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. 1997 Oct;147(2):467–478. doi: 10.1093/genetics/147.2.467

Genetic Interactions between a Pep7 Mutation and the Pep12 and Vps45 Genes: Evidence for a Novel Snare Component in Transport between the Saccharomyces Cerevisiae Golgi Complex and Endosome

G C Webb 1, M Hoedt 1, L J Poole 1, E W Jones 1
PMCID: PMC1208171  PMID: 9335586

Abstract

The PEP7 gene from Saccharomyces cerevisiae encodes a 59-kD hydrophilic polypeptide that is required for transport of soluble vacuolar hydrolase precursors from the TGN to the endosome. This study presents the results of a high-copy suppression analysis of pep7-20 mutant phenotypes. This analysis demonstrated that both VPS45 and PEP12 are allele-specific high-copy suppressors of pep7-20 mutant phenotypes. Overexpression of VPS45 was able to completely suppress the Zn(2+) sensitivity and partially suppress the carboxypeptidase Y deficiency. Overexpression of PEP12 was able to do the same, but to a lesser extent. Vps45p and Pep12p are Sec1p and syntaxin (t-SNARE) homologues, respectively, and are also thought to function in transport between the TGN and endosome. Two additional vacuole pathway SNARE complex homologues, Vps33p (Sec1p) and Pth1p (syntaxin), when overexpressed, were unable to suppress pep7-20 or any other pep7 allele, further supporting the specificity of the interactions of pep7-20 with PEP12 and VPS45. Because several other vesicle docking/fusion reactions take place in the cell without discernible participation of Pep7p homologues, we suggest that Pep7p is a step-specific regulator of docking and/or fusion of TGN-derived transport vesicles onto the endosome.

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Selected References

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  1. Aalto M. K., Ronne H., Keränen S. Yeast syntaxins Sso1p and Sso2p belong to a family of related membrane proteins that function in vesicular transport. EMBO J. 1993 Nov;12(11):4095–4104. doi: 10.1002/j.1460-2075.1993.tb06093.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banfield D. K., Lewis M. J., Rabouille C., Warren G., Pelham H. R. Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains. J Cell Biol. 1994 Oct;127(2):357–371. doi: 10.1083/jcb.127.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bankaitis V. A., Johnson L. M., Emr S. D. Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9075–9079. doi: 10.1073/pnas.83.23.9075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banta L. M., Vida T. A., Herman P. K., Emr S. D. Characterization of yeast Vps33p, a protein required for vacuolar protein sorting and vacuole biogenesis. Mol Cell Biol. 1990 Sep;10(9):4638–4649. doi: 10.1128/mcb.10.9.4638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Becherer K. A., Rieder S. E., Emr S. D., Jones E. W. Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. Mol Biol Cell. 1996 Apr;7(4):579–594. doi: 10.1091/mbc.7.4.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brennwald P., Kearns B., Champion K., Keränen S., Bankaitis V., Novick P. Sec9 is a SNAP-25-like component of a yeast SNARE complex that may be the effector of Sec4 function in exocytosis. Cell. 1994 Oct 21;79(2):245–258. doi: 10.1016/0092-8674(94)90194-5. [DOI] [PubMed] [Google Scholar]
  7. Burd C. G., Peterson M., Cowles C. R., Emr S. D. A novel Sec18p/NSF-dependent complex required for Golgi-to-endosome transport in yeast. Mol Biol Cell. 1997 Jun;8(6):1089–1104. doi: 10.1091/mbc.8.6.1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  9. Clary D. O., Griff I. C., Rothman J. E. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell. 1990 May 18;61(4):709–721. doi: 10.1016/0092-8674(90)90482-t. [DOI] [PubMed] [Google Scholar]
  10. Cowles C. R., Emr S. D., Horazdovsky B. F. Mutations in the VPS45 gene, a SEC1 homologue, result in vacuolar protein sorting defects and accumulation of membrane vesicles. J Cell Sci. 1994 Dec;107(Pt 12):3449–3459. doi: 10.1242/jcs.107.12.3449. [DOI] [PubMed] [Google Scholar]
  11. Eakle K. A., Bernstein M., Emr S. D. Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product. Mol Cell Biol. 1988 Oct;8(10):4098–4109. doi: 10.1128/mcb.8.10.4098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ferro-Novick S., Jahn R. Vesicle fusion from yeast to man. Nature. 1994 Jul 21;370(6486):191–193. doi: 10.1038/370191a0. [DOI] [PubMed] [Google Scholar]
  13. Garcia E. P., Gatti E., Butler M., Burton J., De Camilli P. A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2003–2007. doi: 10.1073/pnas.91.6.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Griff I. C., Schekman R., Rothman J. E., Kaiser C. A. The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein. J Biol Chem. 1992 Jun 15;267(17):12106–12115. [PubMed] [Google Scholar]
  15. Hardwick K. G., Pelham H. R. SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex. J Cell Biol. 1992 Nov;119(3):513–521. doi: 10.1083/jcb.119.3.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hill J., Donald K. A., Griffiths D. E., Donald G. DMSO-enhanced whole cell yeast transformation. Nucleic Acids Res. 1991 Oct 25;19(20):5791–5791. doi: 10.1093/nar/19.20.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  18. Horazdovsky B. F., Busch G. R., Emr S. D. VPS21 encodes a rab5-like GTP binding protein that is required for the sorting of yeast vacuolar proteins. EMBO J. 1994 Mar 15;13(6):1297–1309. doi: 10.1002/j.1460-2075.1994.tb06382.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jacquemin-Faure I., Thomas D., Laporte J., Cibert C., Surdin-Kerjan Y. The vacuolar compartment is required for sulfur amino acid homeostasis in Saccharomyces cerevisiae. Mol Gen Genet. 1994 Sep 1;244(5):519–529. doi: 10.1007/BF00583903. [DOI] [PubMed] [Google Scholar]
  20. Jarvik J., Botstein D. Conditional-lethal mutations that suppress genetic defects in morphogenesis by altering structural proteins. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2738–2742. doi: 10.1073/pnas.72.7.2738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Jones E. W. Proteinase mutants of Saccharomyces cerevisiae. Genetics. 1977 Jan;85(1):23–33. doi: 10.1093/genetics/85.1.23. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones E. W. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:428–453. doi: 10.1016/0076-6879(91)94034-a. [DOI] [PubMed] [Google Scholar]
  23. Jäntti J., Keränen S., Toikkanen J., Kuismanen E., Ehnholm C., Söderlund H., Olkkonen V. M. Membrane insertion and intracellular transport of yeast syntaxin Sso2p in mammalian cells. J Cell Sci. 1994 Dec;107(Pt 12):3623–3633. doi: 10.1242/jcs.107.12.3623. [DOI] [PubMed] [Google Scholar]
  24. Kabcenell A. K., Goud B., Northup J. K., Novick P. J. Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic. J Biol Chem. 1990 Jun 5;265(16):9366–9372. [PubMed] [Google Scholar]
  25. Kacser H., Burns J. A. The molecular basis of dominance. Genetics. 1981 Mar-Apr;97(3-4):639–666. doi: 10.1093/genetics/97.3-4.639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Katagiri H., Terasaki J., Murata T., Ishihara H., Ogihara T., Inukai K., Fukushima Y., Anai M., Kikuchi M., Miyazaki J. A novel isoform of syntaxin-binding protein homologous to yeast Sec1 expressed ubiquitously in mammalian cells. J Biol Chem. 1995 Mar 10;270(10):4963–4966. doi: 10.1074/jbc.270.10.4963. [DOI] [PubMed] [Google Scholar]
  27. Kitamoto K., Yoshizawa K., Ohsumi Y., Anraku Y. Mutants of Saccharomyces cerevisiae with defective vacuolar function. J Bacteriol. 1988 Jun;170(6):2687–2691. doi: 10.1128/jb.170.6.2687-2691.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lian J. P., Ferro-Novick S. Bos1p, an integral membrane protein of the endoplasmic reticulum to Golgi transport vesicles, is required for their fusion competence. Cell. 1993 May 21;73(4):735–745. doi: 10.1016/0092-8674(93)90253-m. [DOI] [PubMed] [Google Scholar]
  29. Mu F. T., Callaghan J. M., Steele-Mortimer O., Stenmark H., Parton R. G., Campbell P. L., McCluskey J., Yeo J. P., Tock E. P., Toh B. H. EEA1, an early endosome-associated protein. EEA1 is a conserved alpha-helical peripheral membrane protein flanked by cysteine "fingers" and contains a calmodulin-binding IQ motif. J Biol Chem. 1995 Jun 2;270(22):13503–13511. doi: 10.1074/jbc.270.22.13503. [DOI] [PubMed] [Google Scholar]
  30. Nasmyth K. A., Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. doi: 10.1016/s0092-8674(80)80051-1. [DOI] [PubMed] [Google Scholar]
  31. Newman A. P., Shim J., Ferro-Novick S. BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol. 1990 Jul;10(7):3405–3414. doi: 10.1128/mcb.10.7.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Novick P., Ferro S., Schekman R. Order of events in the yeast secretory pathway. Cell. 1981 Aug;25(2):461–469. doi: 10.1016/0092-8674(81)90064-7. [DOI] [PubMed] [Google Scholar]
  33. Ohya Y., Ohsumi Y., Anraku Y. Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae. J Gen Microbiol. 1986 Apr;132(4):979–988. doi: 10.1099/00221287-132-4-979. [DOI] [PubMed] [Google Scholar]
  34. Ossig R., Dascher C., Trepte H. H., Schmitt H. D., Gallwitz D. The yeast SLY gene products, suppressors of defects in the essential GTP-binding Ypt1 protein, may act in endoplasmic reticulum-to-Golgi transport. Mol Cell Biol. 1991 Jun;11(6):2980–2993. doi: 10.1128/mcb.11.6.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Piper R. C., Cooper A. A., Yang H., Stevens T. H. VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J Cell Biol. 1995 Nov;131(3):603–617. doi: 10.1083/jcb.131.3.603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Piper R. C., Whitters E. A., Stevens T. H. Yeast Vps45p is a Sec1p-like protein required for the consumption of vacuole-targeted, post-Golgi transport vesicles. Eur J Cell Biol. 1994 Dec;65(2):305–318. [PubMed] [Google Scholar]
  37. Raymond C. K., Howald-Stevenson I., Vater C. A., Stevens T. H. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992 Dec;3(12):1389–1402. doi: 10.1091/mbc.3.12.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rieder S. E., Banta L. M., Köhrer K., McCaffery J. M., Emr S. D. Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Mol Biol Cell. 1996 Jun;7(6):985–999. doi: 10.1091/mbc.7.6.985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Rine J. Gene overexpression in studies of Saccharomyces cerevisiae. Methods Enzymol. 1991;194:239–251. doi: 10.1016/0076-6879(91)94019-9. [DOI] [PubMed] [Google Scholar]
  40. Robinson J. S., Klionsky D. J., Banta L. M., Emr S. D. Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol. 1988 Nov;8(11):4936–4948. doi: 10.1128/mcb.8.11.4936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Rothman J. E. Mechanisms of intracellular protein transport. Nature. 1994 Nov 3;372(6501):55–63. doi: 10.1038/372055a0. [DOI] [PubMed] [Google Scholar]
  42. Rothman J. E. The protein machinery of vesicle budding and fusion. Protein Sci. 1996 Feb;5(2):185–194. doi: 10.1002/pro.5560050201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Rothman J. E., Wieland F. T. Protein sorting by transport vesicles. Science. 1996 Apr 12;272(5259):227–234. doi: 10.1126/science.272.5259.227. [DOI] [PubMed] [Google Scholar]
  44. Rothman J. H., Howald I., Stevens T. H. Characterization of genes required for protein sorting and vacuolar function in the yeast Saccharomyces cerevisiae. EMBO J. 1989 Jul;8(7):2057–2065. doi: 10.1002/j.1460-2075.1989.tb03614.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rothman J. H., Stevens T. H. Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway. Cell. 1986 Dec 26;47(6):1041–1051. doi: 10.1016/0092-8674(86)90819-6. [DOI] [PubMed] [Google Scholar]
  46. Schimmöller F., Riezman H. Involvement of Ypt7p, a small GTPase, in traffic from late endosome to the vacuole in yeast. J Cell Sci. 1993 Nov;106(Pt 3):823–830. doi: 10.1242/jcs.106.3.823. [DOI] [PubMed] [Google Scholar]
  47. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  48. Shim J., Newman A. P., Ferro-Novick S. The BOS1 gene encodes an essential 27-kD putative membrane protein that is required for vesicular transport from the ER to the Golgi complex in yeast. J Cell Biol. 1991 Apr;113(1):55–64. doi: 10.1083/jcb.113.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Singer-Krüger B., Frank R., Crausaz F., Riezman H. Partial purification and characterization of early and late endosomes from yeast. Identification of four novel proteins. J Biol Chem. 1993 Jul 5;268(19):14376–14386. [PubMed] [Google Scholar]
  50. Singer-Krüger B., Stenmark H., Düsterhöft A., Philippsen P., Yoo J. S., Gallwitz D., Zerial M. Role of three rab5-like GTPases, Ypt51p, Ypt52p, and Ypt53p, in the endocytic and vacuolar protein sorting pathways of yeast. J Cell Biol. 1994 Apr;125(2):283–298. doi: 10.1083/jcb.125.2.283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Singer B., Riezman H. Detection of an intermediate compartment involved in transport of alpha-factor from the plasma membrane to the vacuole in yeast. J Cell Biol. 1990 Jun;110(6):1911–1922. doi: 10.1083/jcb.110.6.1911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Stevens T., Esmon B., Schekman R. Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell. 1982 Sep;30(2):439–448. doi: 10.1016/0092-8674(82)90241-0. [DOI] [PubMed] [Google Scholar]
  53. Söllner T., Whiteheart S. W., Brunner M., Erdjument-Bromage H., Geromanos S., Tempst P., Rothman J. E. SNAP receptors implicated in vesicle targeting and fusion. Nature. 1993 Mar 25;362(6418):318–324. doi: 10.1038/362318a0. [DOI] [PubMed] [Google Scholar]
  54. Søgaard M., Tani K., Ye R. R., Geromanos S., Tempst P., Kirchhausen T., Rothman J. E., Söllner T. A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles. Cell. 1994 Sep 23;78(6):937–948. doi: 10.1016/0092-8674(94)90270-4. [DOI] [PubMed] [Google Scholar]
  55. Südhof T. C. The synaptic vesicle cycle: a cascade of protein-protein interactions. Nature. 1995 Jun 22;375(6533):645–653. doi: 10.1038/375645a0. [DOI] [PubMed] [Google Scholar]
  56. Vida T. A., Huyer G., Emr S. D. Yeast vacuolar proenzymes are sorted in the late Golgi complex and transported to the vacuole via a prevacuolar endosome-like compartment. J Cell Biol. 1993 Jun;121(6):1245–1256. doi: 10.1083/jcb.121.6.1245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Wada Y., Kitamoto K., Kanbe T., Tanaka K., Anraku Y. The SLP1 gene of Saccharomyces cerevisiae is essential for vacuolar morphogenesis and function. Mol Cell Biol. 1990 May;10(5):2214–2223. doi: 10.1128/mcb.10.5.2214. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Webb G. C., Zhang J., Garlow S. J., Wesp A., Riezman H., Jones E. W. Pep7p provides a novel protein that functions in vesicle-mediated transport between the yeast Golgi and endosome. Mol Biol Cell. 1997 May;8(5):871–895. doi: 10.1091/mbc.8.5.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Weisman L. S., Wickner W. Molecular characterization of VAC1, a gene required for vacuole inheritance and vacuole protein sorting. J Biol Chem. 1992 Jan 5;267(1):618–623. [PubMed] [Google Scholar]
  60. Wichmann H., Hengst L., Gallwitz D. Endocytosis in yeast: evidence for the involvement of a small GTP-binding protein (Ypt7p). Cell. 1992 Dec 24;71(7):1131–1142. doi: 10.1016/s0092-8674(05)80062-5. [DOI] [PubMed] [Google Scholar]
  61. Wilkie A. O. The molecular basis of genetic dominance. J Med Genet. 1994 Feb;31(2):89–98. doi: 10.1136/jmg.31.2.89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wilson D. W., Wilcox C. A., Flynn G. C., Chen E., Kuang W. J., Henzel W. J., Block M. R., Ullrich A., Rothman J. E. A fusion protein required for vesicle-mediated transport in both mammalian cells and yeast. Nature. 1989 Jun 1;339(6223):355–359. doi: 10.1038/339355a0. [DOI] [PubMed] [Google Scholar]
  63. Woolford C. A., Dixon C. K., Manolson M. F., Wright R., Jones E. W. Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae. Genetics. 1990 Aug;125(4):739–752. doi: 10.1093/genetics/125.4.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yamamoto A., DeWald D. B., Boronenkov I. V., Anderson R. A., Emr S. D., Koshland D. Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast. Mol Biol Cell. 1995 May;6(5):525–539. doi: 10.1091/mbc.6.5.525. [DOI] [PMC free article] [PubMed] [Google Scholar]

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