From protein transport to organelle development

Symposium:
From protein transport to organelle development
Ross Dalbey
YidC, a novel evolutionarily conserved protein, mediates membrane protein
assembly in bacteria
Uener Kolukisaoglu, Burkhard Schulz, Bianca Hust, Ulf-Ingo Fluegge, Ralf Bernd
Kloesgen, Michael Gutensohn
Isolation of an Arabidopsis T-DNA mutant for the chloroplast protein import
receptor atToc33
Johannes Herrmann, Tom Lutz, Stephan Meier, Marc Preuss, Gregor Szyrach,
Frank Baumann
Protein insertion into the inner membrane of mitochondria
Ralf Bernd Klösgen, Bo Hou
The mechanism of deltapH/TAT-dependent protein translocation across the
thylakoid membrane
Mario Jakob, Peter Hanner, Ralf Bernd Klösgen
Hunting for proteins associated with the delta pH / Tat-pathway in chloroplasts
Joao Pedro Marques, Ingrid Dudeck, Martin Schattat, Ralf Bernd Kloesgen
Import and plastid sorting of chimaeric GFP-polypeptides
Joao Pedro Marques, Ingrid Dudeck, Martin Schattat, Ralf Bernd Klösgen
Targeting of chimaeric GFP-polypeptide fusions within chloroplasts
Sabine Molik, Ralf Bernd Klösgen
Transport and assembly of the Rieske Fe/S protein: What happens in the
stromal space?
Nils Wiedemann, Kaye Truscott, Peter Rehling, Chris Meisinger, Nikolaus Pfanner
Mitochondrial translocases for precursor proteins
Tom Rapoport
Protein Transport In and Out of the ER
Colin Robinson
Structure and mechanism of the twin-arginine translocase
Heinrich Jung, Maike Rochon, Corinna Weber-Sparenberg
Targeting of proteins to the flagellar type III secretion system of Escherichia coli

Ross Dalbey
YidC, a novel evolutionarily conserved protein, mediates membrane protein
assembly in bacteria
Membranes contain proteins that catalyze a variety of reactions, which lead to
the selective permeability of the membrane. For membrane proteins to function
as receptors, transporters, channels, and ATPases, they must be targeted to their
correct membrane and inserted into the lipid bilayer. Our goal is to understand
the biogenesis of polytopic membrane proteins in bacteria. While most E. coli
membrane proteins use the Sec YEG pathway for insertion into the plasma
membrane, there are some proteins that insert independent of the Sec pathway.
Recently we identified a new membrane component called YidC that is essential
for insertion of these Sec-independent proteins. YidC is essential for cell viability
and is found in mitochondria and chloroplasts. Depletion of YidC interferes also
with the insertion of Sec-dependent membrane proteins. We find that YidC
directly interacts with membrane proteins during the process of membrane
protein insertion. The chloroplast homolog Albino3 can functionally complement
the bacterial YidC depletion strain, demonstrating that the chloropast and
bacterial YidC homologs are truly functional homologs. The function of YidC for
Sec-independent proteins may be analogous to a chaperone because YidC has
been shown to bind and fold the inserting membrane proteins and not to interact
with the fully synthesized and assembled protein. For sec-dependent proteins,
YidC is proposed to catalyze the movement of hydrophobic regions out of the Sec
channel and into the lipid bilayer.
contact:
Professor Ross Dalbey
Ohio State University
Department of Chemistry
dalbey@chemistry.ohio-state.edu
100 West 18th Ave
43210 Columbus,OH (USA)

Uener Kolukisaoglu, Burkhard Schulz, Bianca Hust, Ulf-Ingo Fluegge, Ralf Bernd
Kloesgen, Michael Gutensohn
Isolation of an Arabidopsis T-DNA mutant for the chloroplast protein import
receptor atToc33
A great number of plastid proteins are nuclear encoded, are synthesized in the
cytosol as precursor with a N-terminal extension (transitpeptide) and have to be
imported into the organelle posttranslationally. Two protein complexes located in
the plastidic envelope membranes are responsible for this transport process. One
of the first steps of this transport is the binding of the precursor protein with its
transitpeptide to one or several receptor proteins, that are exposed on the
cytosolic surface of the plastids. Toc34, a GTP binding component of the import
complex in the outer chloroplast envelope, is one of these receptors and was
shown to directly interact with precursor proteins. To gain more insight into the
in vivo function of this receptor a reverse genetic approach was taken. A
collection of about 32000 Arabidopsis T-DNA lines was sucessfully screened for
insertions in the atToc33 gene, one of the two Arabidopsis Toc34 homologues.
The T-DNA insertion in the isolated mutant is located in the promotor region of
the atToc33 gene. The atToc33 mutant shows a pale yellowish phenotype, that
has also been observed after antisense repression of atToc33, and could be
complemented upon transformation with the wildtype cDNA. Further molecular
and physiological characterisation of the mutant will be presented.
contact:
Dr. Michael Gutensohn
MLU Halle-Wittenberg
Institut für Pflanzenphysiologie
gutensohn@pflanzenphys.uni-halle.de
Weinbergweg 10
06120 Halle/ Saale (Deutschland)

Johannes Herrmann, Tom Lutz, Stephan Meier, Marc Preuss, Gregor Szyrach,
Frank Baumann
Protein insertion into the inner membrane of mitochondria
The inner membrane of mitochondria contains a large number and variety of
proteins. Most of these proteins are synthesized in the cytoplasm and imported
into mitochondria. Inner membrane proteins are inserted on different sorting
pathways. Monotopic proteins can be inserted following a translocation arrest at
the level of the inner membrane. An insertion route from the intermembrane
space is also used by polytopic proteins which have not evolved from bacterial
homologues. In contrast, we found that polytopic proteins derived from the
endosymbiotic ancestors of mitochondria are first completely imported into the
mitochondrial matrix and subsequently inserted into the membrane. This
insertion process depends on the membrane potential and resembles the
insertion of membrane proteins in bacteria in several respects.
The same insertion route is used by inner membrane proteins that are
synthesized on mitochondrial ribosomes. Membrane integration of these proteins
depends on the Oxa1 protein, a component related to bacterial YidC. Oxa1 forms
an oligomeric complex in the inner membrane and contains a matrix-exposed
domain which has the ability to bind translating mitochondrial ribosomes and is
required for efficient insertion of nascent polypeptides. This suggests that
cotranslational membrane insertion of mitochondrial translation products is
achieved by a physical contact of translation complexes with the OXA translocase
in the inner membrane.
contact:
Dr. Johannes Herrmann
Universität München
Institut für Physiologische Chemie
hannes.herrmann@bio.med.uni-muenchen.de
Butenandtstrasse 5
81377 München (Germany)

Ralf Bernd Klösgen, Bo Hou
The mechanism of deltapH/TAT-dependent protein translocation across the
thylakoid membrane
Protein translocation into or across the thylakoid membrane is accomplished by
at least four distinct pathways: the Sec-dependent pathway, the SRP-dependent
pathway, the pH/TAT-dependent pathway and the spontaneous protein
insertion pathway. The pH/TAT-dependent protein translocation pathway is the
sole system that is capable of transporting both folded and unfolded proteins
across the thylakoid membrane. Translocation of precursors by this pathway
does not need stromal factors or nucleoside triphosphates, but is strictly
dependent on the thylakoidal pH. In order to analyze the mechanism of
pH/TAT-dependent protein translocation, we have constructed a chimeric 16/23
protein, which consists of the transit peptide of the 16 kDa protein and the
mature part of the 23 kDa protein. Like the two corresponding authentic
precursor proteins (pre-16 kDa and pre-23 kDa, respectively), the chimeric
16/23 is targeted by pH/TAT-dependent pathway. During thylakoid import, the
chimeric protein is retarded in the pH/TAT-dependent translocation machinery,
resulting in transmembrane translocation intermediates. Two distinct steps of the
transport process have been identified, which are characterized by different
topologies of the translocation intermediates. Taking advantage of this finding,
we could make detailed studies on the mechanism of each processing step. Data
on the differences between these two steps, e.g. considering their dependence
upon pH/TAT-translocase or the energy requirement, will be presented.
contact:
Master Bo Hou
Martin-Luther-Universität Halle-Wittenberg
Institut für Pflazenphysiologie
hou@pflanzenphys.uni-halle.de
Weibergweg 10
06120 Halle (Saale) (Germany)

