Skip to main content
SpringerLink
Log in

env1 Mutant of VPS35 gene exhibits unique protein localization and processing phenotype at Golgi and lysosomal vacuole in Saccharomyces cerevisiae

  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

The yeast vacuole is functionally and structurally equivalent to the mammalian lysosome. Delivery of resident and cargo proteins to the lysosome is vital for proper cellular operations, and failure to correctly target proteins to the organelle is correlated with the development of neurodegenerative and lysosomal storage diseases. We previously reported a novel mutant screen for vacuolar trafficking defects in yeast Saccharomyces cerevisiae that resulted in the isolation of env1, an allelic mutant of VPS35. As a member of the retromer complex, Vps35p binds directly to cargos and facilitates their retrograde transport to trans Golgi from endosomes. Our previous studies established that env1 exhibits unique pleiotropic phenotype in comparison to other tested VPS35 alleles including severe growth sensitivity to hygromycin B and internal accumulation of the precursor form of the vacuolar enzyme carboxypeptidase Y. Here, through a combination of sub-cellular fractionation and indirect immunofluorescence microscopy, we confirm and extend the unique phenotype of env1 to processing and localization of additional proteins within the vacuolar trafficking pathway. In comparative studies with a null and an allelic mutant of VPS35, env1 exhibited unique processing defects of retromer-independent vacuolar membrane enzyme alkaline phosphatase at the vacuole and significant Golgi localization of retromer cargos Vps10p and Kex2p despite compromised trafficking at the Golgi and late endosome interface.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

References

  1. Jones EW, Webb GC, Hiller MA (1997) Molecular biology of the yeast Saccharomyces cerevisiae, vol III. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 363–469

    Google Scholar 

  2. Katzmann DJ, Odorizzi G, Emr SD (2002) Receptor downregulation and multivesicular-body sorting. Nature Rev Mol Cell Biol 3(12):893–905

    Article  CAS  Google Scholar 

  3. Pelham HRB (2002) Insights from yeast endosomes. Curr Opin Cell Biol 14(4):454–462

    Article  CAS  PubMed  Google Scholar 

  4. Bowers K, Stevens TH (2005) Protein transport from the late Golgi to the vacuole in the yeast Saccharomyces cerevisiae. Biochim Biophys Acta 1744(3):438–454

    Article  CAS  PubMed  Google Scholar 

  5. 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(Pt 5):1088–1091

    CAS  PubMed  Google Scholar 

  6. Mijaljica D, Prescott M, Klionsky DJ, Devenish RJ (2007) Autophagy and vacuole homeostasis: a case for self-degradation? Autophagy 3(5):417–421

    CAS  PubMed  Google Scholar 

  7. Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793(4):650–663

    Article  CAS  PubMed  Google Scholar 

  8. Huang J, Klionsky DJ (2007) Autophagy and human disease. Cell Cycle 6(15):1837–1849

    Article  CAS  PubMed  Google Scholar 

  9. He C, Klionsky DJ (2009) Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet 43:67–93

    Article  CAS  PubMed  Google Scholar 

  10. 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

    Article  CAS  PubMed  Google Scholar 

  11. Jones EW (1977) Proteinase mutants of Saccharomyces cerevisiae. Genetics 85:23–33

    CAS  PubMed  Google Scholar 

  12. Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci USA 83:9075–9079

    Article  CAS  PubMed  Google Scholar 

  13. 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

    Article  CAS  PubMed  Google Scholar 

  14. 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

    CAS  PubMed  Google Scholar 

  15. Wada Y, Ohsumi Y, Anraku Y (1992) Genes for directing vacuolar morphogenesis in Saccharomyces cerevisiae. I. Isolation and characterization of two classes of vam mutants. J Biol Chem 267:18665–18670

    CAS  PubMed  Google Scholar 

  16. Takahashi MK, Frost C, Oyadomari K, Pinho M, Sao D, Chima-Okereke O, Gharakhanian E (2008) A novel immunodetection screen for vacuolar defects identifies a unique allele of VPS35 in S. cerevisiae. Mol Cell Biochem 311:121–136

    Article  CAS  PubMed  Google Scholar 

  17. Coudreuse DY, Roël G, Betist MC, Destrée O, Korswagen HC (2006) Wnt gradient formation requires retromer function in Wnt-producing cells. Science 312:921–924

    Article  CAS  PubMed  Google Scholar 

  18. George A, Leahy H, Zhou J, Morin PJ (2007) The vacuolar-ATPase inhibitor bafilomycin and mutant VPS35 inhibit canonical Wnt signaling. Neurobiol Dis 26(1):125–133

    Article  CAS  PubMed  Google Scholar 

  19. Belenkaya TY, Wu Y, Tang X, Zhou B, Cheng L, Sharma YV, Yan D, Selva EM, Lin X (2008) The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network. Dev Cell 14:120–131

    Article  CAS  PubMed  Google Scholar 

  20. Franch-Marro X, Wendler F, Griffith J, Maurice MM, Vincent JP (2008) In vivo role of lipid adducts on Wingless. J Cell Sci 121(Pt 10):1587–1589

    Article  CAS  PubMed  Google Scholar 

  21. Pan CL, Baum PD, Gu M, Jorgensen EM, Clark SG, Garriga G (2008) C. elegans AP-2 and retromer control Wnt signaling by regulating mig-14/Wntless. Dev Cell 14:132–139

    Article  CAS  PubMed  Google Scholar 

  22. Port F, Kuster M, Herr P, Furger E, Bänziger C, Hausmann G, Basler K (2008) Wingless secretion promotes and requires retromer-dependent cycling of Wntless. Nat Cell Biol 10:178–185

    Article  CAS  PubMed  Google Scholar 

  23. Seaman MN, McCaffery JM, Emr SD (1998) A membrane coat complex essential for endosome-to-Golgi retrograde transport in yeast. J Cell Biol 142:665–681

    Article  CAS  PubMed  Google Scholar 

  24. Nothwehr SF, Bruinsma P, Strawn LA (1999) Distinct domains within Vps35p mediate the retrieval of two different cargo proteins from the yeast prevacuolar/endosomal compartment. Mol Cell Biol 10:875–890

    CAS  Google Scholar 

  25. Nothwehr SF, Bruinsma P (2000) Sorting of yeast membrane proteins into an endosome-to Golgi pathway involves direct interaction of their cytosolic domains with Vps35p. J Cell Biol 151:297–309

    Article  CAS  PubMed  Google Scholar 

  26. Nothwehr SF, Hindes AE (1997) The yeast VPS5/GRD2 gene encodes a sorting nexin-1-like protein required for localizing membrane proteins to the late Golgi. J Cell Sci 110(9):1063–1072

    CAS  PubMed  Google Scholar 

  27. Seaman MN, Marcusson EG, Cereghino JL, Emr SD (1997) Endosome to Golgi retrieval of the vacuolar protein sorting receptor, Vps10p, requires the function of the Vps29, Vps30, and Vps35 gene products. J Cell Biol 137:79–92

    Article  CAS  PubMed  Google Scholar 

  28. Robinson JS, Klionsky DJ, Banta LM, Emr SD (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol Cell Biol 8:4936–4948

    CAS  PubMed  Google Scholar 

  29. Paravicini G, Horazdovsky BF, Emr SD (1992) Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell 3:415–427

    CAS  PubMed  Google Scholar 

  30. Banta LM, Robinson JS, Klionsky DJ, Emr SD (1988) Organelle assembly in yeast, characterization of yeast mutants defective in vacuolar biogenesis and protein sorting. J Cell Biol 107:1369–1383

    Article  CAS  PubMed  Google Scholar 

  31. Anand VC, Daboussi L, Lorenz TC, Payne GS (2009) Genome-wide analysis of AP-3-dependent protein transport in yeast. Mol Biol Cell 5:1592–1604

    Article  Google Scholar 

  32. Roberts CJ, Raymond CK, Yamahiro CT, Stevens TH (1991) Methods for studying the yeast vacuole. Methods Enzymol 194:644–661

    Article  CAS  PubMed  Google Scholar 

  33. Rothstein R (1991) Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol 194:281–301

    Article  CAS  PubMed  Google Scholar 

  34. Cooper AA, Stevens TH (1996) Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J Cell Biol 133:529–541

    Article  CAS  PubMed  Google Scholar 

  35. Seeger M, Payne GS (1992) A role for clathrin in the sorting of vacuolar proteins in the Golgi complex of yeast. EMBO J 11(8):2811–2818

    CAS  PubMed  Google Scholar 

  36. Seeger M, Payne GS (1992) Selective and immediate effects of clathrin heavy chain mutations on Golgi membrane protein retention in Saccharomyces cerevisiae. J Cell Biol 118(3):531–540

    Article  CAS  PubMed  Google Scholar 

  37. Cowles CR, Snyder WB, Burd CG, Emr SD (1997) Novel Golgi to vacuole delivery pathway in yeast: identification of a sorting determinant and required transport component. EMBO J 16:2769–2782

    Article  CAS  PubMed  Google Scholar 

  38. Strathern JN, Jones EW, Broach JR (1981) The molecular biology of the yeast Saccharomyces life cycle and inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 147–160

    Google Scholar 

  39. Julius D, Schekman R, Thorner J (1984) Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathway. Cell 36(2):309–318

    Article  CAS  PubMed  Google Scholar 

  40. Fuller RS, Sterne RE, Thorner J (1988) Enzymes required for yeast prohormone processing. Annu Rev Physiol 50:345–362

    Article  CAS  PubMed  Google Scholar 

  41. 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(2):207–218

    Article  CAS  PubMed  Google Scholar 

  42. 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(1):69–83

    Article  CAS  PubMed  Google Scholar 

  43. Banuelos MG, Moreno DE, Olson DK, Nguyen Q, Ricarte F, Aguilera-Sandoval CR, Gharakhanian E (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(2):121–137

    Article  CAS  PubMed  Google Scholar 

  44. Cowles CR, Odorizzi G, Payne GS, Emr SD (1997) The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell 91:109–118

    Article  CAS  PubMed  Google Scholar 

  45. Darsow T, Rieder SE, Emr SD (1997) A multispecificity syntaxin homologue, Vam3p, essential for autophagic and biosynthetic protein transport to the vacuole. J Cell Biol 138(3):517–529

    Article  CAS  PubMed  Google Scholar 

  46. Deloche O, Schekman RW (2002) Vps10p cycles between the TGN and the late endosome via the plasma membrane in clathrin mutants. Mol Biol Cell 13(12):4296–4307

    Article  CAS  PubMed  Google Scholar 

  47. Hierro A, Rojas AL, Rojas R, Murthy N, Effantin G, Kajava AV, Steven AC, Bonifacino JS, Hurley JH (2007) Functional architecture of the retromer cargo-recognition complex. Nature 449:1063–1067

    Article  CAS  PubMed  Google Scholar 

  48. Restrepo R, Zhao X, Peter H, Zhang BY, Arvan P, Nothwehr SF (2007) Structural features of vps35p involved in interaction with other subunits of the retromer complex. Traffic 8:1841–1853

    Article  CAS  PubMed  Google Scholar 

  49. Seaman MN, Williams HP (2002) Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p. Mol Biol Cell 13:2826–2840

    Article  CAS  PubMed  Google Scholar 

  50. Vergés M, Luton F, Gruber C, Tiemann F, Reinders LG, Huang L, Burlingame AL, Haft CR, Mostov KE (2004) The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor. Nat Cell Biol 6(8):763–769

    Article  PubMed  Google Scholar 

  51. 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

    CAS  PubMed  Google Scholar 

  52. Chow C, Palecek SP (2004) Enzyme encapsulation in permeabilized Saccharomyces cerevisiae cells. Biotechnol Prog 20(2):449–456

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We are grateful to G. Payne and his laboratory for continuous support, guidance, and key reagents; we also thank the Emr laboratory for gifts of strains. This work was supported by National Institutes of Health-MBRS-SCORE grant #GM63119-02 to EG. Confocal microscopy was supported by National Science Foundation Major Research Instrumentation grant #DBI0722757. OC and MKT were partially supported by the NIH-MBRS-SCORE grant. DKO was supported by a Beckman Scholars award.

Author information

Author notes

  1. M. Kathleen Takahashi

    Present address: Biology Department, Santa Ana College, Santa Ana, CA, 92706, USA

Authors and Affiliations

  1. Department of Biological Sciences, California State University at Long Beach, 1250 Bellflower Blvd, Long Beach, CA, 90840, USA

    Editte Gharakhanian, Onyinyechi Chima-Okereke, Daniel K. Olson, Christopher Frost & M. Kathleen Takahashi

Authors
  1. Editte Gharakhanian
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Onyinyechi Chima-Okereke
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel K. Olson
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Christopher Frost
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. M. Kathleen Takahashi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Editte Gharakhanian.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gharakhanian, E., Chima-Okereke, O., Olson, D.K. et al. 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 (2011). https://doi.org/10.1007/s11010-010-0604-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11010-010-0604-5

Keywords

Search

Navigation