Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface – ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY). Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes – MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.
Luzio JP, Bright NA, Pryor PR (2007) The role of calcium and other ions in sorting and delivery in the late endocytic pathway. Biochem Soc Trans 35: 1088–1091.
[4]
Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793: 650–663.
[5]
Mullins C, Bonifacino JS (2001) The molecular machinery for lysosome biogenesis. Bioessays 23: 333–343.
[6]
Luzio JP, Poupon V, Lindsay MR, Mullock BM, Piper RC, et al. (2003) Membrane dynamics and the biogenesis of lysosomes. Mol Membr Biol 20: 141–154.
[7]
Nothwehr SF, Stevens TH (1994) Sorting of membrane proteins in the yeast secretory pathway. J Biol Chem 269: 10185–10188.
[8]
Horazdovsky BF, DeWald DB, Emr SD (1995) Protein transport to the yeast vacuole. Curr Opin Cell Biol 7: 544–551.
[9]
Bryant NJ, Piper RC, Weisman LS, Stevens TH (1998) Retrograde traffic out of the yeast vacuole to the TGN occurs via the prevacuolar/endosomal compartment. J Cell Biol 142: 651–663.
[10]
Conibear E, Stevens TH (1998) Multiple sorting pathways between the late Golgi and the vacuole in yeast. Biochim Biophys Acta 1404: 211–230.
[11]
Bowers K, Stevens TH (2005) Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1744: 438–454.
[12]
Piper RC, Katzmann DJ (2007) Biogenesis and function of multivesicular bodies. Annu Rev Cell Dev Biol 23: 519–547.
[13]
Levine B (2005) Eating oneself and uninvited guests: autophagy-related pathways in cellular defense. Cell 120: 159–162.
[14]
Huang J, Klionsky DJ (2007) Autophagy and human disease. Cell Cycle 6: 1837–1849.
[15]
Mijaljica D, Prescott M, Klionsky DJ, Devenish RJ (2007) Autophagy and vacuole homeostasis: a case for self-degradation? Autophagy 3: 417–421.
[16]
Simonsen A, Tooze SA (2009) Coordination of membrane events during autophagy by multiple class III PI3-kinase complexes. J Cell Biol 186: 773–782.
[17]
Ostrowicz CW, Meiringer CT, Ungermann C (2008) Yeast vacuole fusion: a model system for eukaryotic endomembrane dynamics. Autophagy 4: 5–19.
[18]
Wickner W (2010) Membrane fusion: five lipids, four SNAREs, three chaperones, two nucleotides, and a Rab, all dancing in a ring on yeast vacuoles. Annu Rev Cell Dev Biol 26: 115–136.
[19]
Mellman I (1996) Endocytosis and molecular sorting. Annu Rev Cell Dev Biol 12: 575–625.
[20]
Futter CE, Pearse A, Hewlett LJ, Hopkins CR (1996) Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes. J Cell Biol 132: 1011–1023.
[21]
Bright NA, Reaves BJ, Mullock BM, Luzio JP (1997) Dense core lysosomes can fuse with late endosomes and are re-formed from the resultant hybrid organelles. J Cell Sci 110(Pt 17): 2027–2040.
[22]
Wickner W (2002) Yeast vacuoles and membrane fusion pathways. Embo J 21: 1241–1247.
[23]
Luzio JP, Pryor PR, Gray SR, Gratian MJ, Piper RC, et al. (2005) Membrane traffic to and from lysosomes. Biochem Soc Symp 77–86.
[24]
Fratti RA, Wickner W (2007) Distinct targeting and fusion functions of the PX and SNARE domains of yeast vacuolar Vam7p. J Biol Chem 282: 13133–13138.
[25]
Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD (1994) The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77: 579–586.
[26]
Jones EW (1977) Proteinase mutants of Saccharomyces cerevisiae. Genetics 85: 23–33.
[27]
Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A 83: 9075–9079.
[28]
Rothman JH, Stevens TH (1986) Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell 47: 1041–1051.
[29]
Wada Y, Anraku Y (1992) Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. II. VAM7, a gene for regulating morphogenic assembly of the vacuoles. J Biol Chem 267: 18671–18675.
[30]
Bonangelino CJ, Chavez EM, Bonifacino JS (2002) Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13: 2486–2501.
[31]
Takahashi MK, Frost C, Oyadomari K, Pinho M, Sao D, et al. (2008) A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae. Mol Cell Biochem 311: 121–136.
[32]
Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH (1992) Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell 3: 1389–1402.
[33]
Ammerer G, Hunter CP, Rothman JH, Saari GC, Valls LA, et al. (1986) PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol 6: 2490–2499.
[34]
Banuelos MG, Moreno DE, Olson DK, Nguyen Q, Ricarte F, et al. (2010) Genomic analysis of severe hypersensitivity to hygromycin B reveals linkage to vacuolar defects and new vacuolar gene functions in Saccharomyces cerevisiae. Curr Genet 56: 121–137.
[35]
Schluter C, Lam KK, Brumm J, Wu BW, Saunders M, et al. (2008) Global analysis of yeast endosomal transport identifies the vps55/68 sorting complex. Mol Biol Cell 19: 1282–1294.
[36]
Entian KD, Schuster T, Hegemann JH, Becher D, Feldmann H, et al. (1999) Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach. Mol Gen Genet 262: 683–702.
[37]
Yang M, Jensen LT, Gardner AJ, Culotta VC (2005) Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family. Biochem J 386: 479–487.
[38]
Sesaki H, Southard SM, Hobbs AE, Jensen RE (2003) Cells lacking Pcp1p/Ugo2p, a rhomboid-like protease required for Mgm1p processing, lose mtDNA and mitochondrial structure in a Dnm1p-dependent manner, but remain competent for mitochondrial fusion. Biochem Biophys Res Commun 308: 276–283.
[39]
Herlan M, Vogel F, Bornhovd C, Neupert W, Reichert AS (2003) Processing of Mgm1 by the rhomboid-type protease Pcp1 is required for maintenance of mitochondrial morphology and of mitochondrial DNA. J Biol Chem 278: 27781–27788.
[40]
Gan X, Kitakawa M, Yoshino K, Oshiro N, Yonezawa K, et al. (2002) Tag-mediated isolation of yeast mitochondrial ribosome and mass spectrometric identification of its new components. Eur J Biochem 269: 5203–5214.
[41]
Wu J, Tolstykh T, Lee J, Boyd K, Stock JB, et al. (2000) Carboxyl methylation of the phosphoprotein phosphatase 2A catalytic subunit promotes its functional association with regulatory subunits in vivo. Embo J 19: 5672–5681.
[42]
Santos B, Snyder M (2000) Sbe2p and sbe22p, two homologous Golgi proteins involved in yeast cell wall formation. Mol Biol Cell 11: 435–452.
[43]
Pathak R, Bogomolnaya LM, Guo J, Polymenis M (2004) Gid8p (Dcr1p) and Dcr2p function in a common pathway to promote START completion in Saccharomyces cerevisiae. Eukaryot Cell 3: 1627–1638.
[44]
Guo J, Polymenis M (2006) Dcr2 targets Ire1 and downregulates the unfolded protein response in Saccharomyces cerevisiae. EMBO Rep 7: 1124–1127.
[45]
Jonikas MC, Collins SR, Denic V, Oh E, Quan EM, et al. (2009) Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum. Science 323: 1693–1697.
[46]
Chang YW, Chuang YC, Ho YC, Cheng MY, Sun YJ, et al. (2010) Crystal structure of Get4-Get5 complex and its interactions with Sgt2, Get3, and Ydj1. J Biol Chem 285: 9962–9970.
[47]
Planta RJ, Mager WH (1998) The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast 14: 471–477.
[48]
Lecompte O, Ripp R, Thierry JC, Moras D, Poch O (2002) Comparative analysis of ribosomal proteins in complete genomes: an example of reductive evolution at the domain scale. Nucleic Acids Res 30: 5382–5390.
[49]
Westfall PJ, Ballon DR, Thorner J (2004) When the stress of your environment makes you go HOG wild. Science 306: 1511–1512.
[50]
Brewster JL, de Valoir T, Dwyer ND, Winter E, Gustin MC (1993) An osmosensing signal transduction pathway in yeast. Science 259: 1760–1763.
[51]
Han J, Lee JD, Bibbs L, Ulevitch RJ (1994) A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265: 808–811.
[52]
Raingeaud J, Gupta S, Rogers JS, Dickens M, Han J, et al. (1995) Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 270: 7420–7426.
[53]
Broach JR (1991) Ras-regulated signaling processes in Saccharomyces cerevisiae. Curr Opin Genet Dev 1: 370–377.
[54]
Julius D, Schekman R, Thorner J (1984) Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell 36: 309–318.
[55]
Fuller RS, Sterne RE, Thorner J (1988) Enzymes required for yeast prohormone processing. Annu Rev Physiol 50: 345–362.
[56]
Graham TR, Emr SD (1991) Compartmental organization of Golgi-specific protein modification and vacuolar protein sorting events defined in a yeast sec18 (NSF) mutant. J Cell Biol 114: 207–218.
[57]
Roberts CJ, Nothwehr SF, Stevens TH (1992) Membrane protein sorting in the yeast secretory pathway: evidence that the vacuole may be the default compartment. J Cell Biol 119: 69–83.
[58]
Gharakhanian E, Chima-Okereke O, Olson DK, Frost C, Kathleen Takahashi M (2011) env1 Mutant of VPS35 gene exhibits unique protein localization and processing phenotype at Golgi and lysosomal vacuole in Saccharomyces cerevisiae. Mol Cell Biochem 346: 187–195.
[59]
Vida TA, Emr SD (1995) A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeast. J Cell Biol 128: 779–792.
[60]
Sturgill TW, Cohen A, Diefenbacher M, Trautwein M, Martin DE, et al. (2008) TOR1 and TOR2 have distinct locations in live cells. Eukaryot Cell 7: 1819–1830.
[61]
Gustavsson M, Barmark G, Larsson J, Muren E, Ronne H (2008) Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function. Mol Genet Genomics 280: 233–248.
[62]
Huh WK, Falvo JV, Gerke LC, Carroll AS, Howson RW, et al. (2003) Global analysis of protein localization in budding yeast. Nature 425: 686–691.
[63]
Stairs DB, Perry Gardner H, Ha SI, Copeland NG, Gilbert DJ, et al. (1998) Cloning and characterization of Krct, a member of a novel subfamily of serine/threonine kinases. Hum Mol Genet 7: 2157–2166.
[64]
Ligos JM, Gerwin N, Fernandez P, Gutierrez-Ramos JC, Bernad A (1998) Cloning, expression analysis, and functional characterization of PKL12, a member of a new subfamily of ser/thr kinases. Biochem Biophys Res Commun 249: 380–384.
[65]
Berson AE, Young C, Morrison SL, Fujii GH, Sheung J, et al. (1999) Identification and characterization of a myristylated and palmitylated serine/threonine protein kinase. Biochem Biophys Res Commun 259: 533–538.
[66]
Belyaeva OV, Korkina OV, Stetsenko AV, Kim T, Nelson PS, et al. (2005) Biochemical properties of purified human retinol dehydrogenase 12 (RDH12): catalytic efficiency toward retinoids and C9 aldehydes and effects of cellular retinol-binding protein type I (CRBPI) and cellular retinaldehyde-binding protein (CRALBP) on the oxidation and reduction of retinoids. Biochemistry 44: 7035–7047.
[67]
Lussier M, White AM, Sheraton J, di Paolo T, Treadwell J, et al. (1997) Large scale identification of genes involved in cell surface biosynthesis and architecture in Saccharomyces cerevisiae. Genetics 147: 435–450.
[68]
Chen S, Tarsio M, Kane PM, Greenberg ML (2008) Cardiolipin mediates cross-talk between mitochondria and the vacuole. Mol Biol Cell 19: 5047–5058.
[69]
Hailey DW, Rambold AS, Satpute-Krishnan P, Mitra K, Sougrat R, et al. (2010) Mitochondria supply membranes for autophagosome biogenesis during starvation. Cell 141: 656–667.
[70]
Wang L, Seeley ES, Wickner W, Merz AJ (2002) Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle. Cell 108: 357–369.
[71]
Costanzo M, Baryshnikova A, Bellay J, Kim Y, Spear ED, et al. (2010) The genetic landscape of a cell. Science 327: 425–431.
[72]
Liu XF, Supek F, Nelson N, Culotta VC (1997) Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene. J Biol Chem 272: 11763–11769.
[73]
Sullivan JA, Lewis MJ, Nikko E, Pelham HR (2007) Multiple interactions drive adaptor-mediated recruitment of the ubiquitin ligase rsp5 to membrane proteins in vivo and in vitro. Mol Biol Cell 18: 2429–2440.
[74]
Jensen LT, Carroll MC, Hall MD, Harvey CJ, Beese SE, et al. (2009) Down-regulation of a manganese transporter in the face of metal toxicity. Mol Biol Cell 20: 2810–2819.
[75]
Soulard A, Cohen A, Hall MN (2009) TOR signaling in invertebrates. Curr Opin Cell Biol 21: 825–836.
[76]
Stanfel MN, Shamieh LS, Kaeberlein M, Kennedy BK (2009) The TOR pathway comes of age. Biochim Biophys Acta 1790: 1067–1074.
[77]
Kapahi P, Chen D, Rogers AN, Katewa SD, Li PW, et al. (2010) With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging. Cell Metab 11: 453–465.
[78]
Neufeld TP, Arsham AM (2010) tRNA trafficking along the TOR pathway. Cell Cycle 9: 3146–3147.
[79]
Hall MN, Tamanoi F (2010) The Enzymes: Structure, Function and Regulation of TOR Complexes from Yeasts to Mammals. Maryland Heights, MO: Academic Press.
[80]
Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, et al. (2008) The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 320: 1496–1501.
[81]
Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, et al. (2010) Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141: 290–303.
[82]
Flinn RJ, Backer JM (2010) mTORC1 signals from late endosomes: taking a TOR of the endocytic system. Cell Cycle 9: 1869–1870.
[83]
Sherman F (2002) Getting started with yeast. Methods Enzymol 350: 3–41.
[84]
Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793–1808.
[85]
Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122: 19–27.
[86]
Johnson LM, Bankaitis VA, Emr SD (1987) Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell 48: 875–885.
[87]
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76: 4350–4354.