Mario Jakob, Peter Hanner, Ralf Bernd Klösgen
Hunting for proteins associated with the delta pH / Tat-pathway in chloroplasts
Among the four protein transport pathways which have been identified at the
thylakoid membrane of chloroplasts, the delta pH/Tat-pathway is of particular
interest because of its unusual mechanism: it does not require soluble factors nor
nucleoside triphosphates but depends strictly and exclusively on the proton
gradient that is generated during photosynthesis across the thylakoid membrane.
Furthermore, like its bacterial counterpart it is able to transport folded
polypeptide chains. Three components of the thylakoidal delta pH/Tat-transport
machinery have been identified so far (Tat A, B, C). During transport of a protein
across the thylakoid membrane, these subunits apparently form multimeric
complexes of approximately 560 kDa and 620 kDa.
The goal of the project is to find out whether Tat A, B, and C interact
permanently with each other in the thylakoid membrane, or whether they
assemble into a common complex only during the actual transport process.
Furthermore, we want to examine whether additional thylakoidal proteins which
would be promising candidates for peripheral and/or modulating components of
the thylakoidal Tat-translocase can be found attached to Tat A, B, or C,
respectively. For this purpose, antibodies specifically recognizing either of the
three Tat-components were immobilized by various methods and used for affinity
chromatography of solubilized thylakoid preparations from Arabidopsis thaliana.
Stepwise elution protocols were developed in order to separate potential binding
partners with different degrees of binding affinity. Subsequent analysis by
MALDI-TOF mass spectrometry is performed in order to characterize the
interacting proteins. First results of this approach will be presented.
contact:
Dr. Mario Jakob
Martin-Luther-Universität Halle-Wittenberg
Pflanzenphysiologie
jakob@pflanzenphys.uni-halle.de
Weinbergweg 10
06120 Halle (Deutschland)

Joao Pedro Marques, Ingrid Dudeck, Martin Schattat, Ralf Bernd Kloesgen
Import and plastid sorting of chimaeric GFP-polypeptides
In order to get further insights into the transport specificity of the different
thylakoid import pathways we have performed in vitro import experiments using
protein fusions among the GFP protein and several thylakoid transit peptides. The
later were part of proteins assigned to both the delta pH/TAT (16 and 23 kDa
subunits of the water splicing complex of PS2) and Sec pathways (plastocyanin
and 33 kDa subunit of the water splicing complex of PS2).
In the presence of isolated pea thylakoids we were able to demonstrate the
efficient and specific targeting of 16/GFP and 23/GFP fusion proteins by the ?pH
pathway to the thylakoid lumen. In contrast, even after the addition of isolated
stroma fraction, no lumenal import of the PC/GFP and 33/GFP could be observed
in isolated pea thylakoids. In the presence of intact pea and spinach chloroplasts,
however, besides an efficient import of the 16/GFP and 23/GFP fusions, a low
level of 33/GFP import into the thylakoid lumen could also be detected.
The data obtained suggest a preferential import of GFP fusions into the thylakoid
lumen via the delta pH pathway. The explanation for this behaviour might reside
in the transport specificity of the delta pH pathway for folded proteins. In order
to assay the folding status of GFP during the course of plastid import and internal
sorting, transgenic Arabidopsis lines expressing GFP fusions are currently being
produced.
contact:
Doktor Joao Pedro Marques
Martin-Luther-Universitat Halle
Institut für Pflanzenphysiologie
marques@pflanzenphys.uni-halle.de
Weinbergweg 10
06120 Halle (Saale) (Germany)

Joao Pedro Marques, Ingrid Dudeck, Martin Schattat, Ralf Bernd Klösgen
Targeting of chimaeric GFP-polypeptide fusions within chloroplasts
In order to get further insights into the transport specificity of the different
thylakoid import pathways, we have performed in vitro import experiments using
protein fusions among the GFP protein and several thylakoid transit peptides. The
later were part of proteins assigned to both the delta pH/TAT (16 and 23 kDa
subunits of the water splicing complex of PS2) and Sec pathways (plastocyanin
and 33 kDa subunit of the water splicing complex of PS2).
In the presence of isolated pea thylakoids we were able to demonstrate the
efficient and specific targeting of 16/GFP and 23/GFP fusion proteins by the delta
pH pathway to the thylakoid lumen. In contrast, even after the addition of
isolated stroma fraction, no lumenal import of the PC/GFP and 33/GFP could be
observed in isolated pea thylakoids. In the presence of intact pea and spinach
chloroplasts, however, besides an efficient import of the 16/GFP and 23/GFP
fusions, a low level of 33/GFP import into the thylakoid lumen could also be
noticed.
The data obtained suggest a preferential import of GFP fusions into the thylakoid
lumen via the delta pH pathway. The explanation for this behaviour might reside
in the transport specificity of the delta pH pathway for folded proteins. In order
to assay the folding status of GFP during the course of plastid import and internal
sorting, transgenic Arabidopsis lines expressing GFP fusions are currently being
produced.
contact:
Doktor Joao Pedro Marques
MLU-Halle
Institut für Pflanzenphysiologie
marques@pflanzenphys.uni-halle.de
Weinbergweg 10
06120 Halle (Saale) (Germany)

Sabine Molik, Ralf Bernd Klösgen
Transport and assembly of the Rieske Fe/S protein: What happens in the stromal
space?
The Rieske Fe/S protein is a nuclear-encoded subunit of the cytochrom b6/f
complex in chloroplasts. It is synthesised in the cytosol as a precursor molecule
with an N-terminal transit peptide, post-translationally transported into the
organelle and subsequently targeted via the delta pH/TAT pathway to the
thylakoids. In contrast to all other thylakoid proteins analysed so far, transport of
the Rieske protein is retarded in the stromal space leading to the accumulation of
the protein in several complexes of high molecular weight. Using various chimeric
and mutant Rieske proteins, complexes interacting specifically with either the
hydrophilic lumenal domain or the thylakoid targeting signal which is provided by
the membrane anchor can be distinguished. Among the latter is the cpn60
system, the well known folding machinery in the chloroplast. Other stromal
complexes might be responsible for Fe/S cofactor insertion, because delta
pH/TAT pathway is capable of transporting folded proteins. In the case of the
Rieske protein, correct folding and/or assembly of the iron-sufur cluster seems
even to be a prerequisite for translocation across the thylakoid membrane.
contact:
Sabine Molik
Martin-Luther-Universität Halle-Wittenberg
Institut für Pflanzenphysiologie
molik@pflanzenphys.uni-halle.de
Weinbergweg 10
06120 Halle (Saale) (Germany)

Nils Wiedemann, Kaye Truscott, Peter Rehling, Chris Meisinger, Nikolaus
Pfanner
Mitochondrial translocases for precursor proteins
Mitochondria import hundreds of different proteins from the cytosol. Three major
translocase complexes have been identified in the mitochondrial membranes. The
translocase of the outer membrane (TOM complex) transport all kinds of
precursor proteins. It consists of three receptor proteins, a general import pore
and assembly factors. The inner membrane contains two translocases. The
presequence translocase (TIM23 complex) transports preproteins with
amino-terminal signal sequences (presequences), while the carrier translocase
(TIM22 complex) is responsible for insertion of polytopic membrane proteins with
multiple internal targeting signals. All Tom and Tim proteins themselves are
encoded by nuclear genes and synthesized as precursor proteins in the cytosol.
The precursors must be imported into mitochondria and assembled into
functional translocase complexes.
Literature
Pfanner, N., and Geissler, A. (2001). Versatility of the mitochondrial protein
import machinery. Nature Rev. Mol. Cell Biol. 2, 339-349
contact:
Prof. Nikolaus Pfanner
Universität Freiburg
Institut für Biochemie und Molekularbiologie
Nikolaus.Pfanner@biochemie.uni-freiburg.de
Hermann-Herder-Straße 7
79104 Freiburg (D)

