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.

Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Jul;15(7):3635–3643. doi: 10.1128/mcb.15.7.3635

Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae.

H G Dohlman 1, D Apaniesk 1, Y Chen 1, J Song 1, D Nusskern 1
PMCID: PMC230601  PMID: 7791771

Abstract

Genetic analysis of cell-cell signaling in Saccharomyces cerevisiae has led to the identification of a novel factor, known as Sst2p, that promotes recovery after pheromone-induced growth arrest (R. K. Chan and C. A. Otte, Mol. Cell. Biol. 2:11-20, 1982). Loss-of-function mutations lead to increased pheromone sensitivity, but this phenotype is partially suppressed by overexpression of the G protein alpha subunit gene (GPA1). Suppression is allele specific, however, suggesting that there is direct interaction between the two gene products. To test this model directly, we isolated and characterized several dominant gain-of-function mutants of SST2. These mutations block the normal pheromone response, including a loss of pheromone-stimulated gene transcription, cell cycle growth arrest, and G protein myristoylation. Although the SST2 mutations confer a pheromone-resistant phenotype, they do not prevent downstream activation by overexpression of G beta (STE4), a constitutively active G beta mutation (STE4Hpl), or a disruption of GPA1. None of the SST2 alleles affects the expression or stability of G alpha. These results point to the G protein alpha subunit as being the direct target of Sst2p action and underscore the importance of this novel desensitization factor in G-protein-mediated signaling.

Full Text

The Full Text of this article is available as a PDF (736.4 KB).

Selected References

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

  1. Benovic J. L., DeBlasi A., Stone W. C., Caron M. G., Lefkowitz R. J. Beta-adrenergic receptor kinase: primary structure delineates a multigene family. Science. 1989 Oct 13;246(4927):235–240. doi: 10.1126/science.2552582. [DOI] [PubMed] [Google Scholar]
  2. Blinder D., Jenness D. D. Regulation of postreceptor signaling in the pheromone response pathway of Saccharomyces cerevisiae. Mol Cell Biol. 1989 Sep;9(9):3720–3726. doi: 10.1128/mcb.9.9.3720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumer K. J., Thorner J. Beta and gamma subunits of a yeast guanine nucleotide-binding protein are not essential for membrane association of the alpha subunit but are required for receptor coupling. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4363–4367. doi: 10.1073/pnas.87.11.4363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boone C., Davis N. G., Sprague G. F., Jr Mutations that alter the third cytoplasmic loop of the a-factor receptor lead to a constitutive and hypersensitive phenotype. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9921–9925. doi: 10.1073/pnas.90.21.9921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  6. Burkholder A. C., Hartwell L. H. The yeast alpha-factor receptor: structural properties deduced from the sequence of the STE2 gene. Nucleic Acids Res. 1985 Dec 9;13(23):8463–8475. doi: 10.1093/nar/13.23.8463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Chan R. K., Otte C. A. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a factor and alpha factor pheromones. Mol Cell Biol. 1982 Jan;2(1):21–29. doi: 10.1128/mcb.2.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ciejek E., Thorner J. Recovery of S. cerevisiae a cells from G1 arrest by alpha factor pheromone requires endopeptidase action. Cell. 1979 Nov;18(3):623–635. doi: 10.1016/0092-8674(79)90117-x. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. Cole G. M., Stone D. E., Reed S. I. Stoichiometry of G protein subunits affects the Saccharomyces cerevisiae mating pheromone signal transduction pathway. Mol Cell Biol. 1990 Feb;10(2):510–517. doi: 10.1128/mcb.10.2.510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Conklin B. R., Bourne H. R. Structural elements of G alpha subunits that interact with G beta gamma, receptors, and effectors. Cell. 1993 May 21;73(4):631–641. doi: 10.1016/0092-8674(93)90245-l. [DOI] [PubMed] [Google Scholar]
  14. Courchesne W. E., Kunisawa R., Thorner J. A putative protein kinase overcomes pheromone-induced arrest of cell cycling in S. cerevisiae. Cell. 1989 Sep 22;58(6):1107–1119. doi: 10.1016/0092-8674(89)90509-6. [DOI] [PubMed] [Google Scholar]
  15. Dietzel C., Kurjan J. Pheromonal regulation and sequence of the Saccharomyces cerevisiae SST2 gene: a model for desensitization to pheromone. Mol Cell Biol. 1987 Dec;7(12):4169–4177. doi: 10.1128/mcb.7.12.4169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dietzel C., Kurjan J. The yeast SCG1 gene: a G alpha-like protein implicated in the a- and alpha-factor response pathway. Cell. 1987 Sep 25;50(7):1001–1010. doi: 10.1016/0092-8674(87)90166-8. [DOI] [PubMed] [Google Scholar]
  17. Dohlman H. G., Goldsmith P., Spiegel A. M., Thorner J. Pheromone action regulates G-protein alpha-subunit myristoylation in the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9688–9692. doi: 10.1073/pnas.90.20.9688. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dohlman H. G., Thorner J., Caron M. G., Lefkowitz R. J. Model systems for the study of seven-transmembrane-segment receptors. Annu Rev Biochem. 1991;60:653–688. doi: 10.1146/annurev.bi.60.070191.003253. [DOI] [PubMed] [Google Scholar]
  19. Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. doi: 10.1016/0076-6879(83)01013-7. [DOI] [PubMed] [Google Scholar]
  20. Hagen D. C., McCaffrey G., Sprague G. F., Jr Evidence the yeast STE3 gene encodes a receptor for the peptide pheromone a factor: gene sequence and implications for the structure of the presumed receptor. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1418–1422. doi: 10.1073/pnas.83.5.1418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hausdorff W. P., Caron M. G., Lefkowitz R. J. Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J. 1990 Aug;4(11):2881–2889. [PubMed] [Google Scholar]
  22. Hoffman C. S., Winston F. A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene. 1987;57(2-3):267–272. doi: 10.1016/0378-1119(87)90131-4. [DOI] [PubMed] [Google Scholar]
