Skip to main content
NCBI home page
Genetics logoLink to Genetics
. 1998 Mar;148(3):947–961. doi: 10.1093/genetics/148.3.947

Substitutions in the pheromone-responsive Gbeta protein of Saccharomyces cerevisiae confer a defect in recovery from pheromone treatment.

E Li 1, E Meldrum 1, H F Stratton 1, D E Stone 1
PMCID: PMC1460049  PMID: 9539416

Abstract

The pheromone-responsive Galpha protein of Saccharomyces cerevisiae, Gpa1p, stimulates an adaptive mechanism that downregulates the mating signal. In a genetic screen designed to identify signaling elements required for Gpa1p-mediated adaptation, a large collection of adaptive-defective (Adp-) mutants were recovered. Of the 49 mutants characterized thus far, approximately three-quarters exhibit a dominant defect in the negative regulation of the pheromone response. Eight of the dominant Adp- mutations showed tight linkage to the gene encoding the pheromone-responsive Gbeta, STE4. Sequence analysis of the STE4 locus in the relevant mutant strains revealed seven novel STE4 alleles, each of which was shown to disrupt proper regulation of the pheromone response. Although the STE4 mutations had only minor effects on basal mating pathway activity, the mutant forms of Gbeta dramatically affected the ability of the cell to turn off the mating response after exposure to pheromone. Moreover, the signaling activity of the aberrant Gbetagamma subunits was suppressed by G322E, a mutant form of Gpa1p that blocks the pheromone response by sequestering Gbetagamma, but not by E364K, a hyperadaptive form of Gpa1p. On the basis of these observations, we propose that Gpa1p-mediated adaptation involves the binding of an unknown negative regulator to Gbetagamma.

Full Text

The Full Text of this article is available as a PDF (2.1 MB).

