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

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-
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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.
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