| Research Interests:
I am interested in understanding the mechanisms
that underlie the basic organization of the early secretory
pathway. In particular, my laboratory focuses on the
determinants that are responsible for maintaining the
identities of the endoplasmic reticulum (ER) and the
Golgi apparatus. At present, it is not clear why some
proteins are exported from the ER to the Golgi apparatus
by COPII coated vesicles, while others remain behind
in the ER. We are using a combination of in vitro reconstituted
biochemical assays, yeast genetics and microscopy to
approach this problem.
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Stefan
Otte, Assistant Professor
Postdoctoral Training
Barlowe Lab, Dartmouth Medical
School,
Hanover, NH
1999 - 2004
Ph. D. in Biochemistry
Goettingen University and
Max Planck Institute for Experimental Medicine,
Goettingen, Germany 1999
Diploma in Biology
Goettingen University 1996
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According to the bulk flow hypothesis,
proteins leave the ER by default and ER
resident proteins are then retrieved by a
receptor from the Golgi. However, there is
mounting vidence that at least some
secretory proteins are packaged into
budding COPII vesicles by specific cargo
receptors that cycle between these two
compartments. Other possible mechanisms
may be retention of ER resident proteins by interactions
with an ER matrix, or exclusion from budding vesicles.
It seems likely that several or all of these mechanisms
operate in concert to achieve organelle identity. ER
resident proteins may be retained while cargo is specifically
exported, and the retrieval mechanism may then return
ER residents that escaped retention and exclusion. My
goal is to understand the roles and the hierarchy of
these processes.
In the beginning of this work, I embarked on a systematic
analysis of the protein components of yeast COPII vesicles.
Vesicles were isolated in biochemical quantities, and
their protein constituents were resolved on SDS-PAGE
and identified by mass spectrometry. This approach identified
several known vesicle proteins as well as several uncharacterized
ones, which were termed ER Vesicle (Erv) roteins (Otte
et al., 2001). Several Erv proteins turned out to be
required for the packaging of different cargo molecules
into budding vesicles. Importantly, Erv29p is essential
for the efficient export of a model cargo molecule,
the alpha factor pheromone precursor (Belden and Barlowe,
Science 294: 1528-1531, 2001). My working hypothesis
is that Erv29p acts as a sorting receptor that loads
a subset of cargo molecules into budding COPII vesicles.
The Erv proteins have highly conserved orthologs in
higher eukaryotes (Otte et al., 2001). In a collaboration
with Lelio Orci and colleagues at the University of
Geneva, it was demonstrated by immunogold electron microscopy
and immunofluorescence experiments that mammalian Erv46
localizes to equivalent compartments, and is likely
to play a similar role in secretory processes as yeast
Erv46p (Orci et al., 2003).
Erv41p and Erv46p, two Erv proteins of 41 and 46kDa
respectively, form a complex which cycles between the
ER and Golgi. I have used this complex as a model to
study ER export signals on transmembrane proteins (Otte
and Barlowe, 2002). As a result, the specific packaging
of this type of cargo is now better understood: hydrophobic
signal motifs on the cytoplasmically exposed domains
of these proteins are necessary for packaging and binding
to the COPII subunits during vesicle ormation. Surprisingly,
export of the complex requires signals on both subunits
that must be presented to the
COPII machinery in a specific orientation to result
in efficient packaging.
In contrast, the mechanisms responsible for the sorting
of soluble cargo molecules into COPII vesicles are unknown.
We are currently using the alpha factor precursor as
a model cargo and Erv29p as its putative receptor to
study this process. By introducing specific mutations
on the cargo molecule and using the mutant protein in
reconstituted budding assays, a potential ER export
motif has been defined. It remains to be determined
how this receptor mediated export from the ER is regulated,
and how cargo dissociates from its receptor upon delivery
to the Golgi. Moreover, it needs to be established whether
similar mechanisms operate in export of other secretory
proteins, including several clinically important molecules.
To determine whether receptor mediated ER export is
dominant over any retention or exclusion mechanism,
we are using various chimeric proteins containing alpha
factor targeting information fused to different ER resident
proteins in a new molecular 'tug-o'-war' assay.
Selected Publications:
Otte S. and Barlowe C., 2004: Sorting signals can
direct receptor-mediated export of soluble proteins
into COPII vesicles. Nature Cell Biol., online publication
ahead of print October 31st, 2004.
Breuza L., Halbeisen R, Jeno P., Otte S., Barlowe C.,
Hong W. and Hauri H. P., 2004: Proteomics of ERGIC membranes
from brefeldin A-treated HepG2 cells identifies ERGIC-32,
a new cycling protein that interacts with human Erv46.
J. Biol. Chem. 279: 47242 - 47253.
Orci L., Ravazzola M., Mack G. J., Barlowe C. and Otte
S., 2003: Mammalian Erv46 localizes to the endoplasmic
reticulum - Golgi intermediate compartment and to cis-Golgi
cisternae. Proc. Natl. Acad. Sci. USA 100: 4586 - 4591.
Otte S. and Barlowe C., 2002: The Erv41p-Erv46p complex:
Multiple export signals are required in trans for COPII-dependent
transport from the ER. EMBO J. 21: 6095 - 6104.
Otte S., Belden W. J., Heidtman M., Liu J., Jensen
O. N., and Barlowe C., 2001: Erv41p and Erv46p: new
components of COPII vesicles involved in transport between
the ER and Golgi complex. J. Cell Biol. 152: 503 - 517.
Kroll K. A., Otte S., Hirschfeld G., Barnikol-Watanabe
S., Goetz H., Sternbach H., Kratzin H. D., Barnikol
H. U. and Hilschmann N., 1999: Heterologous overexpression
of human NEFA and studies on the two EF-hand calcium-binding
sites. Biochem. Biophys. Res. Commun. 260: 1 - 8.
Otte S., Barnikol-Watanabe S. and Hilschmann N., 1999:
Direct dye terminator sequencing of Lambda DNA and phage
suspensions. Anal. Biochem. 270: 332 - 334.
Otte S., Barnikol-Watanabe S., Vorbrueggen G. and
Hilschmann N., 1999: NUCB1, the Drosophila melanogaster
homolog of the mammalian EF-hand proteins NEFA and nucleobindin.
Mech. Devel. 86: 155 - 158 |
