Skip to main content
Genetics logoLink to Genetics
. 2000 Sep;156(1):105–122. doi: 10.1093/genetics/156.1.105

Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae.

A Srivastava 1, C A Woolford 1, E W Jones 1
PMCID: PMC1461249  PMID: 10978279

Abstract

Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional mutants. Characterization of mutants revealed that pep3(ts) mutants are defective in the endosomal and nonendosomal Golgi to vacuole transport pathways, in the cytoplasm to vacuole targeting pathway, in recycling from the endosome back to the late Golgi, and in endocytosis. PEP3 interacts genetically with two members of the endosomal SNARE complex, PEP12 (t-SNARE) and PEP7 (homologue of mammalian EEA1); Pep3p and Pep5p associate physically with Pep7p as revealed by two-hybrid analysis. Our results suggest that a core Pep3p/Pep5p complex promotes vesicular docking/fusion reactions in conjunction with SNARE proteins at multiple steps in transport routes to the vacuole. We propose that this complex may be responsible for tethering transport vesicles on target membranes.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. 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]
  2. Bennett M. K., Scheller R. H. The molecular machinery for secretion is conserved from yeast to neurons. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):2559–2563. doi: 10.1073/pnas.90.7.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burd C. G., Babst M., Emr S. D. Novel pathways, membrane coats and PI kinase regulation in yeast lysosomal trafficking. Semin Cell Dev Biol. 1998 Oct;9(5):527–533. doi: 10.1006/scdb.1998.0255. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Cao X., Ballew N., Barlowe C. Initial docking of ER-derived vesicles requires Uso1p and Ypt1p but is independent of SNARE proteins. EMBO J. 1998 Apr 15;17(8):2156–2165. doi: 10.1093/emboj/17.8.2156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen Y. J., Stevens T. H. The VPS8 gene is required for localization and trafficking of the CPY sorting receptor in Saccharomyces cerevisiae. Eur J Cell Biol. 1996 Aug;70(4):289–297. [PubMed] [Google Scholar]
  7. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. Darsow T., Rieder S. E., Emr S. D. A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J Cell Biol. 1997 Aug 11;138(3):517–529. doi: 10.1083/jcb.138.3.517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Fuller R. S., Sterne R. E., Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol. 1988;50:345–362. doi: 10.1146/annurev.ph.50.030188.002021. [DOI] [PubMed] [Google Scholar]
  13. Grenson M. Inactivation-reactivation process and repression of permease formation regulate several ammonia-sensitive permeases in the yeast Saccharomyces cerevisiae. Eur J Biochem. 1983 Jun 1;133(1):135–139. doi: 10.1111/j.1432-1033.1983.tb07438.x. [DOI] [PubMed] [Google Scholar]
  14. Götte M., Gallwitz D. High expression of the yeast syntaxin-related Vam3 protein suppresses the protein transport defects of a pep12 null mutant. FEBS Lett. 1997 Jul 7;411(1):48–52. doi: 10.1016/s0014-5793(97)00575-9. [DOI] [PubMed] [Google Scholar]
  15. Götte M., von Mollard G. F. A new beat for the SNARE drum. Trends Cell Biol. 1998 Jun;8(6):215–218. doi: 10.1016/s0962-8924(98)01272-0. [DOI] [PubMed] [Google Scholar]
  16. Hawthorne D C, Mortimer R K. Chromosome Mapping in Saccharomyces: Centromere-Linked Genes. Genetics. 1960 Aug;45(8):1085–1110. doi: 10.1093/genetics/45.8.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hein C., André B. A C-terminal di-leucine motif and nearby sequences are required for NH4(+)-induced inactivation and degradation of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae. Mol Microbiol. 1997 May;24(3):607–616. doi: 10.1046/j.1365-2958.1997.3771735.x. [DOI] [PubMed] [Google Scholar]
  18. Hicke L., Zanolari B., Pypaert M., Rohrer J., Riezman H. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein. Mol Biol Cell. 1997 Jan;8(1):13–31. doi: 10.1091/mbc.8.1.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Horazdovsky B. F., Cowles C. R., Mustol P., Holmes M., Emr S. D. A novel RING finger protein, Vps8p, functionally interacts with the small GTPase, Vps21p, to facilitate soluble vacuolar protein localization. J Biol Chem. 1996 Dec 27;271(52):33607–33615. doi: 10.1074/jbc.271.52.33607. [DOI] [PubMed] [Google Scholar]
  20. Horazdovsky B. F., Emr S. D. The VPS16 gene product associates with a sedimentable protein complex and is essential for vacuolar protein sorting in yeast. J Biol Chem. 1993 Mar 5;268(7):4953–4962. [PubMed] [Google Scholar]
  21. James P., Halladay J., Craig E. A. Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics. 1996 Dec;144(4):1425–1436. doi: 10.1093/genetics/144.4.1425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jauniaux J. C., Grenson M. GAP1, the general amino acid permease gene of Saccharomyces cerevisiae. Nucleotide sequence, protein similarity with the other bakers yeast amino acid permeases, and nitrogen catabolite repression. Eur J Biochem. 1990 May 31;190(1):39–44. doi: 10.1111/j.1432-1033.1990.tb15542.x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Klionsky D. J., Cueva R., Yaver D. S. Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway. J Cell Biol. 1992 Oct;119(2):287–299. doi: 10.1083/jcb.119.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ljungdahl P. O., Gimeno C. J., Styles C. A., Fink G. R. SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast. Cell. 1992 Oct 30;71(3):463–478. doi: 10.1016/0092-8674(92)90515-e. [DOI] [PubMed] [Google Scholar]
  26. Loayza D., Tam A., Schmidt W. K., Michaelis S. Ste6p mutants defective in exit from the endoplasmic reticulum (ER) reveal aspects of an ER quality control pathway in Saccharomyces cerevisiae. Mol Biol Cell. 1998 Oct;9(10):2767–2784. doi: 10.1091/mbc.9.10.2767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Martin T. F. Phosphoinositide lipids as signaling molecules: common themes for signal transduction, cytoskeletal regulation, and membrane trafficking. Annu Rev Cell Dev Biol. 1998;14:231–264. doi: 10.1146/annurev.cellbio.14.1.231. [DOI] [PubMed] [Google Scholar]
  28. Muhlrad D., Hunter R., Parker R. A rapid method for localized mutagenesis of yeast genes. Yeast. 1992 Feb;8(2):79–82. doi: 10.1002/yea.320080202. [DOI] [PubMed] [Google Scholar]
  29. Mulholland J., Konopka J., Singer-Kruger B., Zerial M., Botstein D. Visualization of receptor-mediated endocytosis in yeast. Mol Biol Cell. 1999 Mar;10(3):799–817. doi: 10.1091/mbc.10.3.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. doi: 10.1016/0076-6879(83)01017-4. [DOI] [PubMed] [Google Scholar]
  31. Peterson M. R., Burd C. G., Emr S. D. Vac1p coordinates Rab and phosphatidylinositol 3-kinase signaling in Vps45p-dependent vesicle docking/fusion at the endosome. Curr Biol. 1999 Feb 11;9(3):159–162. doi: 10.1016/s0960-9822(99)80071-2. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Prescianotto-Baschong C., Riezman H. Morphology of the yeast endocytic pathway. Mol Biol Cell. 1998 Jan;9(1):173–189. doi: 10.1091/mbc.9.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Preston R. A., Manolson M. F., Becherer K., Weidenhammer E., Kirkpatrick D., Wright R., Jones E. W. Isolation and characterization of PEP3, a gene required for vacuolar biogenesis in Saccharomyces cerevisiae. Mol Cell Biol. 1991 Dec;11(12):5801–5812. doi: 10.1128/mcb.11.12.5801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Rieder S. E., Emr S. D. A novel RING finger protein complex essential for a late step in protein transport to the yeast vacuole. Mol Biol Cell. 1997 Nov;8(11):2307–2327. doi: 10.1091/mbc.8.11.2307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Roberg K. J., Rowley N., Kaiser C. A. Physiological regulation of membrane protein sorting late in the secretory pathway of Saccharomyces cerevisiae. J Cell Biol. 1997 Jun 30;137(7):1469–1482. doi: 10.1083/jcb.137.7.1469. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Robinson J. S., Graham T. R., Emr S. D. A putative zinc finger protein, Saccharomyces cerevisiae Vps18p, affects late Golgi functions required for vacuolar protein sorting and efficient alpha-factor prohormone maturation. Mol Cell Biol. 1991 Dec;11(12):5813–5824. doi: 10.1128/mcb.11.12.5813. [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. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  43. STATE D. Segmental colon resection in the treatment of congenital megacolon (Hirschsprung's disease). Am J Surg. 1963 Jan;105:93–101. doi: 10.1016/0002-9610(63)90272-1. [DOI] [PubMed] [Google Scholar]
  44. Sevrioukov E. A., He J. P., Moghrabi N., Sunio A., Krämer H. A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila. Mol Cell. 1999 Oct;4(4):479–486. doi: 10.1016/s1097-2765(00)80199-9. [DOI] [PubMed] [Google Scholar]
  45. Simonsen A., Lippé R., Christoforidis S., Gaullier J. M., Brech A., Callaghan J., Toh B. H., Murphy C., Zerial M., Stenmark H. EEA1 links PI(3)K function to Rab5 regulation of endosome fusion. Nature. 1998 Jul 30;394(6692):494–498. doi: 10.1038/28879. [DOI] [PubMed] [Google Scholar]
  46. Stanbrough M., Magasanik B. Transcriptional and posttranslational regulation of the general amino acid permease of Saccharomyces cerevisiae. J Bacteriol. 1995 Jan;177(1):94–102. doi: 10.1128/jb.177.1.94-102.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tall G. G., Hama H., DeWald D. B., Horazdovsky B. F. The phosphatidylinositol 3-phosphate binding protein Vac1p interacts with a Rab GTPase and a Sec1p homologue to facilitate vesicle-mediated vacuolar protein sorting. Mol Biol Cell. 1999 Jun;10(6):1873–1889. doi: 10.1091/mbc.10.6.1873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. VanRheenen S. M., Cao X., Sapperstein S. K., Chiang E. C., Lupashin V. V., Barlowe C., Waters M. G. Sec34p, a protein required for vesicle tethering to the yeast Golgi apparatus, is in a complex with Sec35p. J Cell Biol. 1999 Nov 15;147(4):729–742. doi: 10.1083/jcb.147.4.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Wada Y., Nakamura N., Ohsumi Y., Hirata A. Vam3p, a new member of syntaxin related protein, is required for vacuolar assembly in the yeast Saccharomyces cerevisiae. J Cell Sci. 1997 Jun;110(Pt 11):1299–1306. doi: 10.1242/jcs.110.11.1299. [DOI] [PubMed] [Google Scholar]
  51. Webb G. C., Hoedt M., Poole L. J., Jones E. W. 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. Genetics. 1997 Oct;147(2):467–478. doi: 10.1093/genetics/147.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. Weber T., Zemelman B. V., McNew J. A., Westermann B., Gmachl M., Parlati F., Söllner T. H., Rothman J. E. SNAREpins: minimal machinery for membrane fusion. Cell. 1998 Mar 20;92(6):759–772. doi: 10.1016/s0092-8674(00)81404-x. [DOI] [PubMed] [Google Scholar]
  54. Wilcox C. A., Redding K., Wright R., Fuller R. S. Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole. Mol Biol Cell. 1992 Dec;3(12):1353–1371. doi: 10.1091/mbc.3.12.1353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wilsbach K., Payne G. S. Vps1p, a member of the dynamin GTPase family, is necessary for Golgi membrane protein retention in Saccharomyces cerevisiae. EMBO J. 1993 Aug;12(8):3049–3059. doi: 10.1002/j.1460-2075.1993.tb05974.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Woolford C. A., Bounoutas G. S., Frew S. E., Jones E. W. Genetic interaction with vps8-200 allows partial suppression of the vestigial vacuole phenotype caused by a pep5 mutation in Saccharomyces cerevisiae. Genetics. 1998 Jan;148(1):71–83. doi: 10.1093/genetics/148.1.71. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. 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]
  58. 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]
  59. Zubenko G. S., Park F. J., Jones E. W. Mutations in PEP4 locus of Saccharomyces cerevisiae block final step in maturation of two vacuolar hydrolases. Proc Natl Acad Sci U S A. 1983 Jan;80(2):510–514. doi: 10.1073/pnas.80.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES