Skip to main content

Some NLM-NCBI services and products are experiencing heavy traffic, which may affect performance and availability. We apologize for the inconvenience and appreciate your patience. For assistance, please contact our Help Desk at info@ncbi.nlm.nih.gov.

Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1986 May;83(10):3248–3252. doi: 10.1073/pnas.83.10.3248

Overproduction-induced mislocalization of a yeast vacuolar protein allows isolation of its structural gene.

J H Rothman, C P Hunter, L A Valls, T H Stevens
PMCID: PMC323490  PMID: 3517855

Abstract

Using an immunological screening procedure that allows the detection of yeast cells aberrantly secreting vacuolar proteins, we have isolated a cloned DNA fragment containing the structural gene for the vacuolar enzyme proteinase A (PrA; EC 3.4.23.6). A large portion of PrA is misdirected to the cell surface in cells harboring the PrA structural gene on a multicopy plasmid. This mislocalized PrA traverses the late stages of the secretory pathway and differs slightly in apparent molecular weight from the vacuolar form. A deletion in the genomic copy of the PrA structural gene eliminates immunoreactive PrA as well as the enzymatic activities of at least three other vacuolar hydrolases. In the case of the vacuolar enzyme carboxypeptidase Y (EC 3.4.16.1), the lack of activity is due to the absence of proteolytic activation of the zymogen. Thus, PrA may be required for in vivo processing of a number of yeast vacuolar hydrolases.

Full text

PDF
3248

Images in this article

Selected References

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

  1. 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]
  2. Esmon B., Novick P., Schekman R. Compartmentalized assembly of oligosaccharides on exported glycoproteins in yeast. Cell. 1981 Aug;25(2):451–460. doi: 10.1016/0092-8674(81)90063-5. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  6. Lyons S., Nelson N. An immunological method for detecting gene expression in yeast colonies. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7426–7430. doi: 10.1073/pnas.81.23.7426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Mechler B., Müller M., Müller H., Wolf D. H. In vivo biosynthesis of vacuolar proteinases in proteinase mutants of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1982 Aug;107(3):770–778. doi: 10.1016/0006-291x(82)90590-3. [DOI] [PubMed] [Google Scholar]
  9. Mechler B., Wolf D. H. Analysis of proteinase A function in yeast. Eur J Biochem. 1981 Dec;121(1):47–52. doi: 10.1111/j.1432-1033.1981.tb06427.x. [DOI] [PubMed] [Google Scholar]
  10. Meussdoerffer F., Tortora P., Holzer H. Purification and properties of proteinase A from yeast. J Biol Chem. 1980 Dec 25;255(24):12087–12093. [PubMed] [Google Scholar]
  11. Mitchell J. K., Fonzi W. A., Wilkerson J., Opheim D. J. A particulate form of alkaline phosphatase in the yeast, Saccharomyces cerevisiae. Biochim Biophys Acta. 1981 Feb 13;657(2):482–494. doi: 10.1016/0005-2744(81)90333-8. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Zubenko G. S., Jones E. W. Protein degradation, meiosis and sporulation in proteinase-deficient mutants of Saccharomyces cerevisiae. Genetics. 1981 Jan;97(1):45–64. doi: 10.1093/genetics/97.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Zubenko G. S., Park F. J., Jones E. W. Genetic properties of mutations at the PEP4 locus in Saccharomyces cerevisiae. Genetics. 1982 Dec;102(4):679–690. doi: 10.1093/genetics/102.4.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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 Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES