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

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
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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
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