Tom Rapoport
Protein Transport In and Out of the ER
Protein transport across the ER membrane occurs through a protein-conducting
channel formed from the heterotrimeric Sec61p complex. The channel itself is
passive; it needs to associate with partners that provide the driving force for
translocation and determine directionality. Translocation into the ER can occur
co- or post-translationally. Recent structural studies have given us a better view
of the channel and how it connects with the ribosome during co-translational
translocation. Proteins can also be translocated from the ER back into the cytosol
(retro-translocation). To study the first steps in retro-translocation we have used
cholera toxin. We have found that protein disulfide isomerase (PDI) in the ER
lumen disassembles and unfolds the toxin once its A-chain has been cleaved. PDI
acts as a redox-driven chaperone: in the reduced state, it binds to the A-chain
and in the oxidized state it releases it. We have also started to look at the last
step in retro-translocation, the release of proteins from the ER membrane into
the cytosol. Our results show that poly-ubiquitination is required for
retro-translocation. An AAA ATPase family member, Cdc48p/p97, and its partner
proteins Ufd1 and Npl4p, are subsequently involved in extracting proteins out of
the membrane.
contact:
Professor Tom Rapoport
HHMI/Harvard Medical School
Dept. of Cell Biology
tom_rapoport@hms.harvard.edu
240 Longwood Avenue
02115-6091 Boston, MA (USA)

Colin Robinson
Structure and mechanism of the twin-arginine translocase
The twin-arginine translocation (Tat) system operates in the bacterial plasma
membrane and the thylakoid membrane of plant chloroplasts. The system
recognises proteins bearing N- terminal signal peptides that contain an invariant
twin-arginine motif, and has the unique ability to translocate fully-folded proteins
across tightly sealed membranes. To date, three important tat genes have been
identified in bacteria (tatABC) and homologous genes are present in plants. We
have purified a Tat complex from Escherichia coli and hsow that it contains only
TatABC, with no evidenmce for the presence of hitherto unidentified proteins.
TatB and TatC form the core of the complex and are present in a 1:1 ratio. We
further show that a translational fusion between these proteins is fully active,
indicating that they form a structural and functional unit within the Tat complex.
Preliminary electron microscopy images reveal a stable complex of TatBC that
becomes considerably enlarged in the presence of TatA; the architecture of this
complex will be discussed in the light of models for the mechanism of this
system. The structures and functions of other bacterial Tat systems will also be
discussed.
contact:
Prof. Colin Robinson
University of Warwick
Department of Biological Sciences
CRobinson@bio.warwick.ac.uk
Coventry (GB)

Heinrich Jung, Maike Rochon, Corinna Weber-Sparenberg
Targeting of proteins to the flagellar type III secretion system of Escherichia coli
A number of Gram-negative animal and plant pathogenic bacteria use type III
secretion systems to inject bacterial effector proteins into eukaryotic cells. The
type III secretory pathway is also required for transport of flagellar structural
proteins beyond the cytoplasmic membrane whereby playing an essential role in
the biogenesis of the bacterial flagella. The molecular mechanism underlying this
process is still enigmatic. Studies on type III systems involved in bacterial
virulence suggest that protein substrates are marked for secretion via a mRNA
mediated signal and by their cognate chaperones. Alternatively substrates may
be recognized by a N-terminal amino acid sequence 1 .
To investigate substrate recognition during flagella biogenesis, we used FlgD, a
scaffolding protein in flagellar hook assembly, as a model substrate. Type III
system-dependent export of a FlgD-PhoA hybrid into the periplasm of E. coli was
determined enzymatically as well as by Western blotting. Truncation-analysis of
FlgD reveals that the N-terminal 83 amino acids are crucial for export.
Furthermore, frame shift mutations altering the sequence of amino acids 2 to 20
of FlgD prevent export of FlgD-PhoA, whereas replacement of N-terminal amino
acids with an amphipathic sequence stimulates the transport of the hybrid
protein. Further studies showed that the amphipathic sequence is essential but
not sufficient for FlgD-PhoA transport.
Literature
1 S.A. Lloyd, A. Forsberg, H. Wolf-Watz, M.S. Francis (2001) Trends Microbiol. 9,
367-71.
contact:
Corinna Weber-Sparenberg
Universität Osnabrück
Mikrobiologie
weber-sparenberg@biologie.uni-osnabrueck.de
Barbarastr. 11
49069 Osnabrück (Germany)
additional information
PD Dr. Heinrich Jung
Universität Osnabrück
Mikrobiologie
Barbarastrasse 11
49069 Osnabrück
Germany
jung_h@biologie.uni-osnabrueck.de