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. 1991 Dec;10(13):4081–4088. doi: 10.1002/j.1460-2075.1991.tb04984.x

Saccharomyces cerevisiae a- and alpha-agglutinin: characterization of their molecular interaction.

C Cappellaro 1, K Hauser 1, V Mrśa 1, M Watzele 1, G Watzele 1, C Gruber 1, W Tanner 1
PMCID: PMC453156  PMID: 1756718

Abstract

An O-glycosylated protein of approximately 18 kDa responsible for mating type specific agglutination has been isolated from Saccharomyces cerevisiae a cells, purified to homogeneity and via peptide sequences the gene was cloned by PCR. An open reading frame codes for a protein of 69 amino acids. A minimum of five serine and five threonine residues of the mature protein are glycosylated. alpha-Agglutinin is a highly N-glycosylated protein of approximately 250 kDa. Both purified agglutinins form a specific 1:1 complex in vitro. Pretreatment of alpha-agglutinin, but not of alpha-agglutinin, with diethylpyrocarbonate (DEPC) prevents formation of the complex; treatment of alpha-agglutinin in the presence of alpha-agglutinin protects the former from DEPC inactivation. By carboxy terminal shortening of the alpha-agglutinin gene and by replacing three of its eight histidyl residues by arginine, the active region of alpha-agglutinin for interaction with alpha-agglutinin has been defined. Neither the N- nor the O-linked saccharides of the two agglutinins seem to be essential for their interaction.

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Selected References

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  1. Achstetter T. Regulation of alpha-factor production in Saccharomyces cerevisiae: a-factor pheromone-induced expression of the MF alpha 1 and STE13 genes. Mol Cell Biol. 1989 Oct;9(10):4507–4514. doi: 10.1128/mcb.9.10.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Botstein D., Falco S. C., Stewart S. E., Brennan M., Scherer S., Stinchcomb D. T., Struhl K., Davis R. W. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene. 1979 Dec;8(1):17–24. doi: 10.1016/0378-1119(79)90004-0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  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. Cryer D. R., Eccleshall R., Marmur J. Isolation of yeast DNA. Methods Cell Biol. 1975;12:39–44. doi: 10.1016/s0091-679x(08)60950-4. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Domdey H., Apostol B., Lin R. J., Newman A., Brody E., Abelson J. Lariat structures are in vivo intermediates in yeast pre-mRNA splicing. Cell. 1984 Dec;39(3 Pt 2):611–621. doi: 10.1016/0092-8674(84)90468-9. [DOI] [PubMed] [Google Scholar]
  9. Dunn S. D. Effects of the modification of transfer buffer composition and the renaturation of proteins in gels on the recognition of proteins on Western blots by monoclonal antibodies. Anal Biochem. 1986 Aug 15;157(1):144–153. doi: 10.1016/0003-2697(86)90207-1. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Hauser K., Tanner W. Purification of the inducible alpha-agglutinin of S. cerevisiae and molecular cloning of the gene. FEBS Lett. 1989 Sep 25;255(2):290–294. doi: 10.1016/0014-5793(89)81108-1. [DOI] [PubMed] [Google Scholar]
  13. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  14. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  15. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Jentoft N. Why are proteins O-glycosylated? Trends Biochem Sci. 1990 Aug;15(8):291–294. doi: 10.1016/0968-0004(90)90014-3. [DOI] [PubMed] [Google Scholar]
  18. Kamboj R. K., Gariepy J., Siu C. H. Identification of an octapeptide involved in homophilic interaction of the cell adhesion molecule gp80 of dictyostelium discoideum. Cell. 1989 Nov 17;59(4):615–625. doi: 10.1016/0092-8674(89)90007-x. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Lipke P. N., Wojciechowicz D., Kurjan J. AG alpha 1 is the structural gene for the Saccharomyces cerevisiae alpha-agglutinin, a cell surface glycoprotein involved in cell-cell interactions during mating. Mol Cell Biol. 1989 Aug;9(8):3155–3165. doi: 10.1128/mcb.9.8.3155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Marsh J. L., Erfle M., Wykes E. J. The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene. 1984 Dec;32(3):481–485. doi: 10.1016/0378-1119(84)90022-2. [DOI] [PubMed] [Google Scholar]
  22. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Miles E. W. Modification of histidyl residues in proteins by diethylpyrocarbonate. Methods Enzymol. 1977;47:431–442. doi: 10.1016/0076-6879(77)47043-5. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. 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]
  28. 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]
  29. Roy A., Lu C. F., Marykwas D. L., Lipke P. N., Kurjan J. The AGA1 product is involved in cell surface attachment of the Saccharomyces cerevisiae cell adhesion glycoprotein a-agglutinin. Mol Cell Biol. 1991 Aug;11(8):4196–4206. doi: 10.1128/mcb.11.8.4196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sijmons P. C., Nederbragt A. J., Klis F. M., Van den Ende H. Isolation and composition of the constitutive agglutinins from haploid Saccharomyces cerevisiae cells. Arch Microbiol. 1987 Sep;148(3):208–212. doi: 10.1007/BF00414813. [DOI] [PubMed] [Google Scholar]
  32. Strahl-Bolsinger S., Tanner W. Protein O-glycosylation in Saccharomyces cerevisiae. Purification and characterization of the dolichyl-phosphate-D-mannose-protein O-D-mannosyltransferase. Eur J Biochem. 1991 Feb 26;196(1):185–190. doi: 10.1111/j.1432-1033.1991.tb15802.x. [DOI] [PubMed] [Google Scholar]
  33. Strazdis J. R., MacKay V. L. Induction of yeast mating pheromone a-factor by alpha cells. Nature. 1983 Oct 6;305(5934):543–545. doi: 10.1038/305543a0. [DOI] [PubMed] [Google Scholar]
  34. Terrance K., Heller P., Wu Y. S., Lipke P. N. Identification of glycoprotein components of alpha-agglutinin, a cell adhesion protein from Saccharomyces cerevisiae. J Bacteriol. 1987 Feb;169(2):475–482. doi: 10.1128/jb.169.2.475-482.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Turner B. M. Use of alkaline phosphatase-conjugated antibodies for detection of protein antigens on nitrocellulose filters. Methods Enzymol. 1986;121:848–855. doi: 10.1016/0076-6879(86)21081-2. [DOI] [PubMed] [Google Scholar]
  37. Watzele M., Klis F., Tanner W. Purification and characterization of the inducible a agglutinin of Saccharomyces cerevisiae. EMBO J. 1988 May;7(5):1483–1488. doi: 10.1002/j.1460-2075.1988.tb02966.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  39. Yen P. H., Ballou C. E. Partial characterization of the sexual agglutination factor from Hansenula wingei Y-2340 type 5 cells. Biochemistry. 1974 May 21;13(11):2428–2437. doi: 10.1021/bi00708a030. [DOI] [PubMed] [Google Scholar]
  40. von Heijne G. Signal sequences. The limits of variation. J Mol Biol. 1985 Jul 5;184(1):99–105. doi: 10.1016/0022-2836(85)90046-4. [DOI] [PubMed] [Google Scholar]

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