Skip to main content
PMC home page
The EMBO Journal logoLink to The EMBO Journal
. 1988 May;7(5):1483–1488. doi: 10.1002/j.1460-2075.1988.tb02966.x

Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae.

M Watzele 1, F Klis 1, W Tanner 1
PMCID: PMC458399  PMID: 3044782

Abstract

A cell surface glycoprotein induced by the mating pheromone alpha factor in Saccharomyces cerevisiae a cells has been purified to homogeneity. At 4 x 10(-9) M it strongly inhibits mating-type-specific agglutination between a and alpha cells. The protein is solely O-glycosylated. It consists of 29% carbohydrate and its apparent molecular mass is 22 kd on SDS gels. After HF treatment it behaves like a protein of 13 kd; therefore its true molecular mass probably is close to 18 kd. Mild periodate treatment destroys the biological activity of the purified protein. The protein contains one cysteine, no arginine, and 27% of the amino acids are serine and threonine residues, two thirds of which are glycosylated. With a polyclonal antibody the glycoprotein can already be detected at the cell surface 15 min after pheromone addition. The inducible antigen is not expressed in a specific phase of the cell cycle; it first appears exclusively on the growing bud. Mother cells express the antigen on their surface only after the daughter cells have separated; it is then localized at the tip of the pear-shaped 'shmoo'. Using the secretory ts-mutant sec 18 is shown that a mannosylated precursor of a agglutinin accumulates at the endoplasmic reticulum.

Full text

PDF
1483

Images in this article

1484Image
on p.1484
1485Image
on p.1485
1486Image
on p.1486
1487Image
on p.1487

Selected References

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

  1. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  2. Burke D., Mendonça-Previato L., Ballou C. E. Cell-cell recognition in yeast: purification of Hansenula wingei 21-cell sexual agglutination factor and comparison of the factors from three genera. Proc Natl Acad Sci U S A. 1980 Jan;77(1):318–322. doi: 10.1073/pnas.77.1.318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  4. Chan R. K., Otte C. A. Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):11–20. doi: 10.1128/mcb.2.1.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crandall M. A., Brock T. D. Molecular basis of mating in the yeast hansenula wingei. Bacteriol Rev. 1968 Sep;32(3):139–163. doi: 10.1128/br.32.3.139-163.1968. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Doi S., Suzuki Y., Yoshimura M. Induction of sexual cell agglutinability of A mating type cells by alpha-factor in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1979 Dec 14;91(3):849–853. doi: 10.1016/0006-291x(79)91957-0. [DOI] [PubMed] [Google Scholar]
  7. Fehrenbacher G., Perry K., Thorner J. Cell-cell recognition in Saccharomyces cerevisiae: regulation of mating-specific adhesion. J Bacteriol. 1978 Jun;134(3):893–901. doi: 10.1128/jb.134.3.893-901.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hagiya M., Yoshida K., Yanagishima N. The release of sex-specific substances responsible for sexual agglutination from haploid cells of Saccharomyces cerevisiae. Exp Cell Res. 1977 Feb;104(2):263–272. doi: 10.1016/0014-4827(77)90090-8. [DOI] [PubMed] [Google Scholar]
  9. Haselbeck A., Tanner W. O-glycosylation in Saccharomyces cerevisiae is initiated at the endoplasmic reticulum. FEBS Lett. 1983 Jul 25;158(2):335–338. doi: 10.1016/0014-5793(83)80608-5. [DOI] [PubMed] [Google Scholar]
  10. Hurn B. A., Chantler S. M. Production of reagent antibodies. Methods Enzymol. 1980;70(A):104–142. doi: 10.1016/s0076-6879(80)70044-7. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Lechner J., Wieland F., Sumper M. Biosynthesis of sulfated saccharides N-glycosidically linked to the protein via glucose. Purification and identification of sulfated dolichyl monophosphoryl tetrasaccharides from halobacteria. J Biol Chem. 1985 Jan 25;260(2):860–866. [PubMed] [Google Scholar]
  13. Lipke P. N., Taylor A., Ballou C. E. Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells. J Bacteriol. 1976 Jul;127(1):610–618. doi: 10.1128/jb.127.1.610-618.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Manney T. R. Expression of the BAR1 gene in Saccharomyces cerevisiae: induction by the alpha mating pheromone of an activity associated with a secreted protein. J Bacteriol. 1983 Jul;155(1):291–301. doi: 10.1128/jb.155.1.291-301.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Moore S. Amino acid analysis: aqueous dimethyl sulfoxide as solvent for the ninhydrin reaction. J Biol Chem. 1968 Dec 10;243(23):6281–6283. [PubMed] [Google Scholar]
  16. Morrissey J. H. Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem. 1981 Nov 1;117(2):307–310. doi: 10.1016/0003-2697(81)90783-1. [DOI] [PubMed] [Google Scholar]
  17. Mort A. J., Lamport D. T. Anhydrous hydrogen fluoride deglycosylates glycoproteins. Anal Biochem. 1977 Oct;82(2):289–309. doi: 10.1016/0003-2697(77)90165-8. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Orlean P., Ammer H., Watzele M., Tanner W. Synthesis of an O-glycosylated cell surface protein induced in yeast by alpha factor. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6263–6266. doi: 10.1073/pnas.83.17.6263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pierce M., Ballou C. E. Cell-cell recognition in yeast. Characterization of the sexual agglutination factors from Saccharomyces kluyveri. J Biol Chem. 1983 Mar 25;258(6):3576–3582. [PubMed] [Google Scholar]
  21. Sprague G. F., Jr, Blair L. C., Thorner J. Cell interactions and regulation of cell type in the yeast Saccharomyces cerevisiae. Annu Rev Microbiol. 1983;37:623–660. doi: 10.1146/annurev.mi.37.100183.003203. [DOI] [PubMed] [Google Scholar]
  22. Tanner W., Lehle L. Protein glycosylation in yeast. Biochim Biophys Acta. 1987 Apr 27;906(1):81–99. doi: 10.1016/0304-4157(87)90006-2. [DOI] [PubMed] [Google Scholar]
  23. Terrance K., Lipke P. N. Sexual agglutination in Saccharomyces cerevisiae. J Bacteriol. 1981 Dec;148(3):889–896. doi: 10.1128/jb.148.3.889-896.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yoshida K., HAGIYA M., Yanagishima N. Isolation and purification of the sexual agglutination substance of mating type a cells in Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1976 Aug 23;71(4):1085–1094. doi: 10.1016/0006-291x(76)90765-8. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES