6 that this protein interacts with MIC1 and
MIC4 and serve as specific receptor for sorting into
the micronemes. Biochemical evidence also supports
the genetic data and lead to the conclusion that MIC1,
MIC4 and MIC6 are building a complex in the early
compartment of the secretory pathway, which is prerequisite
for the accurate sorting of the two soluble
MICs. Analyses of deletion mutants of MIC6 indicate
that the targeting signal to the micronemes is
localized within the cytoplasmic tail domain of the
protein. Confirming these results, the cytoplasmic
tail of MIC2 contains identical sorting signals including
a conserved tyrosine based motif, which likely
involves interaction with a member of the adaptor
complexes. MIC4 behaves as a neutral cargo protein
whereas MIC1 appears to be involved in a quality
control mechanism. In mutant parasites where MIC1
gene has been disrupted by homologous recombination,
both MIC4 and MIC6 are retained in the ER and
Golgi. After discharge by the micronemes, MIC6 is
distributed at the surface of the parasite and is rapidly
released from the plasma membrane by proteolytic
cleavage upstream of the transmembrane spanning
domain. The multiple processing events occurring
on the parasite surface might play a key role in
disrupting the tight interaction between parasite and
host cell membranes once invasion is achieved.
III. A controlled gene expression system
M. Meißner
An inducible control of individual gene expression is
prerequisite to the study of essential processes such
as host cell invasion by an obligate intracellular parasite.
The straightforward strategy would be to import
the tetracycline-repressor TetR or transactivator tTA.
A modified synthetic gene coding for tTA and tetRep
gene in which the codon usage is remodeled in favor
of a more GC-rich content, was kindly provided by
the group of Dr. Bujard. Both synthetic tetRep and
tTA were faithfully expressed as stable transgenes
in T. gondii tachyzoites. We confirmed by gel shift
retardation assay that both proteins have the capacity
to bind to tetO sequences and in a tetracycline
dependent fashion. All reporter plasmids tested so far
failed to measure transactivation by tTA. In contrast
a 10-fold repression of reporter gene expression was
observed in presence of tetRep using a tubulin promoter
where two tetO sequences have been introduced
at the site of transcriptional initiation. The
present results suggest that the VP16 activating
domain fails to interact with the parasite transcription
machinery. In contrast, the repressor system might
provide with a system to manipulate the level of
gene expression in T. gondii in a controlled manner
although optimization of the inducibility is still indispensable.
External Funding
During the period reported our research was supported
by grants from the Deutsche Forschungsgemeinschaft
DFG (SFB 544 “Kontrolle tropischer
Infektionskrankheiten”, Schwerpunkt “Molekulare
Motoren”, Schwerpunkt “Voraussetzungen und
molekulare Mechanismen der Persistenz von Para–
siten im Wirt”, Graduiertenkolleg “Kontrolle der Genexpression
in pathogenen Mikroorganismen”, Projekt-’Sachbeihilfen’)
and from the Humboldt Foundation.
PUBLICATIONS
Soldati, D., Lassen, A., Dubremetz, J.F. and Boothroyd,
J.C. (1998). Processing of Toxoplasma ROP1
protein in nascent rhoptries. Mol. Biochem. Parasitol.
96, 37-48.
Striepen, B., He, C.Y., Matrajt, M., Soldati, D. and
Roos, D.S. (1998). Expression, selection, and organellar
targeting of the green fluorescent protein in
Toxoplasma gondii. Mol. Biochem. Parasitol. 92,
325-338.
Soëte, M., Hettman, C. and Soldati, D. (1999). The
importance of reverse genetics in determining gene
function in apicomplexan parasites. Parasitol. 118,
S53-61.
Di Cristina, M., Ghouze, F., Kocken, C.H., Naitza,
S., Cellini, P., Soldati, D., Thomas, A.W. and Crisanti,
A. (1999). Transformed Toxoplasma gondii
tachyzoites expressing the circumsporozoite protein
of Plasmodium knowlesi elicit a specific immune
response in rhesus monkeys. Infect. Immun. 67,
1677-1682.
Brecht, S., Erdhart, H., Soete, M. and Soldati, D.
(1999). Genome engineering of Toxoplasma gondii
using the site-specific recombinase Cre. Gene 234,
239-247.
Jomaa, H., Wiesner, J., Sanderbrand, S., Altincicek,
135

B., Weidemeyer, C., Hintz, M., Turbachova, I., Eberl,
M., Zeidler, J., Lichtenthaler, H.K. Soldati, D. and
Beck, E. (1999). Inhibitors of the nonmevalonate
pathway of isoprenoid biosynthesis as antimalarial
drugs. Science 285, 1573-1576.
Mattsson, J.G. and Soldati, D. (1999). MPS1: a small,
evolutionarily conserved zinc finger protein from
the protozoan Toxoplasma gondii. FEMS Microbiol.
Lett. 180, 235-239.
Hettmann, C. and Soldati, D. (1999). Cloning and
analysis of a Toxoplasma gondii histone acetyltransferase:
a novel chromatin remodelling factor
in Apicomplexan parasites. Nucleic Acids Res. 27,
4344-4352.
Hettmann, C. Herm, A., Frank, B, Schwarz, E. Geiter,
A. Soldati, T. and Soldati, D. (2000). A dibasic motif
in the tail of a class XIV apicomplexan myosin is an
essential determinant of plasma membrane localisation.
Mol. Biol. Cell, 11, 1385-1400.
Ding, M., Clayton C., and Soldati D. (2000). Toxoplasma
gondii catalase: are there peroxisomes in
Apicomplexa? J. Cell Sci., 113, 2409-2419.
Di Cristina, M., Spaccapelo, R., Soldati, D., Bistoni,
F., and Crisanti, A. (2000). Two conserved amino acid
motifs mediate protein targeting to the micronemes of
the apicomplexan parasite Toxoplasma gondii. Mol.
Cell. Biol. (in press).
Tomley, F. and Soldati, D. (2000). Mix and Match
Modules: Structure and Function of Microneme Proteins
in Apicomplexan Parasites. Parasitology Today
(in press).
136
Ferguson, D.J.P., Brecht, S. and Soldati, D. (2000).
The microneme protein MIC4, or an MIC4-like protein,
is expressed within the macrogamete and associated
with oocyst wall formation in Toxoplasma
gondii. Int. J. Parasitol (in press).
THESES
Diploma
Reiß, Matthias (1998). Identification and characterization
of an invasion factor in Toxoplasma gondii.
Geiter, Ariane (1998). Subcellular distribution of
Toxoplasma gondii myosins.
Ding, Martina (1999). Characterization of organelles
in Toxoplasma gondii.
Herm, Angelika (1999). Purification and characterization
of recombinant myosins from Toxoplasma
gondii.
Viebig, Nicola (1999). Characterization of a sorting
receptor for regulated soluble secretory proteins of
Toxoplasma gondii.
Sänger, Astrid (2000). Analysis and characterization
of Toxoplasma gondii myosins B and C.
Dissertations
Brecht, Susan (2000). Characterization of a Toxoplasma
gondii adhesin and establishment of Cre-lox
recombinase system for T. gondii genome engineering.
Hettmann, Christine (2000). Identification of a chromatin
remodelling factor in Toxoplasma gondii and
myosins as molecular motors for the invasion of host
cells by the Apicomplexa.
STRUCTURE OF THE GROUP
E-mail: soldati@sun0.urz.uni-heidelberg.de
Group leader Soldati, Dominique, Dr.
Postdoctoral
fellows Soëte, Martine*
Hernandez, Carmen *
Delbac, Frederic*
Ph.D. students Brecht, Susan, Dipl.-Biol.
Hettmann, Christine, Dipl.-Biol.
Reiss, Matthias, Dipl.-Biol *
Meißner, Markus, Dipl.-Biol *
Diploma students Geiter, Ariane*
Herm, Angelika*
Viebig, Nicola*
Sänger, Astrid*
Opitz, Corinna*
Techn. assistant Jäkle, Ursula*
* part of the time reported
137

138
Central Facilities
Biomolecular Chemistry
The chemical synthesis, structural analysis and the
characterization of structure-activity relationships of
biopolymers such as proteins and nucleic acids represent
an important link between organic chemistry,
physical chemistry and molecular biology. The chemical
core facility operates in this interface region
making biomolecular chemistry available to all scientists
at the ZMBH and to numerous associated groups
in the surrounding institutes. In particular for the following
techniques instrumentation and practical support
are available:
- micropreparative isolation of proteins and peptides
- synthesis and purification of peptides
- DNA sequence analysis
- mass spectrometrical analysis of biopolymers.
In cooperation with biologists these techniques are
continuously updated. That makes the newest methods
available for the solution of biological problems.
Similarly, the close proximity of members of the core
facility and the biological research groups leads to frequent
scientific exchange. Early interaction has proved
to be very valuable.
During the last years the market has changed considerably.
Companies are now offering services for the synthesis
of oligonucleotides at low prices. Therefore, we
discontinued the synthesis of oligonucleotides since
we could not compete with these specialized companies.
We rather direct now our main focus on the analysis
of proteins and the synthesis of peptides. The still
growing number of complete sequences of procaryotic
and eukaryotic genomes results in an increasing
demand for protein technology. The catchword is
proteome analysis. The facility is equipped with a
MALDI instrument (matrix assisted laser desorption
ionisation) and an electrospray ionisation (ESI) ion
trap mass spectrometer. This enables us to characterize
proteins, which have been separated by one- or
two-dimensional gel electrophoresis and assign them
to the corresponding genes.
Although the majority of ordered peptides is in the
range of 10-20 amino acids, we are able to synthesize
peptides longer than 100 amino acids (longest peptide
115 amino acids) including certain additional chemical
modifications.
In parallel to the service function of the group several
collaborative projects are pursued concerning the analysis
of protein primary structure and protein function.
These projects are supplemented by our studies on the
chemistry and instrumentation for synthesis and analysis
of biopolymers. Our goal is to provide the members
of the institute with state of the art technology.
Richard Herrmann
139

Biocomputing
Computing and communication via the internet are
absolutely essential for the modern biological Sciences.
Accordingly, the ZMBH is equipped with a
powerful computer network that comprises by now
three central Macintosh Servers, ≈180 Macintoshs for
endusers, two Sun workstations, 20 Windows com–
puters and 20 printers that are connected with each
other through an Ethernet and a Local talk system.
A considerable amount of computer-controlled equipment,
such as microscopes, fluor- and phosphoimagers,
oligonucleotide- and polypeptide synthesisers, as
well as DNA-sequencing machines, are also integrated
into the network.
The ZMBH employs a team of three computer specialists,
complemented by one or more students,
who maintain and up-date the hardware and software
and give a comprehensive support to all users. This
includes, for example, administration of the network
and servers, set up of computers according to the
individual user‘s requirements and the creation of
customised databases for sophisticated, user-specific
sequence analysis with UNIX-based software. In addition,
the computer group provides software training
in small groups or on an individual basis for a broad
range of applications.
140
Raphael Mosbach
Animal House and Transgene Lab 1
The ZMBH animal house is a central division of the
institute that provides research groups with the opportunity
to breed experimental animals and perform
animal experiments. For this purpose, the animal house
maintains quantities of mice, rats, rabbits and African
clawed toads (Xenopus laevis).
Covering a total area of 560 m 2 there are rooms for
housing the animals, preparation rooms, storage areas
and a large enclosure with cage-washing facilities and
autoclave. The animal house is run as a SPF-unit. That
is to say, the animals are kept under conditions that are
strictly isolated from the outside environment. Careful
controls ensure that animals introduced into the facility
are free of any of the pathogens typically found in
the species.
Experimental protocols indicating immunizations,
blood samplings etc. are performed by the animal
house staff as a service to the research groups. Methods
employed, for example, for treatment applications,
body fluid samplings and surgery, adhere to contemporary
standards in experimental animal science. Moreover
the methods used are under continuous assessment
and improved techniques are introduced whenever
possible.
The Transgene-Laboratory offers the generation of
transgenic mice and rats and mouse-chimeras, as well
as offering in-vitro-fertilization, ovary-transfer and
cryopreservation of embryos of both species. During
1998/99 224 pronucleus- and ES-cell-injections were
performed with mice, which delivered a total of over
1.300 founders and chimeras respectively - an average
of 6 positive animals per experiment. Also, 49
transgenic mouse lines were cryopreserved, and 2 lines
were revitalized. Finally, 2 invitro-fertilizations were
performed and 11 microbilogically contaminated lines
from other facilities were rederived by embryotransfer.
Moreover, courses on transgenic techniques and reproductive
biology were held for graduate students, and
special training programs were offered for transgene
technicians.
1 For more information use the link „Biogroup“ of the ZMBH-homepage
Jürgen Weiß
Teaching Facilities
The ZMBH has invested considerable funds and effort
in building and maintaining adequate facilities for
student laboratory courses in molecular biology, cell
biology, biochemistry and microbiology. This teaching
facility is organized as an independent unit consisting
of a laboratory providing space for 14 students,
with labs for special equipment, a darkroom and a
kitchen for cleaning glassware and for media preparation.
Major equipment includes: scintillation counters,
spectrophotometers, ultracentrifuges and computers.
In addition, there is all the equipment required
for work with radioactive compounds and biological
materials at the S1 safety level. The facilities are
scheduled each year for the 10 months of the regular
curriculum of the Faculty of Biology. For the remaining
time, the laboratory is used for postgraduate training.
Our teaching secretariat provides administrative support
to all the teaching activities and to the development
of curricula. It also keeps records of all ZMBH
students.
In the academic year 1998/99 we had some 80 graduates
and around 300 undergraduates participating in
the institute‘s programs in molecular biology and cell
biology. These two programs have been chosen by
70% of the biology students after their „Vordiplom“ in
1998/1999.
Hans Peter Blaschkowski
Richard Herrmann
141

Library
The library of the ZMBH offers a number of services
to support the research groups of the ZMBH. Subscriptions
to 43 scientific journals and 23 monographic
series are being entertained and the access to
online journals has been increased to 193 journals via
the central library of the university. While the online
journals can be accessed from every lab in the institute,
two computers within the library allow in addition
to use our collection of CD-ROMs which include
e. g. „Current Protocols in Molecular Biology“ and
„Current Protocols in Protein Science“. Finally, access
to articles from way out journals or backissues is provided
via the University‘s copy service.
Besides all the hard- and software support, the library
is a place for quiet and undisturbed study away from
the sometimes hectic activity at the bench.
142
Hermann Bujard
Documentation Service
The ZMBH documentation service supply all type of
visual communication supports. This service includes
computer graphics, photography and graphic arts activities.
The service provides drawings and figures destined
for publications, poster sessions or conferences. The
visual supports are produced as black and white or
colour prints, as overhead transparencies or as slides.
The computerized equipment of the service is based
on two Apple Computers connected to a desktop colour
scanner, a slide scanner, a 35 mm film exposure
unit and InkJet photo quality colour printers A4 to
A2 size. Software used are Canvas and Illustrator
for drawing, Photoshop for photographic touchingup,
QuarkXPress for DTP work and PowerPoint for
slide production.
Scientific photography is increasingly handled by
computerized processes-which give high quality results
in shorter times. For those who want to learn the
use of these new imaging methods, the documentation
service provides advice and courses.
Finally other products of this service lab include any
kind of photographic shots during ZMBH symposia
and the desktop publishing and layout of the annual
report as well as other documents that the ZMBH
edits.
Yves Cully
Central Administration
The management of all personnel, of finances and of
various administrative and technical facilities of the
ZMBH lies within the responsibilities of our institute‘s
central administration.
During 1998/99, about 250 individuals have been
working at the ZMBH. They include approx. 70 Ph.D.
students, approx. 40 postdoctoral scientists and scholarship
holders from 15 nations. In addition we have 3
foreign group-leaders and many guest scientists from
foreign countries. As usual, we had a high turnover
rate of approx. 100 per year.
The ZMBH‘s financial structure is quite complex. The
state of Baden-Württemberg has, for the past years,
been the only basic financial supporter of the institute,
which has allowed an effective and efficient management
of the ZMBH‘s financial resources. However,
the institute has also been affected by the drastic
financial cuts in the public sector. In real terms, this
means the loss of 5 staff positions until 2002 in the
central infrastructure. Such personnel reductions can
only be made by restructuring the work profile in the
central services for maximum efficiency. To compensate
for the job cuts, the ZMBH has been given the
assurance of basic financial support for the coming
years.
From 1998-1999, the development of financial support
outside the public sector corresponded to the number
of occupied research group leader positions.Due to the
high staff fluctuation on this level as well as the complicated
proceedings in the appointment of a professorship,
there has been a noticeable drop in external
funding for this period as compared with the previous
period from 1996-1997. This trend is expected to even
out when all the vacant research group leader positions
are filled.
In 1998, the central administration introduced a data
base system in the areas of finance and personnel.
With this system, all research group leaders, secretaries
as well as staff members holding key functions
are able to obtain in varying detail important financial
and personnel information online. The next step is the
coordination of this system with the SAP-data base
system used by the main administration of the University
of Heidelberg. The setting up and maintenance of
the data base at the ZMBH plays a central role in
the ZMBH administration by optimising and linking
the administrative tasks as well as the wide range of
support services available to the staff members for
research and teaching.
Jürgen Auer
143

Electrical and Mechanical Workshop
Experimental research requires functional equipment
and technical facilities around the clock. The main
task of the workshop is, therefore, the maintenance
and repair of instruments and technical help in emergencies,
whether it be the breakdown of an autoclave,
a cold room, a freezer or a laminar flow hood. Custom-made
equipment is another focus of the workshop‘s
services, for example, electrophoretic equipment
in the „ZMBH-design“, produced in a limited
series, relieves some strain on the budgets of research
groups. In the past two years, the workshop has fulfilled
requests for about 1500 repair jobs, 700 emergency
calls and has produced around 2500 pieces of
equipment or parts thereof.
144
Matthias Pawlitschko
Gert Stegmüller
Head Technicians
The ZMBH scientific staff includes two head technicians
who support the research groups by undertaking
organisational and technical tasks. Their responsibilities
are partly assigned at the level of individual storeys,
and partly towards the entire building; they also
act as safety officers for all areas including radioactive
and biological safety. Each of these scientists is supported
by a technician.
Tasks at „storey“ level are:
- Discussing problems with individual research
groups
- Organisation of, and taking minutes for, floor
meetings; implementing decisions
- Administrating the floor budget
- Purchase and maintenance of major communal
equipment
- Organisation of the washing-up kitchens
- Central purchasing of certain dangerous materials
such as radioactivity
- Assisting in moving operations when groups arrive
or depart
Tasks for the whole ZMBH are:
* Radioactive safety
- Organisation and monitoring of the various special
radioactive labs
- Central disposal of waste
- Regular radioactive safety courses for ZMBH employees
* Biological safety
- Making sure that the work is conducted in accordance
with the biological safety laws
- Giving advice to people writing biological safety
applications
- Regular biological safety courses for ZMBH employees
* Other safety precautions
* Disposal of chemical waste
* Taking care of the central stores of spare apparatus
Head Technician: Baumm, Axel
1. and 2. floor (radioactive safety,
other safety precautions)
Assistant: Merx, Alexander
Head Technician: Sawruk, Erich, Dr.
3. and 4. floor (biological safety,
chemical waste, central stores of
spare apparatus)
Assistant: Fabian, Gerd
Axel Baumm
Erich Sawruk
145

146
The Graduate Programme
The graduate programme involves all diploma and
PhD students at the ZMBH. The programme aims to
promote contact among the graduate students and also
between the scientific groups. A major goal is to expose
and train the students to perform the many additional
tasks expected from a scientist, like writing
grant applications, preparing oral presentations, etc. In
addition, talks, discussions and special method courses
are offered by specialists from the house e.g. microscopic
techniques, creation of transgenic animals, the
use of software for the analysis of data and their presentation.
The programme is organized by an elected committee
of three students and two liason professors.
At the beginning of their curriculum the graduate students
obtain help to orientate themselves at the ZMBH
and to complete the necessary administration procedures.
Additionally, organised parties and sport events
serve to promote a sense of community.
Weekend seminar in Schmitten
Twice a year a small symposium organized by the
graduate students takes place at a seminar centre in
the Taunus. The students can develop their presentation
skills by giving a research seminar in an informal
setting.
Our main philosophy is that science should be exciting
and fun.
147

ZMBH-Lehrprogramm im Grund- und Hauptstudium
Studienjahr 1998/99
Molekularbiologie und Zellbiologie im Grundstudium
Grundvorlesung
Biologie III, Teil Zellbiologie WS B. Dobberstein, D. Görlich,W. Huttner
(16.1. - 13.2.98)
Grundpraktika
Grundpraktikum C Teil 3 SS R. Herrmann und Forschungsgruppenleiter
(14 gleiche Wochenblöcke) des ZMBH
Seminare
Einführung in das Studium der Biologie WS H. Bujard
Einführung in das Studium der Biologie WS B. Dobberstein, P. Mayinger, C. Hölscher
Einführung in das Studium der Biologie WS G. Multhaup, P. Prior
Einführung in das Studium der Biologie WS R. Paro, G. Cavalli
Einführung in die Neurowissenschaften SS K.-A. Nave
148
Molekularbiologie und Zellbiologie im Hauptstudium
Vorlesungen
Molekularbiologie I WS K. Beyreuther, H. Bujard
Molekularbiologie II: Genetik, Entwicklungs- SS S. Freundlieb, R. Jansen, K.-A. Nave R. Paro
biologie und Differenzierung
Seminar zur Vorlesung Genetik, Entwicklungs- SS H. Bujard, R. Jansen, R. Paro
biologie und Differenzierung
Molekularbiologie III (Allgemeine Mikrobiologie) WS H.U. Schairer, B. Hauer, W. Plaga
Seminar zur Vorlesung Molekularbiologie III WS H.U. Schairer, B. Hauer, W. Plaga
(Allgemeine Mikrobiologie)
Molekularbiologie IV: Virologie SS H. Schaller, U. Protzer, S. Urban
Seminar zur Vorlesung Molekularbiologie IV SS H. Schaller, U. Protzer, S. Urban
Molekulare Zellbiologie I WS B. Dobberstein, H. Herrmann-Lerdon, W.W.
Franke, E. Hurt, D. Görlich
Seminar begleitend zur Vorlesung WS B. Dobberstein, G. Layh-Schmitt,
Molekulare Zellbiologie I D. Görlich, C. Harter
Seminar begleitend zur Vorlesung: WS K. Simons, E. Hurt, H. Herrmann-Lerdon,
Molekulare Zellbiologie U. Kutay
Zellbiologie IV: Molekulare und Zelluläre SS W.B. Huttner zus. mit K. Beyreuther, M.
Neurobiologie Brand, R. Brandt, H.-H. Gerdes, D. Langosch,
K.-A. Nave, B. Sakmann, P. Seeburg,
J. Trotter, K. Unsicher
Transposon und DNA-Rekombination: WS D.-H. Lankenau, R. Paro
Schrittmacher in der Evolution und Genetik
Transposonbiologie: Begleitseminar zur Vorlesung WS D.-H. Lankenau, R. Paro
Grundzüge der Immunologie SS P. Knolle, H. Schaller
von Infektionskrankheiten
149

Seminare
Pathogene Mikroorganismen WS H. Schaller, G. Darai, S. Urban, U. Protzer
Pathogene Mikroorganismen Bakterien, WS C. Clayton, D. Soldati, NN
Eukaryonten
Kontrolle der Genaktivität auf posttrans- WS C. Clayton, R.-P. Jansenkriptioneller
Ebene
Molekulare Mechanismen zellulärer WS R.-P. Jansen, F. Sauer, K.-A. Nave
Differenzierung
Genetische Grundlagen neurologischer WS K.-A. Nave, M. Sereda
Erkrankungen
Proteingene und ihre Funktion WS G. Multhaup, N.N.
Mikrobielle Entwicklungs- und Regulations- SS H.U. Schairer, W. Plagamechanismen
Mechanismen bakterieller Virulenz SS H.U. Schairer
Mechanismen der Genregulation in Eukaryonten SS G. Merdes, R. Paro
Biologie und Pathologie von Malariaparasiten und SS H. Bujard, H.-M. Müller
neue Strategien zur Bekämpfung von Malaria tropica
Moderne Methoden in der Proteinchemie SS G. Multhaup, L. Hesse
Organell-Biogenese SS C. Clayton, D. Görlich
Protein/Protein-Erkennung SS D. Langosch, S. Dübel, G. Multhaup, R.
Brandt
Neurobiologie des Lernens SS K.-A. Nave, A. Bartholomä, M. Rossner
Entwicklungsneurobiologie SS W.B. Huttner, zusammen mit M. Brand, R.
Brandt, R. Klein, K.-A. Nave, J. Trotter, K.
Unsicker
Zell-Regulation und Signaltransduktion SS B. Dobberstein, I. Hoffmann
Moderne Methoden in der Proteinchemie SS G. Multhaup, D. Beher
150
Hauptpraktika
Methoden der Molekularbiologie, 3-wöchig, WS R. Herrmann, G. Gounari, U. Kutay
ganztägig
Molekularbiologie A1, 3-wöchig, ganztägig WS K.-A. Nave, M. Rossner, D. Soldati
Zellbiologie A1 WS Die Dozenten der Zellbiologie
Molekularbiologie A2, 3-wöchig, ganztägig WS T. Hartmann, S. Lichtenthaler, K. Paliga, P.
Prior, A. Weidemann, K. Beyreuther
Molekularbiologie A2, 3-wöchig, ganztägig WS H. U. Schairer, W. Plaga, P. Prior, K.
Willwand, K. Beyreuther
Molekulare Untersuchungen der Drosophila WS R. Paro
Entwicklung, 3-wöchig, ganztägig
Begleitvorlesung zum Hauptpraktikum: Molekulare WS R. Paro
Untersuchungen der Drosophila- Entwicklung,
3-wöchig, ganztägig
Molekularbiologie: Mechanismen der Transkriptions- SS H. Bujard, S. Freundlieb, H.-M. Müller und
kontrolle, 3 Wochen, ganztägig Mitarbeiter
Methoden der Virusdiagnostik, SS H. Schaller, P. Knolle, C. Kuhn, S. Urban
3 Wochen, ganztägig,
Molekularbiologie: Mikrobiologie, SS R. Herrmann, H. Schaller, B. Hauer,
6 Wochen, ganztägig, C.H. Schröder, H. Zentgraf
Mikrobiologie für Nebenfächler und Lehramt SS R. Herrmann, C.H. Schröder,
3 Wochen, ganztägig,
Molekularbiologie: Laborpraktika , Die Forschungsgruppenleiter des ZMBH
3-6 wöchig, ganztägig,
Hauptpraktikum Zellbiologie: Proteintransport in SS B. Dobberstein, P. Mayinger, M. Pool,
vivo und in vitro, 3 Wochen ganztägig M. Seedorf
Molekulare Parasitologie, 3-wöchig, ganztägig. SS C. Clayton, D. Soldati, G. Layh-Schmitt, M.
Lanzer
Zellbiologie: Laborpraktika, Die Forschungsgruppenleiter des ZMBH
3-6 wöchig, ganztägig
151

Studienprogramm für Graduierte des ZMBH
Wintersemester 1998/99
ZMBH-Kolloquium C. Clayton und die Forschungsgruppenleiter des ZMBH
Cell Biology Lectures B. Dobberstein, F. Wieland
(Graduiertenkolleg 230 und SFB 352)
Arbeitsberichte für Mitglieder des C. Clayton und die anderen Dozenten des Grad.Kollegs
Graduiertenkollegs Kontrolle der Genexpression
bei pathogenen Organismen
Seminare
Regulation der Genaktivität H. Bujard
Spezielle Arbeiten der Tropenmedizin H. Bujard
Aktuelle Veröffentlichungen in der Entwick- R. Paro, F. Sauer
lungsgenetik
Neue Veröffentlichungen in der Zellbiologie B. Dobberstein, P. Mayinger, D. Görlich
Neuere Arbeiten aus der Molekularbiologie H. Schaller, P. Knolle
Literaturseminar H.U. Schairer, R. Herrmann
Literaturseminar: RNA C. Clayton, R.-P. Jansen
Neue Ergebnisse der Molekularbiologie M. Eilers, K.-A. Nave, C. Clayton
Aktuelle Probleme der Molekular- und K. Beyreuther, G. Multhaup, T. Hartmann, P. Jäkälä,
Neurobiologie K. Paliga P. Prior, S. Lichtenthaler, A. Weidemann
Neuere Literatur der Molekularen Neurobiologie K.-A. Nave
Neuere Arbeiten der Molekularen Zellbiologie H. Bujard, S. Freundlieb, D. Soldati
Molekulare Entwicklungsbiologie R. Paro, F. Sauer
Proteintransport B. Dobberstein und Mitarbeiter
Intrazellulärer Proteintransport D. Görlich
Hepatitis B Viren und Retroviren H. Schaller
152
Molekularbiologie mikrobieller Regulation H.U. Schairer, W. Plaga
und Entwicklung
Mitarbeiterseminar R. Herrmann
Molekulare Biologie und Zellbiologie von C. Clayton
Trypanosomen
Molekulare Parasitologie C. Clayton, D. Soldati
Molekulare Motoren D. Soldati, R.P. Jansen, T. Soldati, M. Manstein
Molekularbiologie und Genetik der K. Beyreuther, G. Multhaup, T. Hartmann, P. Jäkälä,
Alzheimer Krankheit K. Paliga, P. Prior, S. Lichtenthaler, A. Weidemann
Neurogenetik K.-A. Nave, A. Bartholomä, M. Rossner, E.-M. Krämer
Mechanismen der Genexpression F. Sauer
Messenger RNA-Lokalisierung R.-P. Jansen
Methodenkurse
Sequenzanalyse Kurs 1: LASERGENE B. Reiner
Sequenzanalyse Kurs 2: Internet, World Wide Web B. Reiner
Literatursuche und -verarbeitung B. Reiner
Dokumentationstechniken Y. Cully
Grundlagen der Mikroskopie A. Baumm
Immunisierung von Versuchstieren J. Weiss, R. Frank
Herstellung transgener Mäuse und damit J. Weiss, F. Zimmermann, F. Sprengel
verbundener Techniken
153

Sommersemester 1999
ZMBH-Kolloquium C. Clayton, H. Schaller und die Forschungsgruppenleiter
des ZMBH
Neurobiology Lectures mit Gastrednern W.B. Huttner, R. Brandt, D. Langosch, K.-A. Nave und die
anderen Forschungsgruppenleiter der Neurobiologie
Kolloquium des Graduiertenkollegs Molekulare W.B. Huttner, K.-A. Nave und die anderen Dozenten des
und zelluläre Neurobiologie mit Gastrednern Graduiertenkollegs Neurobiologie
Methodische Weiterbildung für Mitglieder des W.B. Huttner, K.-A. Nave und die anderen Dozenten des
Graduiertenkollegs Molekulare und zelluläre Graduiertenkollegs Neurobiologie
Neurobiologie
Seminare
Regulation der Genaktivität H. Bujard, S. Freundlieb
Spezielle Aspekte der Tropenmedizin H. Bujard, D. Soldati und Mitarbeiter
Aktuelle Veröffentlichungen in der R. Paro, F. Sauer
Entwicklungsgenetik
Literaturseminar: Molecular cell biology B. Dobberstein und Mitarbeiter, D. Görlich
Neuere Arbeiten aus der Molekularbiologie H. Schaller, P. Knolle
Literaturseminar R. Herrmann, H.U. Schairer
Aktuelle Probleme der Genetik, Molekular-, K. Beyreuther, G. Multhaup, T. Hartmann, C. Bergsdorf,
Neuro- und Zellbiologie C. Bergmann, P. Jäkälä, K. Ishii, K. Paliga, P. Prior,
A. Weidemann
Neuere Arbeiten aus dem Gebiet der R. Herrmann, H.U. Schairer und Mitarbeiter
biologischen Regulation
Neuere Literatur der Molekularen Neurobiologie K.-A. Nave
Neue Ergebnisse der Forschungsarbeiten H. Bujard, S. Freundlieb und Mitarbeiter
Molekulare Entwicklungsbiologie R. Paro, F. Sauer
Hepatitis B Viren und Retroviren H. Schaller und Mitarbeiter
Forschungsseminar: Protein targeting and sorting B. Dobberstein und Mitarbeiter
Molekularbiologie von Myxobakterien H.U. Schairer und Mitarbeiter
Mitarbeiterseminar R. Herrmann
154
Molekularbiologie und Zellbiologie bei C. Clayton
Trypanosomen
Molekulare Parasitologie C. Clayton, D. Soldati
Neurogenetik K.-A. Nave
Molekularbiologie und Genetik der K. Beyreuther, G. Multhaup, T. Hartmann, C. Bergsdorf,
Alzheimer Krankheit SC. Bergmann, P. Jäkälä, K. Ishii, K. Paliga, P. Prior,
A. Weidemann
Methoden der Molekularbiologie und Genetik K. Beyreuther, G. Multhaup, T. Hartmann, C. Bergsdorf,
der Alzheimer Krankheit C. Bergmann, P. Jäkälä, K. Ishii, K. Paliga, P. Prior,
A. Weidemann
Modelle neuronaler Entwicklung und Regeneration T. Hartmann, P. Jäkälä, C. Bergmann, K. Ishii, P. Prior,
K. Beyreuther
Proteingene bei der Alzheimer Krankheit G. Multhaup, L. Hesse, J. Kinter, C. Elle
Methoden und Ergebnisse der molekularen D. Bohmann, M. Brand, M. Hassel, W. Huttner, T. Leitz,
Entwicklungsbiologie B. Mechler, W. Müller, K.-A. Nave, C. Niehrs, R. Paro,
E. Pollerberg, F. Sauer, G. Schütz
Methodenkurse
Sequenzanalyse Kurs 1: LASERGENE B. Reiner
Sequenzanalyse Kurs 2: Internet, World Wide Web
Literatursuche und -verarbeitung
Dokumentation Y. Cully
Grundlagen der Mikroskopie A. Baumm
Herstellung transgener Tiere und damit J. Weiss, F. Zimmermann, S. Dlugosz
zusammenhängende Techniken
155

156
Anhang / Appendix
157

ZZMBH-Colloquia, -Seminars and Cell
Biology Lectures 1998/99 — Invited
Speakers
Acquati, F. (Milano, Italy)
Gene hunting in the chromosome 21 Down Region at
21q22.3
Aktories, A. (Freiburg, Germany)
Activation of Rho GTPases by bacterial protein
toxins
Allshire, R. (Edinburgh, UK)
Utilising transcriptional silencing to dissect fission
yeast centromere structure and function
Asai, K. (Nara, Japan
Disruption and transcription analysis of genes around
oric in Bacillus subtilis
Attardi, G. (Pasadena, USA)
Genetic and functional thresholds in mitochondrial
diseases: How much is enough?
Bartenschlager, R. (Mainz, Germany)
Molecular analysis of Hepatitis C virus replication
Bauerle, R. (Basel, Schweiz)
Strategies for regulation of metabolic pathways: the
paradigm of differentially regulated isozymes in aromatic
biosynthesis
Berberich, C. (Medellin, Kolumbien)
Strategies of control, diagnosis and vaccination against
Leishmaniasis in an endemic region: The example
Colombia, South America
Bereswill, S. (Freiburg, Germany)
Metal-dependent gene regulation in the stomach pathogen
Helicobacter pylori
158
Bergeron, J.M. (Montreal, Canada)
The role of calnexin in glycoprotein folding and quality
control
Berkhout, B. (Amsterdam, The Netherlands)
Analysis of HIV-1 transcription with mutant/ revertant
viruses
Blumer, K. (München, Germany)
Tierversuche - zum Wohle der Menschen? Ethische
Aspekte zu einem kontroversen Thema
Bonifacino, J. (Bethesda, USA)
Signal-adaptor interactions in internalization and lysosomal
targeting
Brady, S.T. (Dallas, USA)
Sculpting the functional architecture of the neuron:
Myelinating glia and the axonal cytoskeleton
Breakefield, X. (Charlestown, MA, USA)
Early onset torsion dystonia: A nondegenerative neurologic
disease caused by a dominant mutation in an
HSP-like protein
Brimacombe, R. (Berlin, Germany)
Structure of ribosomal RNA at 13A resolution
Bruss, V. (Göttingen, Germany)
Generation and selection of Hepatitis B virus core
gene mutations which block nucleocapsid envelopment
Bukau, B. (Freiburg, Germany)
The DnaK chaperone system
Casaccia-Bonnefil, P. (New York, USA)
The decision between survival and death in the oligodendroycte
lineage
Cattaneo, R. (Zürich, Switzerland)
Towards therapeutic viruses based on a measles vaccine
Chavrier, P. (Marseille, France)
Function of Rho and ARF GTP-binding proteins in
the regulation of actin cytoskeleton organization
Conzelmann, K. (Tübingen, Germany)
Rhabdoviruses: Targeted gene delivery and antivirals
Cooper, J. A. (Brisbane, Australia)
Mapping of conformational B cell epitopes within
alpha-helical coiled coil proteins
Delbac, F. (Aubiere, France)
Immunological and molecular characterization of
polar tube proteins in the microsporidia Encephalitozoon
cuniculi
Dikstein, R. (Jerusalem, Israel)
TAF II 105 - a coactivator TFIID subunit involved in
cytokine signaling and B cell specific transcription
Dobbelstein, M. (Marburg, Germany)
Adenoviral oncoproteins moonlighting as transporters
Duboule, D. (Geneva, Switzerland)
A genetic approach to vertebrate hox gene regulation
Duesberg, P. (Mannheim, Germany)
Is aneuploidy sufficient to cause cancer and genetic
instability?
Eaton, S. (Heidelberg, Germany)
Planar polarization in Drosophila and vertebrate epithelia
Endo, T. (Nagoya, Japan)
Import and folding of mitochondrial proteins
Erdmann, R. (Bochum, Germany)
Components of the protein import machinery of peroxisomes
Esser, A.F. (Kansas City, USA)
The membrane attack complex of complement: or
how to kill a bug without committing suicide
Estevez, A.M. (Los Angeles, USA)
Development of new tools for the study of RNA editing
in Trypanosomes
Gaul, U. (New York, USA)
Establishment of neural connectivity in the developing
visual system of Drosophila
Gebhardt, R. (Leipzig, Germany)
Positional expression of glutamine synthetase in the
liver: features, regulation and implications
Gorvel, J.-P. (Marseille, France)
Membrane traffic of intracellular pathogens: The
models of Brucella and Salmonella
Grant, S. (Edinburgh, UK)
The NMDA receptor - PSD95 multiprotein complex
regulated by fyn: a central mechanism for synaptic
plasticity and learning?
Graumann, P. (Cambridge, USA)
Arrangement and Active Segregation of Chromosomes
in Bacteria
Greber, U. (Zürich, Switzerland)
Adenovirus entry: Transport of DNA Virus into the
Cell and Viral DNA into the Nucleus
Gripon, P. (Rennes, France)
Effect of PreS1 deletions on L protein retention, translocation
and viral assembly of HBV
Gull, K. (Manchester, UK)
Cell cycle, differentiation and cytoskeleton of African
trypanosomes
Hacker, J. (Würzburg, Germany)
Pathogenicity islands and the evolution of infectious
agents
159

Hegemann, J. (Düsseldorf, Germany)
The Saccharomyces cerevisiae genome - from structure
to function
Heise, T. (Hamburg, Germany)
Control of Hepatitis B Virus RNA stability by host
factors
Hentze, M. (Heidelberg, Germany)
Translational control by RNA binding proteins
Herrlich, P. (Karlsruhe, Germany)
Signalling goes RNA: Regulated alternative splicing
of CD44
Herz, J. (Dallas, USA)
The LDL receptor gene family - from lipid metabolism
to brain development
Hoch, M. (Bonn, Germany)
Control of organogenesis be Wingless and EGF receptor
signaling at ectoderm/endoderm boundaries in the
Drosophila gut
Hoeijmakers, J. (Rotterdam, The Netherlands)
DNA repair in mammals: from in vivo dynamics to
human syndromes
Hoey, T. (San Francisco, USA)
Cytokine signalling during the immune response
Hohn, B. (Basel, Switzerland)
„Gene therapy“ in green eukaryotes
Hopkins, C. (London, U.K.)
Cytoplasmic coat mechanisms operating on the endocytotic
pathways of fibroblasts and epithelial cells
Horrocks, P. (Würzburg, Germany)
Mutational analysis identifies a five base pair cis-acting
sequence essential for GBP130 promoter activity
in Plasmodium falciparum
Hwang, J.-J. (Los Angeles, USA)
Retroviral vectors for regulatable or tissue-specific
160
gene expression
Jacobs, A. (Köln, Germany)
Non-invasive imaging of gene expression by Positron-
Emission-Tomography
Jäckle, H. (Göttingen, Germany)
Segmentation in Drosophila: From gradients to
stripes
Jentsch, S. (Martinsried, Germany)
A novel ubiquitination factor, E4, is involved in multiubiquitin
chain assembly
Johnston, B. (Mountain View, CA, USA)
RNA Padlocks: A new approach to gene inhibition
Kafatos, F. (Heidelberg, Germany)
Mosquito immunity and susceptibility of anopheles to
parasites
Kann, M. (Giessen, Germany)
Nuclear Import of HBV Capsid and Genome
Kennedy, M. (Pasadeny, USA)
Signal transduction molecules at the postsynaptic density
of CNS gutamatergic neurons
Kioussis, D. (London, UK)
Position effect variegation, chromatin structure and
lineage commitment
Kirchhoff, F. (Berlin, Germany)
Transgenic mouse models to study glial physiology
Klinkert, M. (Rom, Italy)
New approaches to anti-schistosomal therapy
Koszinowski, U. (München, Germany)
Viral proteins responsible for immune evasion: The
gene m153 of mouse cytomegalovirus
Langowski, J. (Heidelberg, Germany)
Large-scale structure of DNA and chromatin - modelling
and experiments
Laskey, R. (London, U.K.)
Cell cycle control of DNA replication in normal and
neoplastic cells
Leclerus, D. (Paris, France)
The insecticidal toxins and the virulence factors of
Bacillus thuringiensis
Lee, J. E. (Denver, USA)
Neuro D: a differentiation factor for neuronal and pancreatic
beta cells
Lefranc, M.-P. (Montpellier, France)
The immuno genetics database (IMGT): a necessity
for analysis of the B and T cells repertoire
Lengauer, C. (Baltimore, MD, USA)
Genetic Instability - the Achilles‘ Heel of Human
Cancer
Lind, M. (Uppsala, Sweden)
Cellular iron homeostasis
Lingelbach, K. (Marburg, Germany)
Secretion in Plasmodium falciparum
Lord, M. (Coventry, U.K.)
Toxin entry into mammalian cells: Retrograde transport
and membrane translocation
Luini, A. (Chieti, Italy)
The lipid machinery of membrane fission: The role
of CtBP/BARS and the acylation of lyso-phosphatidic
acid
Mandelkow, E. (Hamburg, Germany)
Tau protein: Phosphorylation, role in intracellular traffic,
and implications for Alzheimer‘s disease
Manson, M.D. (College Station, USA)
Mechanisms of transmembrane signaling and receptor
crosstalk in bacterial chemotaxis
Masters, C. (Melbourne, Australia)
Aß is more important than plaques
McBride, J.S. (Edinburgh, UK)
Human antibody responses to P. falciparum MSP1
Mettenleiter, T.C. (Riems, Germany)
Role of Viral Glycoproteins in Entry and Exit of
Herpes Viruses
Michaeli, S. (Rehovot, Israel)
Novel small RNAs in trypanosomes
Nover, L. (Frankfurt/M., Germany)
Heat stress response: A concert of transcription factors
and chaperones
Pahl, H. (Freiburg, Germany)
Signal transduction and virulence regulation by two
component systems
Peles, E. (Rehovot, Israel)
CASPR in neuron-glia interactions
Pieters, J. (Basel, Switzerland)
A phagosomal coat protein involved in intracellular
survival of Mycobacteria
Pines, J. (Cambridge, UK)
4-dimensional control of mitosis
Plückthun, A. (Zürich, Switzerland)
From molecular interaction screening to directed
molecular evolution
Pohlmeyer, K. (Kiel, Germany)
Solute Transport in Chloroplast
Prunel, B. (Washington, USA)
Science, more than a journal
Quijada, L. (Madrid, Spain)
Regulation of hsp70 gene expression in Leishmania
infantum
Reth, M. (Freiburg, Germany)
Signal transduction from the B-cell antigen receptor
161

Riekinen, P. Jr. (Kuopio, Finland)
Effects of estrogen therapy on memory and Abeta
levels in APP*Ps1 transgenic mice
Robinson, C. (Coventry, U.K.)
Novel pathways for the targeting of proteins in chloroplasts
and bacteria
Römisch, K. (London, U.K.)
Export of protein from the ER lumen to the cytosol
Rohlmann, A. (Göttingen, Germany)
Tissue-specific k.o. of LRP (low-density lipoprotein
receptor-related protein) in liver and brain of mice
using the cre/loxP recombination system
Roos, D.S. (Philadelphia, USA)
Exploring eukaryotic cell biology via the molecular
genetics of Toxoplasma gondii
Rothblatt, J. (Columbia, USA)
Extending the lifespan of C. elegans: What is needed
to live longer?
Sakar, M. (Manchester, U.K.)
Analysis of the cell cycle and differentiation in African
trypanosomes
Saedler, H. (Köln, Germany)
MADS box genes in flower development and evolution
Schibler, U. (Geneva, Switzerland)
Circadian timing in animals and cells
Schliwa, M. (München, Germany)
Organelle movements and motor proteins
Schübeler, D. (Braunschweig, Germany)
Site-specific recombination to express transgenes and
to analyze Cis-acting sequences
Schultz, U. (Freiburg, Germany)
Interferons in HBV infection - studies in the duck
model system
162
Shahi, S. (Dummersdorf, Germany)
Detection of polymorphism in the gene of adipogenic
transcription factor PPAR gamma: Peroxisome proliferator
activated receptor gamma
Silver, P.A. (Harvard, USA)
Movement of macromolecules in and out of the
nucleus
Sinning, I. (Heidelberg, Germany)
Role of SRP GTPases in protein transport
Skoff, R. (Wane State Univ., USA)
Central nervous system myelin protein genes are abundantly
expressed in non-neural cell types including
preimplantation embryos
Smith, J.C. (Heidelberg, Germany)
From sequence to function via structure: Molecular
modelling of proteins, complexes and conformational
change
Spasic, M. (Belgrade, Yugoslavia)
Maturation of penicillin G amidase of Providencia
rettgeri
Speranca, M.A. (Sao Paulo, Brazil)
Construction and use of cdc2-chimeras to investigate
cell cycle control in the human malaria parasite Plasmodium
vivax
Springer, S. (Berkeley, USA)
Specific packaging of membrane proteins into COPII
ER to Golgi transport vesicles
StJohnston, D. (Cambridge, UK)
mRNA localisation and axis formation in Drosophila
Subramani, S. (San Diego, USA)
Mechanisms of peroxisomal protein import and biogenesis
Tafel, A. (Warschau, Poland)
The domain structural of mitochondrial ribosomal
protein NAM9 in yeast
Tamkun, J. (Santa Cruz, USA)
Chromatin remodeling factors and the control of cell
fate in Drosophila
Thoenen, H. (Martinsried, Germany)
Modulatory role of neurotrophins in activity-mediated
neuronal plasticity
Van Meer, G. (Amsterdam, The Netherlands)
Membrane lipid diversity in search of cellular functions
Verma, I. (La Jolla, CA, USA)
Gene Therapy: Problems and Prospects
Vertel, B. (Chicago, USA)
Biosynthesis of cartilage extracellular matrix molecules:
Expected and unexpected pathways
Voigt, H. (Lausanne, Switzerland)
The role of myosin IB in Entamoeba histolytica
Yoshida, K.-C. (Hiroshima, Japan)
Reverse genetics after the whole genome sequencing:
identification of a divergon involved in inositol catabolism
in Bacillus subtilis and its regulation
Watts, C. (Dundee, U.K.)
Capture and processing of antigens for presentation
on MHC molecules
Way, M. (Heidelberg, Germany)
Actin and its role in cell motility
Wimmer, E. (New York, USA)
Molecular Mechanisms of Poliovirus Pathogenesis:
Can Poliovirus be Harnessed for Treatment of Brain
Tumors?
Wimmer, E.A. (Bayreuth, Germany)
Bicoid-independent formation of thoracic segments
in Drosophila and universal insect transgenesis
Wintersberger, U. (Wien, Austria)
Ribonucleases H of Saccharomyces cerevisiae and
their evolutionary relatives from other organisms
Zerial, M. (Heidelberg, Germany)
Rab proteins in the biogenesis and function of endosomes
163

Administrative and Technical Staff (31.12.1999)
Labor für Biomole- N.N. Sekretariate/ Baro, Ina
kulare Chemie Bosserhoff, Armin Secretary Coutinho, Karin
Ellis, Margrit Demuth, Heidemarie
Hudak, Manuela Güth-Köhler, Gerlind
Rami, Jutta
EDV/Biocomputing Mosbach, Raphael, Dipl.Phys. Reinig, Sibylle
Reiner, Berta, Dipl.Math. Schmid, Ingrid
Wingert, Winfried Schwarz, Annette
Wee, Gaik-Pin
Dokumentation Cully, Yves-Michel
Spül-, Medien- Barcinski, Barbara
Versuchstierhaltung/ Weiß, Jürgen, Dr. Küchen/Kitchens Dölling, Kerstin
Transgenlabor Dlugosz, Sascha Dörich, Gerda
Animal Facilities/ Fiala, Sandra Grimm, Martina
Transgen Lab Gärtner, Ulrike Heil, Wilma
Hartmann, Susanne Kienbacher, Ute
Hühn, Martina Kobek, Martina
Mees, Doris Meffert, Ingrid
Michelberger, Yvonne Neuhäuser, Annerose
Pfeffer, Margarita Veit, Gertrud
Wayß, Sybille Weizenwieser, Christa
Zimmermann, Frank Winkler, Helena
Bibliothek/Library delValle, Christine Verwaltung/ Auer, Jürgen, Betriebs-
Administration wirt (VWA)
Lehreinheit/ Herrmann, Richard, Prof. Apfel, Roswitha
Teaching unit Blaschkowski, H.P., Dr. Bier, Gabriela
Kern, Andrea Kröner, Barbara
Reiser, Walter, Dr. Wochlik, Claudia
Zoffer, Roswitha
Werkstätten/ Konrad, Michael
Laborsicherheit, Baumm, Axel, Dipl. Biol. Hausmeister Mächtel, Christian
Entsorgung Sawruk, Erich, Dr. Workshops/ Pawlitschko, Matthias
Biol. Sicherheit/ Fabian, Gerd Caretaker Reis, Karl-Friedrich
Lab safety Merx, Alexander Stegmüller, Gerd
164
Budget 1999
Haushalt des Landes / State Budget
Personalmittel 8.994.850,00
Sachmittel 2.017.700,00
Investitionen 515.400,00
Landeshaushalt gesamt/State Budget Total 11.527.950,00
Drittmittel / Grant Monies
Deutsche Forschungsgemeinschaft (DFG)
SFB 352, SFB 317 und SFB 544 1.753.417,00
Projektmittel aus Einzelförderung 2.227.855,00
Graduiertenkollegs 534.500,00
Bundesministerium für Bildung, Wissenschaft,
Forschung und Technologie (BMBF)
Projektmittel aus Einzelförderung einschl.
Kooperationen im Rahmen der BioRegio 1.788.230,00
Drittmittel überstaatlicher
Organisationen (EU, HFSP u.a.) 291.023,00
Projektmittel aus Einzelförderung
der Industrie 1.314.457,00
Projektmittel aus Einzelförderung
sonstiger (VW-Stiftung etc.) 271.543,00
Drittmittel gesamt/Grant Monies Total 8.181.025,00
Gesamtbetrag des Budgets 1999 / Total 1999 19.708.975,00 DM
Gesamtbetrag des Budgets 1999 / Total 1999 in EURO 10.077.038,90 EURO
165