12/26/01, 4:52 PM ) that, when VBM2 and VBM1 4108 Phosphorylation and vesicle fusion. In wild-type cells (a) v- (Snc precursors that accumulate in vbm mutant cells. Activation Activation cells. mutant vbm in inactivated mutation, by allow cells lacking the v-SNAREs to accumulate grow and secrete Our normally. recent work has that revealed that a protein phosphatase is activated directly sphingolipid by precursors of the phosphatase results in dephosphorylation of the Sso t-SNAREs, resulting in the formation of a t-t SNARE complex that drives fusion(Fig. 1). Thus, phosphorylated SNAREs do not assemble into SNARE complexes, while dephosphorylated SNAREs do.These findings demonstrate that a lipid-activated signaling cascade controls secretion and that SNARE then identified two other genes ( genes twoother identified then 972 8 934 Fig. 1 1,2) and t-SNAREs (Sso1,2 and Sec9) assemble into a fusion complex, resulting in secretion. In snc cells (b), no v-SNAREs present, are Sso t-SNAREs then are hyperphosphorylated, and vesicle fusion is blocked. whenHowever, snc cells treated are with ceramide, sphingoid bases, or by vbm mutation (c), Sso t-SNAREs dephosphorylated are by a phosphatase, allowing it to assemble into a 2:1 fusion complex with Sec9. Fax. 2106 [email protected] [email protected] 972 8 934 Tel. E-mail: Department of Molecular Genetics Department The molecular basis for for basis molecular The SNAREs, secretion: cellular and regulators, SNARE vesicles secretory genes in yeast results in the

as a model system to elucidate the 104 SNC1,2 In order for eukaryotic cells newly synthesized to grow, Originally, we discovered the vesicle SNAREs (Snc1 and 2) the of Deletion Research Objectives Research proteins and lipids are delivered to the cell surface via a process known as exocytosis. My laboratory utilizes the yeast cerevisiae Saccharomyces mechanisms that underlie intracellular membrane trafficking membrane intracellular underlie that mechanisms and exocytosis. In particular, we are interested in the role of SNAREs, fusogenic proteins that are present upon both vesicle and acceptor membranes, in the steps leading to vesicle docking and fusion. One project deals specifically with the role of cell signaling in the control of exocytosis, and concerns the involvement of protein kinases and phosphatases in regulating SNARE functioning. A second project deals with proteins that bind to SNAREs (called SNARE regulators) them and prevent from assembling into the fusion complex. a third project Finally, deals with studying the actual biogenesis of secretory vesicles and(SVs) aims at understanding the molecular requirements necessary for their formation. All three projects are relevant towards understanding the processes of cell growth, hormonal secretion, and neurotransmission that are found in higher eukaryotes, can yet be demonstrated in a simple and elegant yeast. using fashion Findings that are required docking for SV and fusion in yeast. Their mammalian counterparts, or synaptobrevins, known as VAMPs focused have we are required for the fusion of synaptic vesicles with the years, presynaptic membrane. Both are important Sncs and VAMPs recent In 1). because they assemble into a complex with partner t-SNAREs, (Fig. located on the plasma or presynaptic membranes, fusion to drive membrane pathways, transduction signal by SNAREs the of regulation upon as well SNARE-binding as by proteins. These types of regulation are important for determining where and when SNAREs can delivery the regulating thus, complex, fusion the into assemble cell surface. the to proteins lipids and of RoleSNARE of phosphorylation in vesicle fusion accumulation and a block in cellular of SVs secretion. We Galina Gabriely Gurunathan Sangiliyandi Michael Marash Peretz Oshrat Micha Robinson Salzberg Yehuda Adina Weinberger Jeffrey Gerst Gerst Jeffrey

]104 jef_gerst.id jef_gerst.id proteins that preventproteins that assembly. SNAREaffects assembly directly, but also the association of only not to Vsm1. phosphorylation SNARE that We conclude preferentially binds SNAREs, with toassemble unable is which fashion. Thus, Sso,phosphorylation-dependent phosphorylated a in but t-SNAREs, Sso Vsm1 tothe that have shown binds also Recently, we fusion. and docking ofvesicle aregulator as acts surface. cell the from toand transport retrograde and anterograde both confer v-SNAREs these Thus, partners. t-SNARE different vesicles and fusion with ofdocking endosomes, using endocytic in endocytic, as well as exocytic, events. They mediate the Role of exocytic v-SNAREs in endocytosis in v-SNAREs exocytic of Role exocytosis in regulators SNARE of Role processes. growth physical with cycle cell coordinate cascades signaling how aretounderstand efforts our Thus, (i.e., ER-Golgi). pathway secretory ofthe levels atall occurs phosphorylation SNARE that show studies Ongoing fusion. and SVin docking role play animportant dephosphorylation and phosphorylation proteins are exported from the cell via the default LDSV route. default the via cell the from are exported proteins HDSV sorting routes. Under such circumstances, vacuolar and secreted the and sorting protein vacuolar abolish transport Golgi-to-endosome confer that vesicles of fusion or biogenesis the either inhibit that Mutations pathway. sorting (PVC) Golgi-to-endosome intact an require route HDSV the via LDSVs secreted HDSVs. or Proteins byeither surface the to Golgi Fig. 2 Earlier, we identified a v-SNARE-binding protein (Vsm1) protein that av-SNARE-binding Earlier, weidentified We have recently shown that the Snc v-SNAREs participate A model for SV biogenesis. Secreted proteins are trafficked from the the from are trafficked proteins Secreted SV for biogenesis. Amodel 105 Marash, M. and Gerst, J.E. (2002) Phosphorylation-dependent Phosphorylation-dependent (2002) J.E. Gerst, and M. Marash, J.E. Gerst, and A., M., Weinberger, S., Marash, Gurunathan, and Dynamin (2002) J.E. D., Gerst, S., David, and Gurunathan, dephosphorylation t-SNARE (2001) J.E. Gerst, and M. Marash, D., Trajkovic, S., and S., Chapman-Shimshoni, Gurunathan, Yeast (1999) V. J.E. VSM1 Gerst, encodes and Lustgarten, in regulators SNARE and SNAREs (1999) J.E. Gerst, David, D., Sundarababu, S., and Gerst, J.E. (1998) Gerst, J.E. (1997) Conserved (1997) J.E. Conserved Gerst, Selected Publications biogenesis requires both clathrin and dynamin. HDSV that We show also 2). (Fig. direct not is and compartment intermediate an through transport requires HDSV biogenesis also block HDSV transport Golgi-to-endosome production. Thus, block that mutations that We show now membrane. plasma the with tofuse areunable vesicles the when cells in accumulate SVs (HDSVs) SVs high-density and (LDSVs) low-density Both content. and density both in ofSVs differ types that two Yeast produce SVs compartment. that another from may form suggests evidence and demonstrated directly been not has this vesicles of secretory Biogenesis Cancer Research. Cancer in Chair Kaplan Henry the J.E.G. holds Genetics. for Molecular Center Forchheimer the and (Germany), Foundation Minerva Acknowledgements The origin of SVs has been thought to be the Golgi. However, Golgi. the tobe ofSVs thought been origin has The Our work is supported by the Israel Science Foundation, Foundation, Science Israel by the supported is work Our (Submitted). partner. t-SNARE toits regulator ofaSNARE binding phosphatase. (Submitted). protein ofaceramide-activated byactivation ameliorated are mutants t-SNARE v-and yeast in defects Endocytic (2002) press). J., (in EMBO yeast. in vesicles secretory of class ofadistinct biogenesis the for required are clathrin 411-421.J. 20, EMBO yeast. in exocytosis and assembly SNARE promotes 11, Cell Biol. Mol. 3629-3643. endocytosis. confer v-SNAREs Yeast exocytic (2000) J.E. Gerst, 4480-4494. 19, Biol. Cell. Mol. exocytosis. ofconstitutive regulator anegative as may act that protein binding a v-SNARE 707-734. 55, Sci. Life Mol. Cell. exocytosis. and fusion membrane 1167-1182. 143, Biol. J.Cell yeast. in vesicles ofsecretory trafficking the in elongation acid fatty chain oflong Involvement 16591-16598. 272, Chem. J.Biol. function. exocytic mediate ofv-SNAREs family synaptobrevin/VAMP ofthe homologs α -helical segments on yeast yeast on segments -helical

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