  23. Jahng K. Y., Ferguson J., Reed S. I. Mutations in a gene encoding the alpha subunit of a Saccharomyces cerevisiae G protein indicate a role in mating pheromone signaling. Mol Cell Biol. 1988 Jun;8(6):2484–2493. doi: 10.1128/mcb.8.6.2484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Julius D., Blair L., Brake A., Sprague G., Thorner J. Yeast alpha factor is processed from a larger precursor polypeptide: the essential role of a membrane-bound dipeptidyl aminopeptidase. Cell. 1983 Mar;32(3):839–852. doi: 10.1016/0092-8674(83)90070-3. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Kurjan J., Hirsch J. P., Dietzel C. Mutations in the guanine nucleotide-binding domains of a yeast G alpha protein confer a constitutive or uninducible state to the pheromone response pathway. Genes Dev. 1991 Mar;5(3):475–483. doi: 10.1101/gad.5.3.475. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Leberer E., Dignard D., Hougan L., Thomas D. Y., Whiteway M. Dominant-negative mutants of a yeast G-protein beta subunit identify two functional regions involved in pheromone signalling. EMBO J. 1992 Dec;11(13):4805–4813. doi: 10.1002/j.1460-2075.1992.tb05586.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lefkowitz R. J. G protein-coupled receptor kinases. Cell. 1993 Aug 13;74(3):409–412. doi: 10.1016/0092-8674(93)80042-d. [DOI] [PubMed] [Google Scholar]
  30. Lohse M. J., Benovic J. L., Caron M. G., Lefkowitz R. J. Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem. 1990 Feb 25;265(6):3202–3211. [PubMed] [Google Scholar]
  31. MacKay V. L., Armstrong J., Yip C., Welch S., Walker K., Osborn S., Sheppard P., Forstrom J. Characterization of the Bar proteinase, an extracellular enzyme from the yeast Saccharomyces cerevisiae. Adv Exp Med Biol. 1991;306:161–172. doi: 10.1007/978-1-4684-6012-4_21. [DOI] [PubMed] [Google Scholar]
  32. Madura K., Varshavsky A. Degradation of G alpha by the N-end rule pathway. Science. 1994 Sep 2;265(5177):1454–1458. doi: 10.1126/science.8073290. [DOI] [PubMed] [Google Scholar]
  33. Miyajima I., Arai K., Matsumoto K. GPA1Val-50 mutation in the mating-factor signaling pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Jun;9(6):2289–2297. doi: 10.1128/mcb.9.6.2289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Miyajima I., Nakafuku M., Nakayama N., Brenner C., Miyajima A., Kaibuchi K., Arai K., Kaziro Y., Matsumoto K. GPA1, a haploid-specific essential gene, encodes a yeast homolog of mammalian G protein which may be involved in mating factor signal transduction. Cell. 1987 Sep 25;50(7):1011–1019. doi: 10.1016/0092-8674(87)90167-x. [DOI] [PubMed] [Google Scholar]
  35. Moore S. A. Yeast cells recover from mating pheromone alpha factor-induced division arrest by desensitization in the absence of alpha factor destruction. J Biol Chem. 1984 Jan 25;259(2):1004–1010. [PubMed] [Google Scholar]
  36. Nakayama N., Miyajima A., Arai K. Nucleotide sequences of STE2 and STE3, cell type-specific sterile genes from Saccharomyces cerevisiae. EMBO J. 1985 Oct;4(10):2643–2648. doi: 10.1002/j.1460-2075.1985.tb03982.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Nomoto S., Nakayama N., Arai K., Matsumoto K. Regulation of the yeast pheromone response pathway by G protein subunits. EMBO J. 1990 Mar;9(3):691–696. doi: 10.1002/j.1460-2075.1990.tb08161.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Rad M. R., Xu G., Hollenberg C. P. STE50, a novel gene required for activation of conjugation at an early step in mating in Saccharomyces cerevisiae. Mol Gen Genet. 1992 Dec;236(1):145–154. doi: 10.1007/BF00279653. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Schiestl R. H., Gietz R. D. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. doi: 10.1007/BF00340712. [DOI] [PubMed] [Google Scholar]
  41. Sherman F., Hicks J. Micromanipulation and dissection of asci. Methods Enzymol. 1991;194:21–37. doi: 10.1016/0076-6879(91)94005-w. [DOI] [PubMed] [Google Scholar]
  42. Sikorski R. S., Boeke J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 1991;194:302–318. doi: 10.1016/0076-6879(91)94023-6. [DOI] [PubMed] [Google Scholar]
  43. Sikorski R. S., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989 May;122(1):19–27. doi: 10.1093/genetics/122.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Steden M., Betz R., Duntze W. Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor. Mol Gen Genet. 1989 Nov;219(3):439–444. doi: 10.1007/BF00259617. [DOI] [PubMed] [Google Scholar]
  45. Stone D. E., Cole G. M., de Barros Lopes M., Goebl M., Reed S. I. N-myristoylation is required for function of the pheromone-responsive G alpha protein of yeast: conditional activation of the pheromone response by a temperature-sensitive N-myristoyl transferase. Genes Dev. 1991 Nov;5(11):1969–1981. doi: 10.1101/gad.5.11.1969. [DOI] [PubMed] [Google Scholar]
  46. Stone D. E., Reed S. I. G protein mutations that alter the pheromone response in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Sep;10(9):4439–4446. doi: 10.1128/mcb.10.9.4439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. 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]
  48. Weiner J. L., Guttierez-Steil C., Blumer K. J. Disruption of receptor-G protein coupling in yeast promotes the function of an SST2-dependent adaptation pathway. J Biol Chem. 1993 Apr 15;268(11):8070–8077. [PubMed] [Google Scholar]
  49. 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]
  50. Whiteway M., Hougan L., Thomas D. Y. Overexpression of the STE4 gene leads to mating response in haploid Saccharomyces cerevisiae. Mol Cell Biol. 1990 Jan;10(1):217–222. doi: 10.1128/mcb.10.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES