Background Growth and division of Saccharomyces cerevisiae is dependent on the action of SNARE proteins that are required for membrane fusion. SNAREs are regulated, through a poorly understood mechanism, to ensure membrane fusion at the correct time and place within a cell. Although fusion of secretory vesicles with the plasma membrane is important for yeast cell growth, the relationship between exocytic SNAREs and cell physiology has not been established. Methodology/Principal Findings Using genetic analysis, we identified several influences on the function of exocytic SNAREs. Genetic disruption of the V-ATPase, but not vacuolar proteolysis, can suppress two different temperature-sensitive mutations in SEC9. Suppression is unlikely due to increased SNARE complex formation because increasing SNARE complex formation, through overexpression of SRO7, does not result in suppression. We also observed suppression of sec9 mutations by growth on alkaline media or on a non-fermentable carbon source, conditions associated with a reduced growth rate of wild-type cells and decreased SNARE complex formation. Conclusions/Significance Three main conclusions arise from our results. First, there is a genetic interaction between SEC9 and the V-ATPase, although it is unlikely that this interaction has functional significance with respect to membrane fusion or SNAREs. Second, Sro7p acts to promote SNARE complex formation. Finally, Sec9p function and SNARE complex formation are tightly coupled to the physiological state of the cell.
References
[1]
Novick P, Schekman R (1979) Secretion and cell-surface growth are blocked in a temperature-sensitive mutant of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A 76: 1858–1862.
[2]
Field C, Schekman R (1980) Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J Cell Biol 86: 123–128.
[3]
S？llner T, Whiteheart SW, Brunner M, Erdjument-Bromage H, Geromanos S, et al. (1993) SNAP receptors implicated in vesicle targeting and fusion. Nature 362: 318–324.
Gerst JE (1999) SNAREs and SNARE regulators in membrane fusion and exocytosis. Cell Mol Life Sci 55: 707–734.
[6]
Sutton RB, Fasshauer D, Jahn R, Brunger AT (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 ？ resolution. Nature 395: 347–353.
[7]
Novick P, Field C, Schekman R (1980) Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell 21: 205–215.
[8]
Brennwald P, Kearns B, Champion K, Ker？nen S, Bankaitis V, Novick P (1994) Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell 79: 245–258.
[9]
Rossi G, Salminen A, Rice LM, Brünger AT, Brennwald P (1997) Analysis of a yeast SNARE complex reveals remarkable similarity to the neuronal SNARE complex and a novel function for the C terminus of the SNAP-25 homolog, Sec9. J Biol Chem 272: 16610–16617.
[10]
Aalto MK, Ronne H, Ker？nen S (1993) Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. EMBO J 12: 4095–4104.
[11]
Protopopov V, Govindan B, Novick P, Gerst JE (1993) Homologs of the synaptobrevin/VAMP family of synaptic vesicle proteins function on the late secretory pathway in S. cerevisiae. Cell 74: 855–861.
[12]
Kane PM (2006) The where, when, and how of organelle acidification by the yeast vacuolar H+-ATPase. Microbiol Mol Biol Rev 70: 177–191.
[13]
Bayer MJ, Reese C, Buhler S, Peters C, Mayer A (2003) Vacuole membrane fusion: V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel. J Cell Biol 162: 211–222.
[14]
Peters C, Bayer MJ, Bühler S, Andersen JS, Mann M, Mayer A (2001) Trans-complex formation by proteolipid channels in the terminal phase of membrane fusion. Nature 409: 581–588.
[15]
Hiesinger PR, Fayyazuddin A, Mehta SQ, Rosenmund T, Schulze KL, et al. (2005) The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila. Cell 121: 607–620.
[16]
Liégeois S, Benedetto A, Garnier JM, Schwab Y, Labouesse M (2006) The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans. J Cell Biol 173: 949–961.
[17]
Peri F, Nüsslein-Volhard C (2008) Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133: 916–927.
[18]
Galli T, McPherson PS, De Camilli P (1996) The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze thawing sensitive complex. J Biol Chem 271: 2193.
[19]
Lehman K, Rossi G, Adamo JE, Brennwald P (1999) Yeast homologues of tomosyn and lethal giant larvae function in exocytosis and are associated with the plasma membrane SNARE, Sec9. J Cell Biol 146: 125–140.
[20]
Kagami M, Toh-e A, Matsui Y (1998) Sro7p, a Saccharomyces cerevisiae counterpart of the tumor suppressor l(2)gl protein, is related to myosins in function. Genetics 149: 1717–1727.
[21]
Matsui Y, Toh-E A (1992) Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1. Mol Cell Biol 12: 5690–5699.
[22]
Gangar A, Rossi G, Andreeva A, Hales R, Brennwald P (2005) Structurally conserved interaction of Lgl family with SNAREs is critical to their cellular function. Curr Biol 15: 1136–1142.
[23]
Hattendorf DA, Andreeva A, Gangar A, Brennwald PJ, Weis WI (2007) Structure of the yeast polarity protein Sro7 reveals a SNARE regulatory mechanism. Nature 446: 567–571.
[24]
Grosshans BL, Andreeva A, Gangar A, Niessen S, Yates JR, et al. (2006) The yeast lgl family member Sro7p is an effector of the secretory Rab GTPase Sec4p. J Cell Biol 172: 55–66.
[25]
Cheviet S, Bezzi P, Ivarsson R, Renstr？m E, Viertl D, et al. (2006) Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis. J Cell Sci 119: 2912–2920.
[26]
Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD, et al. (2006) Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans. PLoS Biol 4: e261.
[27]
Prelich G (1999) Suppression mechanisms: themes from variations. Trends Genet 15: 261–266.
[28]
Hirata R, Ohsumk Y, Nakano A, Kawasaki H, Suzuki K, Anraku Y (1990) Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae. J Biol Chem 265: 6726–6733.
[29]
Hirata R, Graham LA, Takatsuki A, Stevens TH, Anraku Y (1997) VMA11 and VMA16 encode second and third proteolipid subunits of the Saccharomyces cerevisiae vacuolar membrane H+-ATPase. J Biol Chem 272: 4795–4803.
[30]
Forgac M (2007) Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat Rev Mol Cell Biol 8: 917–929.
[31]
S？rensen SO, van den Hazel HB, Kielland-Brandt MC, Winther JR (1994) pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro. Eur J Biochem 220: 19–27.
[32]
Ammerer G, Hunter CP, Rothman JH, Saari GC, Valls LA, Stevens TH (1986) PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol 6: 2490–2499.
[33]
Nebes VL, Jones EW (1991) Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence. J Biol Chem 266: 22851–22857.
[34]
Zubenko GS, Park FJ, Jones EW (1983) Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc Natl Acad Sci U S A 80: 510–514.
[35]
Martinez-Munoz GA, Kane PM (2008) Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast. J Biol Chem 283: 20309–20319.
[36]
Munson M, Chen X, Cocina AE, Schultz SM, Hughson FM (2000) Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly. Nat Struct Biol 7: 894–902.
[37]
Munson M, Hughson FM (2002) Conformational regulation of SNARE assembly and disassembly in vivo. J Biol Chem 277: 9375–9381.
[38]
Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153: 163–168.
[39]
Sherman F (2002) Getting started with yeast. Methods Enzymol 350: 3–41.
[40]
Longtine MS, McKenzie A, Demarini DJ, Shah NG, Wach A, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
[41]
Carr CM, Grote E, Munson M, Hughson FM, Novick PJ (1999) Sec1p binds to SNARE complexes and concentrates at sites of secretion. J Cell Biol 146: 333–344.
[42]
Grote E, Novick PJ (1999) Promiscuity in Rab-SNARE interactions. Mol Biol Cell 10: 4149–4161.