Selected References

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

  1. Blinder D., Bouvier S., Jenness D. D. Constitutive mutants in the yeast pheromone response: ordered function of the gene products. Cell. 1989 Feb 10;56(3):479–486. doi: 10.1016/0092-8674(89)90250-x. [DOI] [PubMed] [Google Scholar]
  2. Clapham D. E., Neer E. J. New roles for G-protein beta gamma-dimers in transmembrane signalling. Nature. 1993 Sep 30;365(6445):403–406. doi: 10.1038/365403a0. [DOI] [PubMed] [Google Scholar]
  3. Cole G. M., Reed S. I. Pheromone-induced phosphorylation of a G protein beta subunit in S. cerevisiae is associated with an adaptive response to mating pheromone. Cell. 1991 Feb 22;64(4):703–716. doi: 10.1016/0092-8674(91)90500-x. [DOI] [PubMed] [Google Scholar]
  4. Crespo P., Cachero T. G., Xu N., Gutkind J. S. Dual effect of beta-adrenergic receptors on mitogen-activated protein kinase. Evidence for a beta gamma-dependent activation and a G alpha s-cAMP-mediated inhibition. J Biol Chem. 1995 Oct 20;270(42):25259–25265. doi: 10.1074/jbc.270.42.25259. [DOI] [PubMed] [Google Scholar]
  5. Cross F. R. DAF1, a mutant gene affecting size control, pheromone arrest, and cell cycle kinetics of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Nov;8(11):4675–4684. doi: 10.1128/mcb.8.11.4675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cross F., Hartwell L. H., Jackson C., Konopka J. B. Conjugation in Saccharomyces cerevisiae. Annu Rev Cell Biol. 1988;4:429–457. doi: 10.1146/annurev.cb.04.110188.002241. [DOI] [PubMed] [Google Scholar]
  7. Doi K., Gartner A., Ammerer G., Errede B., Shinkawa H., Sugimoto K., Matsumoto K. MSG5, a novel protein phosphatase promotes adaptation to pheromone response in S. cerevisiae. EMBO J. 1994 Jan 1;13(1):61–70. doi: 10.1002/j.1460-2075.1994.tb06235.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gaudet R., Bohm A., Sigler P. B. Crystal structure at 2.4 angstroms resolution of the complex of transducin betagamma and its regulator, phosducin. Cell. 1996 Nov 1;87(3):577–588. doi: 10.1016/s0092-8674(00)81376-8. [DOI] [PubMed] [Google Scholar]
  9. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  10. Grishin A. V., Weiner J. L., Blumer K. J. Control of adaptation to mating pheromone by G protein beta subunits of Saccharomyces cerevisiae. Genetics. 1994 Dec;138(4):1081–1092. doi: 10.1093/genetics/138.4.1081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Iñiguez-Lluhi J., Kleuss C., Gilman A. G. The importance of G-protein beta lambda subunits. Trends Cell Biol. 1993 Jul;3(7):230–236. doi: 10.1016/0962-8924(93)90122-h. [DOI] [PubMed] [Google Scholar]
  13. Kao L. R., Peterson J., Ji R., Bender L., Bender A. Interactions between the ankyrin repeat-containing protein Akr1p and the pheromone response pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1996 Jan;16(1):168–178. doi: 10.1128/mcb.16.1.168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Konopka J. B., Jenness D. D., Hartwell L. H. The C-terminus of the S. cerevisiae alpha-pheromone receptor mediates an adaptive response to pheromone. Cell. 1988 Aug 26;54(5):609–620. doi: 10.1016/s0092-8674(88)80005-9. [DOI] [PubMed] [Google Scholar]
  15. Kurjan J. Pheromone response in yeast. Annu Rev Biochem. 1992;61:1097–1129. doi: 10.1146/annurev.bi.61.070192.005313. [DOI] [PubMed] [Google Scholar]
  16. Lambright D. G., Sondek J., Bohm A., Skiba N. P., Hamm H. E., Sigler P. B. The 2.0 A crystal structure of a heterotrimeric G protein. Nature. 1996 Jan 25;379(6563):311–319. doi: 10.1038/379311a0. [DOI] [PubMed] [Google Scholar]
  17. Lee E., Taussig R., Gilman A. G. The G226A mutant of Gs alpha highlights the requirement for dissociation of G protein subunits. J Biol Chem. 1992 Jan 15;267(2):1212–1218. [PubMed] [Google Scholar]
  18. Liu M., Simon M. I. Regulation by cAMP-dependent protein kinease of a G-protein-mediated phospholipase C. Nature. 1996 Jul 4;382(6586):83–87. doi: 10.1038/382083a0. [DOI] [PubMed] [Google Scholar]
  19. MacKay V. L., Welch S. K., Insley M. Y., Manney T. R., Holly J., Saari G. C., Parker M. L. The Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to pepsin. Proc Natl Acad Sci U S A. 1988 Jan;85(1):55–59. doi: 10.1073/pnas.85.1.55. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marcus S., Xue C. B., Naider F., Becker J. M. Degradation of a-factor by a Saccharomyces cerevisiae alpha-mating-type-specific endopeptidase: evidence for a role in recovery of cells from G1 arrest. Mol Cell Biol. 1991 Feb;11(2):1030–1039. doi: 10.1128/mcb.11.2.1030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Orr-Weaver T. L., Szostak J. W., Rothstein R. J. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 1983;101:228–245. doi: 10.1016/0076-6879(83)01017-4. [DOI] [PubMed] [Google Scholar]
  22. Pryciak P. M., Hartwell L. H. AKR1 encodes a candidate effector of the G beta gamma complex in the Saccharomyces cerevisiae pheromone response pathway and contributes to control of both cell shape and signal transduction. Mol Cell Biol. 1996 Jun;16(6):2614–2626. doi: 10.1128/mcb.16.6.2614. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Reed S. I., Hadwiger J. A., Lörincz A. T. Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc Natl Acad Sci U S A. 1985 Jun;82(12):4055–4059. doi: 10.1073/pnas.82.12.4055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Reneke J. E., Blumer K. J., Courchesne W. E., Thorner J. The carboxy-terminal segment of the yeast alpha-factor receptor is a regulatory domain. Cell. 1988 Oct 21;55(2):221–234. doi: 10.1016/0092-8674(88)90045-1. [DOI] [PubMed] [Google Scholar]
  25. Rothstein R. Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast. Methods Enzymol. 1991;194:281–301. doi: 10.1016/0076-6879(91)94022-5. [DOI] [PubMed] [Google Scholar]
  26. Schreibmayer W., Dessauer C. W., Vorobiov D., Gilman A. G., Lester H. A., Davidson N., Dascal N. Inhibition of an inwardly rectifying K+ channel by G-protein alpha-subunits. Nature. 1996 Apr 18;380(6575):624–627. doi: 10.1038/380624a0. [DOI] [PubMed] [Google Scholar]
  27. Simon M. N., De Virgilio C., Souza B., Pringle J. R., Abo A., Reed S. I. Role for the Rho-family GTPase Cdc42 in yeast mating-pheromone signal pathway. Nature. 1995 Aug 24;376(6542):702–705. doi: 10.1038/376702a0. [DOI] [PubMed] [Google Scholar]
  28. Slater M. R., Craig E. A. Transcriptional regulation of an hsp70 heat shock gene in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1987 May;7(5):1906–1916. doi: 10.1128/mcb.7.5.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sondek J., Bohm A., Lambright D. G., Hamm H. E., Sigler P. B. Crystal structure of a G-protein beta gamma dimer at 2.1A resolution. Nature. 1996 Jan 25;379(6563):369–374. doi: 10.1038/379369a0. [DOI] [PubMed] [Google Scholar]
  30. Spain B. H., Koo D., Ramakrishnan M., Dzudzor B., Colicelli J. Truncated forms of a novel yeast protein suppress the lethality of a G protein alpha subunit deficiency by interacting with the beta subunit. J Biol Chem. 1995 Oct 27;270(43):25435–25444. doi: 10.1074/jbc.270.43.25435. [DOI] [PubMed] [Google Scholar]
  31. Stratton H. F., Zhou J., Reed S. I., Stone D. E. The mating-specific G(alpha) protein of Saccharomyces cerevisiae downregulates the mating signal by a mechanism that is dependent on pheromone and independent of G(beta)(gamma) sequestration. Mol Cell Biol. 1996 Nov;16(11):6325–6337. doi: 10.1128/mcb.16.11.6325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tatchell K., Nasmyth K. A., Hall B. D., Astell C., Smith M. In vitro mutation analysis of the mating-type locus in yeast. Cell. 1981 Nov;27(1 Pt 2):25–35. doi: 10.1016/0092-8674(81)90357-3. [DOI] [PubMed] [Google Scholar]
  33. Trueheart J., Boeke J. D., Fink G. R. Two genes required for cell fusion during yeast conjugation: evidence for a pheromone-induced surface protein. Mol Cell Biol. 1987 Jul;7(7):2316–2328. doi: 10.1128/mcb.7.7.2316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Whiteway M. S., Wu C., Leeuw T., Clark K., Fourest-Lieuvin A., Thomas D. Y., Leberer E. Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p. Science. 1995 Sep 15;269(5230):1572–1575. doi: 10.1126/science.7667635. [DOI] [PubMed] [Google Scholar]
  35. Whiteway M., Clark K. L., Leberer E., Dignard D., Thomas D. Y. Genetic identification of residues involved in association of alpha and beta G-protein subunits. Mol Cell Biol. 1994 May;14(5):3223–3229. doi: 10.1128/mcb.14.5.3223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Whiteway M., Hougan L., Dignard D., Thomas D. Y., Bell L., Saari G. C., Grant F. J., O'Hara P., MacKay V. L. The STE4 and STE18 genes of yeast encode potential beta and gamma subunits of the mating factor receptor-coupled G protein. Cell. 1989 Feb 10;56(3):467–477. doi: 10.1016/0092-8674(89)90249-3. [DOI] [PubMed] [Google Scholar]
  37. Zhao Z. S., Leung T., Manser E., Lim L. Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24. Mol Cell Biol. 1995 Oct;15(10):5246–5257. doi: 10.1128/mcb.15.10.5246. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES