Advertisement

Characterization of the Bar Proteinase, An Extracellular Enzyme from the Yeast Saccharomyces Cerevisiae

  • Vivian L. MacKay
  • Jacque Armstrong
  • Carli Yip
  • Susan Welch
  • Kathy Walker
  • Sherri Osborn
  • Paul Sheppard
  • John Forstrom
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 306)

Abstract

Haploid S. cerevisiae cells of the a mating type constitutively secrete an extracellular proteinase that cleaves the peptide mating pheromone (α-factor) secreted by mating-type α cells. DNA sequence analysis of the BAR1 gene that encodes Bar proteinase demonstrated that the primary translation product of 587 amino acids has strong homology to two-domain aspartic proteinases such as pepsin, chymosin, and others, but contains a unique third domain that is not homologous to these enzymes. When produced by wild-type yeast cells, the Bar enzyme exists as a heterogeneous, heavily glycosylated protein with apparent molecular weight >200,000 Da. By producing the proteinase in mutant yeast strains that are defective in glycosylation, we have been able to purify and characterize a homogeneous species. In this paper, we will describe some of the enzyme’s physical properties and substrate requirements, as well as present data indicating that the third domain is required for secretion of the proteinase to the culture medium.

Keywords

Triose Phosphate Isomerase Aspartyl Proteinase Putative Active Site Scissile Bond Mutant Yeast Strain 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Alber, T., & Kawasaki, G., 1982, Nucleotide sequence of the triose phosphate isomerase gene of Saccharomyces cerevisiae, J. Mol. Appl. Genet., 1: 419.PubMedGoogle Scholar
  2. Ballou, C. E., 1990, Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects, Meth. Enzymol., 185: 440.PubMedCrossRefGoogle Scholar
  3. Bianchi, M., Boigegrain, R. A., Castro, B., & Coletti-Previero, M.-A., 1990, N-terminal domain of pepsin as a model for retroviral dimeric aspartyl protease, Biochem. Biophys. Res. Comm., 167: 339.PubMedCrossRefGoogle Scholar
  4. Chan, R. K., & Otte, C. A., 1982, Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to Gl arrest by a-factor and α-factor pheromones, Mol. Cell. Biol, 2: 11.PubMedGoogle Scholar
  5. Cross, R., Hartwell, L. H., Jackson, C., & Konopka, J. B., 1988, Conjugation in Saccharomyces cerevisiae, Ann. Rev. Cell Biol., 4: 429.PubMedCrossRefGoogle Scholar
  6. Darke, P. L., Leu, C.-T., Davis, L. J., Heimbach, J. C., Diehl, R. E., Hill, W. S., Dixon, R. A. F., & Sigal, I. S., 1989, Human immunodeficiency virus protease: Bacterial expression and characterization of the purified aspartic protease, J. Biol. Chem., 264: 2307.PubMedGoogle Scholar
  7. Dunn, B. M., Jimenez, M., Weidner, J., Pennington, M., Carter, M., & Parten, B., 1987, Kinetic product analysis of aspartyl proteinases utilizing new synthetic substrates and reversed phase HPLC, in: “Proteins,” J. J. L’Italien, ed., Plenum, New York.Google Scholar
  8. Fuller, R. S., Brake, A., & Thorner, J., 1989, Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease, Proc. Natl. Acad. Sci., U.S.A., 86: 1434.PubMedCrossRefGoogle Scholar
  9. Herskowitz, I., 1989, A regulatory hierarchy for cell specialization in yeast, Nature (London), 342: 749.CrossRefGoogle Scholar
  10. Hicks, J. B., & Herskowitz, I., 1976, Evidence for a new diffusible element of mating pheromones in yeast, Nature (London), 260: 246.CrossRefGoogle Scholar
  11. Kurjan, J., & Herskowitz, I., 1982, Structure of a yeast pheromone gene: A putative a-factor precursor contains four tandem copies of mature α-factor, Cell, 30: 933.PubMedCrossRefGoogle Scholar
  12. Kronstad, J. W., Holly, J. A., & MacKay, V. L., 1987, A yeast operator overlaps an upstream activation site, Cell, 50: 369.PubMedCrossRefGoogle Scholar
  13. MacKay, V. L., 1986, Secretion of heterologous proteins in yeast, in: “The Biochemistry and Molecular Biology of Industrial Yeasts,” G. G. Stewart, I. Russell, R. D. Klein, and R. R. Hiebsch, eds., CRC Press, Boca Raton, Florida.Google Scholar
  14. MacKay, V. L., Welch, S. K., Insley, M. I., Manney, T. R., Holly, J., Saari, G. C., & Parker, M. L., 1988, The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin, Proc. Natl. Acad. Sci., U.S.A., 85: 55.PubMedCrossRefGoogle Scholar
  15. MacKay, V. L., Yip, C., Welch, S., Gilbert, T., Seidel, P., Grant, F., & O’Hara, P., 1990, Glycosylation and export of heterologous proteins expressed in yeast, in: “Recombinant Systems in Protein Expression,” K. K. Alitalo, M.-L. Huhtala, J. Knowles, & A. Vaheri, eds., Elsevier, Amsterdam.Google Scholar
  16. Manney, T. R., 1983, Expression of the BAR1 gene in Saccharomyces cerevisiae: Induction by the a mating pheromone of an activity associated with a secreted protein, J. Bacteriol., 155: 291.PubMedGoogle Scholar
  17. Meek, T. D., Dayton, B. D., Metcalf, B. W., Dreyer, G. B., Strickler, J. E., Gorniak, J. G., Rosenberg, M., Moore, M. L., Magaard, V. W., & Debouck, C., 1989, Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease, Proc. Natl. Acad. Sci., U.S.A., 86: 1841.PubMedCrossRefGoogle Scholar
  18. Salovuori, I., Makarow, M., Rauvala, H., Knowles, J., & Kaariainen, L., 1987, Low molecular weight high-mannose type glycans in a secreted protein of the filamentous fungus Trichoderma reesei, Bio/Technology, 5: 152.CrossRefGoogle Scholar
  19. Sprague, G. F., Jr., & Herskowitz, I., 1981, Control of yeast cell type by the mating type locus. II. Identification and control of expression of the a-specific gene BAR1, J. Mol Biol., 153: 305.PubMedCrossRefGoogle Scholar
  20. Steden, M, Betz, R., & Duntze, W., 1989, Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor, Mol Gen. Genet., 219: 439.PubMedCrossRefGoogle Scholar
  21. Tomme, P., Van Tilbeurgh, H., Pettersson, G., Van Damme, J., Vandekerckhove, J., Knowles, J., Teeri, T., & Claeyssens, M., 1988, Studies of the cellulolytic system of Trichoderma reesei QM 9414: Analysis of domain function in two cellobiohydrolases by limited proteolysis, Eur. J. Biochem., 170: 575.PubMedCrossRefGoogle Scholar
  22. von Heinje, G., 1986, A new method for predicting signal sequence cleavage sites, J. Mol Biol, 184: 99.Google Scholar

Copyright information

© Plenum Press, New York 1991

Authors and Affiliations

  • Vivian L. MacKay
    • 1
  • Jacque Armstrong
    • 1
  • Carli Yip
    • 1
  • Susan Welch
    • 1
  • Kathy Walker
    • 1
  • Sherri Osborn
    • 1
  • Paul Sheppard
    • 1
  • John Forstrom
    • 1
  1. 1.ZymoGenetics, IncSeattleUSA

Personalised recommendations