Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Nov;8(11):4936–4948. doi: 10.1128/mcb.8.11.4936

Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases.

J S Robinson 1, D J Klionsky 1, L M Banta 1, S D Emr 1
PMCID: PMC365587  PMID: 3062374

Abstract

Using a selection for spontaneous mutants that mislocalize a vacuolar carboxypeptidase Y (CPY)-invertase fusion protein to the cell surface, we identified vacuolar protein targeting (vpt) mutants in 25 new vpt complementation groups. Additional alleles in each of the eight previously identified vpt complementation groups (vpt1 through vpt8) were also obtained. Representative alleles from each of the 33 vpt complementation groups (vpt1 through vpt33) were shown to exhibit defects in the sorting and processing of several native vacuolar proteins, including the soluble hydrolases CPY, proteinase A, and proteinase B. Of the 33 complementation groups, 19 were found to contain mutant alleles that led to extreme defects. In these mutants, CPY accumulated in its Golgi complex-modified precursor form which was secreted by the mutant cells. Normal protein secretion appeared to be unaffected in the vpt mutants. The lack of significant leakage of cytosolic markers from the vpt mutant cells indicated that the vacuolar protein-sorting defects associated with these mutants do not result from cell lysis. In addition, the observation that the precursor rather than the mature forms of CPY, proteinase A, proteinase B were secreted from the vpt mutants was consistent with the fact that mislocalization occurred at a stage after Golgi complex-specific modification, but before final vacuolar sorting of these enzymes. Vacuolar membrane protein sorting appeared to be unaffected in the majority of the vpt mutants. However, a subset of the vpt mutants (vpt11, vpt16, vpt18, and vpt33) was found to exhibit defects in the sorting of a vacuolar membrane marker enzyme, alpha-mannosidase. Up to 50% of the alpha-mannosidase enzyme activity was found to be mislocalized to the cell surface in these vpt mutants. Seven of the vpt complementation groups (vpt3, vpt11, vpt15, vpt16, vpt18, vpt29, and vpt33) contained alleles that led to a conditional lethal phenotype; the mutants were temperature sensitive for vegetative cell growth. This temperature-sensitive phenotype has been shown to be recessive and to cosegregate with the vacuolar protein-sorting defect in each case. Tetrad analysis showed that vpt3 mapped to the right arm of chromosome XV and that vpt15 mapped to the right arm of chromosome II. Intercrosses with other mutants that exhibited defects in vacuolar protein sorting or function (vpl, sec, pep, and end mutants) revealed several overlaps among these different sets of genes. Together, these data indicate that more than 50 gene products are involved, directly or indirectly, in the process of vacuolar protein sorting.

Full text

PDF

Images in this article

4939Image
on p.4939
4941Image
on p.4941
4942Image
on p.4942
4942Image
on p.4942
4943Image
on p.4943
4943Image
on p.4943

Selected References

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

  1. Ammerer G., Hunter C. P., Rothman J. H., Saari G. C., Valls L. A., Stevens T. H. PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors. Mol Cell Biol. 1986 Jul;6(7):2490–2499. doi: 10.1128/mcb.6.7.2490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Bernstein M., Hoffmann W., Ammerer G., Schekman R. Characterization of a gene product (Sec53p) required for protein assembly in the yeast endoplasmic reticulum. J Cell Biol. 1985 Dec;101(6):2374–2382. doi: 10.1083/jcb.101.6.2374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burgess T. L., Kelly R. B. Constitutive and regulated secretion of proteins. Annu Rev Cell Biol. 1987;3:243–293. doi: 10.1146/annurev.cb.03.110187.001331. [DOI] [PubMed] [Google Scholar]
  5. Chvatchko Y., Howald I., Riezman H. Two yeast mutants defective in endocytosis are defective in pheromone response. Cell. 1986 Aug 1;46(3):355–364. doi: 10.1016/0092-8674(86)90656-2. [DOI] [PubMed] [Google Scholar]
  6. Douglas M. G., Geller B. L., Emr S. D. Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria. Proc Natl Acad Sci U S A. 1984 Jul;81(13):3983–3987. doi: 10.1073/pnas.81.13.3983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Emr S. D., Schekman R., Flessel M. C., Thorner J. An MF alpha 1-SUC2 (alpha-factor-invertase) gene fusion for study of protein localization and gene expression in yeast. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7080–7084. doi: 10.1073/pnas.80.23.7080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Esmon P. C., Esmon B. E., Schauer I. E., Taylor A., Schekman R. Structure, assembly, and secretion of octameric invertase. J Biol Chem. 1987 Mar 25;262(9):4387–4394. [PubMed] [Google Scholar]
  9. Farquhar M. G., Palade G. E. The Golgi apparatus (complex)-(1954-1981)-from artifact to center stage. J Cell Biol. 1981 Dec;91(3 Pt 2):77s–103s. doi: 10.1083/jcb.91.3.77s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Ferro-Novick S., Novick P., Field C., Schekman R. Yeast secretory mutants that block the formation of active cell surface enzymes. J Cell Biol. 1984 Jan;98(1):35–43. doi: 10.1083/jcb.98.1.35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  12. Hasilik A., Tanner W. Biosynthesis of the vacuolar yeast glycoprotein carboxypeptidase Y. Conversion of precursor into the enzyme. Eur J Biochem. 1978 Apr 17;85(2):599–608. doi: 10.1111/j.1432-1033.1978.tb12275.x. [DOI] [PubMed] [Google Scholar]
  13. Hemmings B. A., Zubenko G. S., Hasilik A., Jones E. W. Mutant defective in processing of an enzyme located in the lysosome-like vacuole of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1981 Jan;78(1):435–439. doi: 10.1073/pnas.78.1.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hoffmann W. CAN1-SUC2 gene fusion studies in Saccharomyces cerevisiae. Mol Gen Genet. 1987 Dec;210(2):277–281. doi: 10.1007/BF00325694. [DOI] [PubMed] [Google Scholar]
  15. Johnson L. M., Bankaitis V. A., Emr S. D. Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar protease. Cell. 1987 Mar 13;48(5):875–885. doi: 10.1016/0092-8674(87)90084-5. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Jones E. W., Zubenko G. S., Parker R. R. PEP4 gene function is required for expression of several vacuolar hydrolases in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):665–677. doi: 10.1093/genetics/102.4.665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Klionsky D. J., Banta L. M., Emr S. D. Intracellular sorting and processing of a yeast vacuolar hydrolase: proteinase A propeptide contains vacuolar targeting information. Mol Cell Biol. 1988 May;8(5):2105–2116. doi: 10.1128/mcb.8.5.2105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kornfeld S. Trafficking of lysosomal enzymes in normal and disease states. J Clin Invest. 1986 Jan;77(1):1–6. doi: 10.1172/JCI112262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mechler B., Hirsch H. H., Müller H., Wolf D. H. Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB. EMBO J. 1988 Jun;7(6):1705–1710. doi: 10.1002/j.1460-2075.1988.tb02999.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mechler B., Müller H., Wolf D. H. Maturation of vacuolar (lysosomal) enzymes in yeast: proteinase yscA and proteinase yscB are catalysts of the processing and activation event of carboxypeptidase yscY. EMBO J. 1987 Jul;6(7):2157–2163. doi: 10.1002/j.1460-2075.1987.tb02483.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Mechler B., Müller M., Müller H., Meussdoerffer F., Wolf D. H. In vivo biosynthesis of the vacuolar proteinases A and B in the yeast Saccharomyces cerevisiae. J Biol Chem. 1982 Oct 10;257(19):11203–11206. [PubMed] [Google Scholar]
  23. Moir D., Stewart S. E., Osmond B. C., Botstein D. Cold-sensitive cell-division-cycle mutants of yeast: isolation, properties, and pseudoreversion studies. Genetics. 1982 Apr;100(4):547–563. doi: 10.1093/genetics/100.4.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mortimer R. K., Hawthorne D. C. Genetic mapping in Saccharomyces. Genetics. 1966 Jan;53(1):165–173. doi: 10.1093/genetics/53.1.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Novick P., Field C., Schekman R. Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway. Cell. 1980 Aug;21(1):205–215. doi: 10.1016/0092-8674(80)90128-2. [DOI] [PubMed] [Google Scholar]
  27. Payne G. S., Baker D., van Tuinen E., Schekman R. Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast. J Cell Biol. 1988 May;106(5):1453–1461. doi: 10.1083/jcb.106.5.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Payne G. S., Hasson T. B., Hasson M. S., Schekman R. Genetic and biochemical characterization of clathrin-deficient Saccharomyces cerevisiae. Mol Cell Biol. 1987 Nov;7(11):3888–3898. doi: 10.1128/mcb.7.11.3888. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pfeffer S. R., Rothman J. E. Biosynthetic protein transport and sorting by the endoplasmic reticulum and Golgi. Annu Rev Biochem. 1987;56:829–852. doi: 10.1146/annurev.bi.56.070187.004145. [DOI] [PubMed] [Google Scholar]
  30. Reid G. A. Pulse labeling of yeast cells and spheroplasts. Methods Enzymol. 1983;97:324–329. doi: 10.1016/0076-6879(83)97144-6. [DOI] [PubMed] [Google Scholar]
  31. Reitman M. L., Varki A., Kornfeld S. Fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy are deficient in uridine 5'-diphosphate-N-acetylglucosamine: glycoprotein N-acetylglucosaminylphosphotransferase activity. J Clin Invest. 1981 May;67(5):1574–1579. doi: 10.1172/JCI110189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Riezman H. Endocytosis in yeast: several of the yeast secretory mutants are defective in endocytosis. Cell. 1985 Apr;40(4):1001–1009. doi: 10.1016/0092-8674(85)90360-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Schauer I., Emr S., Gross C., Schekman R. Invertase signal and mature sequence substitutions that delay intercompartmental transport of active enzyme. J Cell Biol. 1985 May;100(5):1664–1675. doi: 10.1083/jcb.100.5.1664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Sly W. S., Fischer H. D. The phosphomannosyl recognition system for intracellular and intercellular transport of lysosomal enzymes. J Cell Biochem. 1982;18(1):67–85. doi: 10.1002/jcb.1982.240180107. [DOI] [PubMed] [Google Scholar]
  36. Stevens T. H., Rothman J. H., Payne G. S., Schekman R. Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol. 1986 May;102(5):1551–1557. doi: 10.1083/jcb.102.5.1551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Valls L. A., Hunter C. P., Rothman J. H., Stevens T. H. Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell. 1987 Mar 13;48(5):887–897. doi: 10.1016/0092-8674(87)90085-7. [DOI] [PubMed] [Google Scholar]
  39. Yoshihisa T., Ohsumi Y., Anraku Y. Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae. J Biol Chem. 1988 Apr 15;263(11):5158–5163. [PubMed] [Google Scholar]
  40. 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 Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES