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dictyNews Volume 34 Number 02
dictyNews
Electronic Edition
Volume 34, number 2
January 15, 2010
Please submit abstracts of your papers as soon as they have been
accepted for publication by sending them to dicty@northwestern.edu
or by using the form at
http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit.
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Abstracts
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Unconventional secretion of Acb1 is mediated by autophagosomes.
Juan M. Duran, Christophe Anjard, Chris Stefan, William F. Loomis,
and Vivek Malhotra
J. Cell Biol, in press
Some secreted proteins lack the N-terminal signal sequence necessary
for entering the Endoplasmic Reticulum (ER). These proteins use
unconventional pathways for release from the cell. We have shown
that one such protein, acyl-CoA binding protein (AcbA), requires the
Golgi associated protein GRASP for release from Dictyostelium cells.
We now report that the yeast Saccharomyces cerevisiae homolog Acb1,
is also released in a GRASP dependent manner. Strains carrying
temperature sensitive mutations that block the classical secretion
pathway released Acb1 at the non-permissive temperature as well as
wild type strains. However, strains carrying mutations in the genes
encoding the fusion protein Sec18p, the plasma membrane T-SNARE,
SSo1, or genes necessary for autophagy failed to release Acb1.
Therefore, secretion of AcbA, while independent of transport through
the ER-Golgi pathway, is mediated by a vesicular intermediate likely
to be derived from autophagosomes that ordinarily transport
intracellular proteins to the vacuole for degradation, but under these
conditions fuse with the plasma membrane.
Submitted by Bill Loomis [wloomis@UCSD.edu]
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Unconventional secretion of Pichia pastoris Acb1 is dependent on
GRASP protein, peroxisomal functions and autophagosome formation
Ravi Manjithaya, Christophe Anjard, William F. Loomis
and Suresh Subramani1*
J. Cell Biol, in press
In contrast to the enormous advances made regarding mechanisms of
conventional protein secretion, mechanistic insights into the unconventional
secretion of proteins are lacking. Acyl-CoA binding protein (AcbA in
Dictyostelium discoideum), an unconventionally secreted protein, is
dependent on GRASP for its secretion. We discovered, surprisingly, that
the secretion, processing and function of an AcbA-derived peptide, SDF2,
are conserved between the yeast Pichia pastoris and D. discoideum. We
show that in yeast, the secretion of SDF2-like activity is GRASP-dependent,
triggered by nitrogen starvation and requires autophagy proteins as well as
medium-chain fatty acyl-CoA generated by peroxisomes. Additionally, a
phospholipase D implicated in SNARE-mediated vesicle fusion at the
plasma membrane is necessary, but neither peroxisome turnover nor
fusion between autophagosomes and the vacuole is essential. Moreover,
yeast Acb1 and several proteins required for its secretion are necessary
for sporulation in P. pastoris. Our studies implicate heretofore unknown,
evolutionarily-conserved pathways in unconventional secretion.
Submitted by Bill Loomis [wloomis@UCSD.edu]
--------------------------------------------------------------------------------
Loss of Dictyostelium ATG9 results in a pleiotropic phenotype affecting
growth, development, phagocytosis and clearance and replication of
Legionella pneumophila.
Sze Man Tung 1, Can Ünal 2, Alexandra Ley 1, Cohue Peña 3,
Budi Tunggal 1, Angelika A. Noegel 1, 4, Oleg Krut 3, 4,
Michael Steinert 2 and Ludwig Eichinger 1, 4*
1 Zentrum für Biochemie, Medizinische Fakultät, Universität zu Köln,
Joseph-Stelzmann-Str. 52, D-50931 Köln
2 Institut für Mikrobiologie, Technische Universität Braunschweig,
Spielmannstr. 7, D-38106 Braunschweig
3 Institut für Medizinische Microbiologie, Immunologie und Hygiene,
Universität zu Köln, Goldenfelsstr. 19-21, D-50935 Köln
4 Zentrum für Molekulare Medizin Köln (ZMMK), Universität zu Köln
D-50931 Köln
+ equal contribution
Cell. Microbiology, in press
Infection of Dictyostelium discoideum with Legionella pneumophila resulted
in a large number of differentially regulated genes among them three core
autophagy genes, ATG8, ATG9, and ATG16 (Farbrother et al., 2006).
Macroautophagy contributes to many physiological and pathological
processes and might also constitute an important mechanism in
cell-autonomous immunity. For further studies we selected the highly
conserved ATG9. In colocalisation studies with GFP-tagged ATG9 and
different organelle marker proteins we neither observed colocalisation with
mitochondria, the ER nor lysosomes. However, there was partial
colocalisation with the Golgi apparatus and many ATG9-GFP containing
vesicles localised alongmicrotubules and accumulated around the microtubule
organising center. ATG9-deficient cells had pleiotropic defects. In addition to
growth defects they displayed severe developmental defects, consistent with
the known role of autophagy in Dictyostelium development. Unexpectedly,
the ATG9 mutant also had a strong phagocytosis defect that was particularly
apparent when infecting the cells with L. pneumophila. However, those
Legionellae that entered the host could multiply better in mutant than in
wild-type cells, due to a less efficient clearance in the early and a more
efficient replication in the late phase of infection. We conclude that ATG9
and hence macroautophagy has a protective role during pathogen infection.
Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de]
--------------------------------------------------------------------------------
A Rap/PI3K pathway controls pseudopod formation.
Arjan Kortholt, Parvin Bolourani, Holger Rehmann, Ineke Keizer-Gunnink,
Gerald Weeks, Alfred Wittinghofer, and Peter J.M. Van Haastert|
Mol Biol Cell, in press
GbpD, a Dictyostelium discoideum guanine exchange factor specific for
Rap1, has been implicated in adhesion, cell polarity and chemotaxis. Cells
overexpressing GbpD are flat, exhibit strongly increased cell-substrate
attachment, and extend many bifurcated and lateral pseudopodia. Phg2,
a serine/threonine-specific kinase, mediates Rap1-regulated cell-substrate
adhesion, but not cell polarity or chemotaxis. In this study we demonstrate
that overexpression of GbpD in pi3k1/2-null cells does not induce the
adhesion and cell morphology phenotype. Furthermore we show that Rap1
directly binds to the Ras binding domain of PI3K, and overexpression of
GbpD leads to strongly enhanced PIP3 levels. Consistently, upon
overexpression of the PIP3 degradating enzyme PTEN in
GbpD-overexpressing cells, the strong adhesion and cell morphology
phenotype is largely lost. These results indicate that a GbpD/Rap/PI3K
pathway controls pseudopod formation and cell polarity. Like in Rap
regulated pseudopod formation in Dictyostelium, mammalian Rap and
PI3K are essential for determining neuronal polarity, suggesting that the
Rap/PI3K pathway is a conserved module regulating the establishment
of cell polarity.
Submitted by Peter Van Haastert [p.j.m.van.haastert@rug.nl]
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[End dictyNews, volume 34, number 2]
