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

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.
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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.
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