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. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= 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] -------------------------------------------------------------------------------- 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] ============================================================== [End dictyNews, volume 34, number 2]