Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Sep;16(9):5194–5209. doi: 10.1128/mcb.16.9.5194

Sst2, a negative regulator of pheromone signaling in the yeast Saccharomyces cerevisiae: expression, localization, and genetic interaction and physical association with Gpa1 (the G-protein alpha subunit).

H G Dohlman 1, J Song 1, D Ma 1, W E Courchesne 1, J Thorner 1
PMCID: PMC231520  PMID: 8756677

Abstract

Sst2 is the prototype for the newly recognized RGS (for regulators of G-protein signaling) family. Cells lacking the pheromone-inducible SST2 gene product fail to resume growth after exposure to pheromone. Conversely, overproduction of Sst2 markedly enhanced the rate of recovery from pheromone-induced arrest in the long-term halo bioassay and detectably dampened signaling in a short-term assay of pheromone response (phosphorylation of Ste4, Gbeta subunit). When the GPA1 gene product (Galpha subunit) is absent, the pheromone response pathway is constitutively active and, consequently, growth ceases. Despite sustained induction of Sst2 (observed with specific anti-Sst2 antibodies), gpa1delta mutants remain growth arrested, indicating that the action of Sst2 requires the presence of Gpa1. The N-terminal domain (residues 3 to 307) of Sst2 (698 residues) has sequence similarity to the catalytic regions of bovine GTPase-activating protein and human neurofibromatosis tumor suppressor protein; segments in the C-terminal domain of Sst2 (between residues 417 and 685) are homologous to other RGS proteins. Both the N- and C-terminal domains were required for Sst2 function in vivo. Consistent with a role for Sst2 in binding to and affecting the activity of Gpa1, the majority of Sst2 was membrane associated and colocalized with Gpa1 at the plasma membrane, as judged by sucrose density gradient fractionation. Moreover, from cell extracts, Sst2 could be isolated in a complex with Gpa1 (expressed as a glutathione S-transferase fusion); this association withstood the detergent and salt conditions required for extraction of these proteins from cell membranes. Also, SST2+ cells expressing a GTPase-defective GPA1 mutant displayed an increased sensitivity to pheromone, whereas sst2 cells did not. These results demonstrate that Sst2 and Gpa1 interact physically and suggest that Sst2 is a direct negative regulator of Gpa1.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. Arkinstall S. J., Papasavvas S. G., Payton M. A. Yeast alpha-mating factor receptor-linked G-protein signal transduction suppresses Ras-dependent activity. FEBS Lett. 1991 Jun 17;284(1):123–128. doi: 10.1016/0014-5793(91)80777-z. [DOI] [PubMed] [Google Scholar]
  2. Arshavsky VYu, Bownds M. D. Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP. Nature. 1992 Jun 4;357(6377):416–417. doi: 10.1038/357416a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ballester R., Marchuk D., Boguski M., Saulino A., Letcher R., Wigler M., Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. doi: 10.1016/0092-8674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  4. Bardwell L., Cook J. G., Inouye C. J., Thorner J. Signal propagation and regulation in the mating pheromone response pathway of the yeast Saccharomyces cerevisiae. Dev Biol. 1994 Dec;166(2):363–379. doi: 10.1006/dbio.1994.1323. [DOI] [PubMed] [Google Scholar]
  5. Baum P., Thorner J., Honig L. Identification of tubulin from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4962–4966. doi: 10.1073/pnas.75.10.4962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Berstein G., Blank J. L., Jhon D. Y., Exton J. H., Rhee S. G., Ross E. M. Phospholipase C-beta 1 is a GTPase-activating protein for Gq/11, its physiologic regulator. Cell. 1992 Aug 7;70(3):411–418. doi: 10.1016/0092-8674(92)90165-9. [DOI] [PubMed] [Google Scholar]
  7. Bhattacharya S., Chen L., Broach J. R., Powers S. Ras membrane targeting is essential for glucose signaling but not for viability in yeast. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2984–2988. doi: 10.1073/pnas.92.7.2984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. 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]
  9. 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]
  10. Blumer K. J., Reneke J. E., Courchesne W. E., Thorner J. Functional domains of a peptide hormone receptor: the alpha-factor receptor (STE2 gene product) of the yeast Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):591–603. doi: 10.1101/sqb.1988.053.01.068. [DOI] [PubMed] [Google Scholar]
  11. Blumer K. J., Reneke J. E., Thorner J. The STE2 gene product is the ligand-binding component of the alpha-factor receptor of Saccharomyces cerevisiae. J Biol Chem. 1988 Aug 5;263(22):10836–10842. [PubMed] [Google Scholar]
  12. 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]
  13. Blumer K. J., Thorner J. Receptor-G protein signaling in yeast. Annu Rev Physiol. 1991;53:37–57. doi: 10.1146/annurev.ph.53.030191.000345. [DOI] [PubMed] [Google Scholar]
  14. Boeke J. D., Trueheart J., Natsoulis G., Fink G. R. 5-Fluoroorotic acid as a selective agent in yeast molecular genetics. Methods Enzymol. 1987;154:164–175. doi: 10.1016/0076-6879(87)54076-9. [DOI] [PubMed] [Google Scholar]
  15. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: conserved structure and molecular mechanism. Nature. 1991 Jan 10;349(6305):117–127. doi: 10.1038/349117a0. [DOI] [PubMed] [Google Scholar]
  16. Bowser R., Novick P. Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle. J Cell Biol. 1991 Mar;112(6):1117–1131. doi: 10.1083/jcb.112.6.1117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Carlson M., Botstein D. Two differentially regulated mRNAs with different 5' ends encode secreted with intracellular forms of yeast invertase. Cell. 1982 Jan;28(1):145–154. doi: 10.1016/0092-8674(82)90384-1. [DOI] [PubMed] [Google Scholar]
  18. Cassel D., Eckstein F., Lowe M., Selinger Z. Determination of the turn-off reaction for the hormone-activated adenylate cyclase. J Biol Chem. 1979 Oct 10;254(19):9835–9838. [PubMed] [Google Scholar]
  19. Chan R. K., Melnick L. M., Blair L. C., Thorner J. Extracellular suppression allows mating by pheromone-deficient sterile mutants of Saccharomyces cerevisiae. J Bacteriol. 1983 Aug;155(2):903–906. doi: 10.1128/jb.155.2.903-906.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. 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]
  22. Chen W. J., Douglas M. G. The role of protein structure in the mitochondrial import pathway. Analysis of the soluble F1-ATPase beta-subunit precursor. J Biol Chem. 1987 Nov 15;262(32):15598–15604. [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. 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]
  27. De Vries L., Mousli M., Wurmser A., Farquhar M. G. GAIP, a protein that specifically interacts with the trimeric G protein G alpha i3, is a member of a protein family with a highly conserved core domain. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11916–11920. doi: 10.1073/pnas.92.25.11916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. 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]
  30. Dohlman H. G., Apaniesk D., Chen Y., Song J., Nusskern D. Inhibition of G-protein signaling by dominant gain-of-function mutations in Sst2p, a pheromone desensitization factor in Saccharomyces cerevisiae. Mol Cell Biol. 1995 Jul;15(7):3635–3643. doi: 10.1128/mcb.15.7.3635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Druey K. M., Blumer K. J., Kang V. H., Kehrl J. H. Inhibition of G-protein-mediated MAP kinase activation by a new mammalian gene family. Nature. 1996 Feb 22;379(6567):742–746. doi: 10.1038/379742a0. [DOI] [PubMed] [Google Scholar]
  34. Evan G. I., Lewis G. K., Ramsay G., Bishop J. M. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. 1985 Dec;5(12):3610–3616. doi: 10.1128/mcb.5.12.3610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Fuller R. S., Brake A. J., Thorner J. Intracellular targeting and structural conservation of a prohormone-processing endoprotease. Science. 1989 Oct 27;246(4929):482–486. doi: 10.1126/science.2683070. [DOI] [PubMed] [Google Scholar]
  36. Fuller R. S., Brake A., Thorner J. Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease. Proc Natl Acad Sci U S A. 1989 Mar;86(5):1434–1438. doi: 10.1073/pnas.86.5.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  38. Graziano M. P., Gilman A. G. Synthesis in Escherichia coli of GTPase-deficient mutants of Gs alpha. J Biol Chem. 1989 Sep 15;264(26):15475–15482. [PubMed] [Google Scholar]
  39. Hartwell L. H. Mutants of Saccharomyces cerevisiae unresponsive to cell division control by polypeptide mating hormone. J Cell Biol. 1980 Jun;85(3):811–822. doi: 10.1083/jcb.85.3.811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Hasson M. S., Blinder D., Thorner J., Jenness D. D. Mutational activation of the STE5 gene product bypasses the requirement for G protein beta and gamma subunits in the yeast pheromone response pathway. Mol Cell Biol. 1994 Feb;14(2):1054–1065. doi: 10.1128/mcb.14.2.1054. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Herskowitz I. MAP kinase pathways in yeast: for mating and more. Cell. 1995 Jan 27;80(2):187–197. doi: 10.1016/0092-8674(95)90402-6. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. 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]
  44. 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]
  45. Jones E. W. Tackling the protease problem in Saccharomyces cerevisiae. Methods Enzymol. 1991;194:428–453. doi: 10.1016/0076-6879(91)94034-a. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. Kleuss C., Raw A. S., Lee E., Sprang S. R., Gilman A. G. Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):9828–9831. doi: 10.1073/pnas.91.21.9828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Koelle M. R., Horvitz H. R. EGL-10 regulates G protein signaling in the C. elegans nervous system and shares a conserved domain with many mammalian proteins. Cell. 1996 Jan 12;84(1):115–125. doi: 10.1016/s0092-8674(00)80998-8. [DOI] [PubMed] [Google Scholar]
  49. Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Kozasa T., Gilman A. G. Purification of recombinant G proteins from Sf9 cells by hexahistidine tagging of associated subunits. Characterization of alpha 12 and inhibition of adenylyl cyclase by alpha z. J Biol Chem. 1995 Jan 27;270(4):1734–1741. doi: 10.1074/jbc.270.4.1734. [DOI] [PubMed] [Google Scholar]
  52. 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]
  53. Kurjan J. The pheromone response pathway in Saccharomyces cerevisiae. Annu Rev Genet. 1993;27:147–179. doi: 10.1146/annurev.ge.27.120193.001051. [DOI] [PubMed] [Google Scholar]
  54. 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]
  55. Landis C. A., Masters S. B., Spada A., Pace A. M., Bourne H. R., Vallar L. GTPase inhibiting mutations activate the alpha chain of Gs and stimulate adenylyl cyclase in human pituitary tumours. Nature. 1989 Aug 31;340(6236):692–696. doi: 10.1038/340692a0. [DOI] [PubMed] [Google Scholar]
  56. Lee B. N., Adams T. H. Overexpression of flbA, an early regulator of Aspergillus asexual sporulation, leads to activation of brlA and premature initiation of development. Mol Microbiol. 1994 Oct;14(2):323–334. doi: 10.1111/j.1365-2958.1994.tb01293.x. [DOI] [PubMed] [Google Scholar]
  57. Levin D. E., Errede B. The proliferation of MAP kinase signaling pathways in yeast. Curr Opin Cell Biol. 1995 Apr;7(2):197–202. doi: 10.1016/0955-0674(95)80028-x. [DOI] [PubMed] [Google Scholar]
  58. Lohse M. J. Molecular mechanisms of membrane receptor desensitization. Biochim Biophys Acta. 1993 Nov 7;1179(2):171–188. doi: 10.1016/0167-4889(93)90139-g. [DOI] [PubMed] [Google Scholar]
  59. 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]
  60. 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]
  61. 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]
  62. Markby D. W., Onrust R., Bourne H. R. Separate GTP binding and GTPase activating domains of a G alpha subunit. Science. 1993 Dec 17;262(5141):1895–1901. doi: 10.1126/science.8266082. [DOI] [PubMed] [Google Scholar]
  63. Marsh L., Herskowitz I. From membrane to nucleus: the pathway of signal transduction in yeast and its genetic control. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):557–565. doi: 10.1101/sqb.1988.053.01.064. [DOI] [PubMed] [Google Scholar]
  64. Marshall M. S., Hill W. S., Ng A. S., Vogel U. S., Schaber M. D., Scolnick E. M., Dixon R. A., Sigal I. S., Gibbs J. B. A C-terminal domain of GAP is sufficient to stimulate ras p21 GTPase activity. EMBO J. 1989 Apr;8(4):1105–1110. doi: 10.1002/j.1460-2075.1989.tb03480.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  66. Martin G. A., Viskochil D., Bollag G., McCabe P. C., Crosier W. J., Haubruck H., Conroy L., Clark R., O'Connell P., Cawthon R. M. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990 Nov 16;63(4):843–849. doi: 10.1016/0092-8674(90)90150-d. [DOI] [PubMed] [Google Scholar]
  67. Masters S. B., Miller R. T., Chi M. H., Chang F. H., Beiderman B., Lopez N. G., Bourne H. R. Mutations in the GTP-binding site of GS alpha alter stimulation of adenylyl cyclase. J Biol Chem. 1989 Sep 15;264(26):15467–15474. [PubMed] [Google Scholar]
  68. Milligan G. Agonist regulation of cellular G protein levels and distribution: mechanisms and functional implications. Trends Pharmacol Sci. 1993 Nov;14(11):413–418. doi: 10.1016/0165-6147(93)90064-Q. [DOI] [PubMed] [Google Scholar]
  69. 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]
  70. 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]
  71. Miyajima I., Nakayama N., Nakafuku M., Kaziro Y., Arai K., Matsumoto K. Suppressors of a gpa1 mutation cause sterility in Saccharomyces cerevisiae. Genetics. 1988 Aug;119(4):797–804. doi: 10.1093/genetics/119.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  72. 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]
  73. 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]
  74. Oehlen B., Cross F. R. Signal transduction in the budding yeast Saccharomyces cerevisiae. Curr Opin Cell Biol. 1994 Dec;6(6):836–841. doi: 10.1016/0955-0674(94)90053-1. [DOI] [PubMed] [Google Scholar]
  75. Papasavvas S., Arkinstall S., Reid J., Payton M. Yeast alpha-mating factor receptor and G-protein-linked adenylyl cyclase inhibition requires RAS2 and GPA2 activities. Biochem Biophys Res Commun. 1992 May 15;184(3):1378–1385. doi: 10.1016/s0006-291x(05)80035-x. [DOI] [PubMed] [Google Scholar]
  76. Pennington S. R. GTP-binding proteins. 1: heterotrimeric G proteins. Protein Profile. 1994;1(3):169–342. [PubMed] [Google Scholar]
  77. Peterson J., Zheng Y., Bender L., Myers A., Cerione R., Bender A. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast. J Cell Biol. 1994 Dec;127(5):1395–1406. doi: 10.1083/jcb.127.5.1395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Pimplikar S. W., Simons K. Regulation of apical transport in epithelial cells by a Gs class of heterotrimeric G protein. Nature. 1993 Apr 1;362(6419):456–458. doi: 10.1038/362456a0. [DOI] [PubMed] [Google Scholar]
  79. Pringle J. R., Adams A. E., Drubin D. G., Haarer B. K. Immunofluorescence methods for yeast. Methods Enzymol. 1991;194:565–602. doi: 10.1016/0076-6879(91)94043-c. [DOI] [PubMed] [Google Scholar]
  80. Redding K., Holcomb C., Fuller R. S. Immunolocalization of Kex2 protease identifies a putative late Golgi compartment in the yeast Saccharomyces cerevisiae. J Cell Biol. 1991 May;113(3):527–538. doi: 10.1083/jcb.113.3.527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  81. 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]
  82. Rogers S., Wells R., Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science. 1986 Oct 17;234(4774):364–368. doi: 10.1126/science.2876518. [DOI] [PubMed] [Google Scholar]
  83. Roush W. Regulating G protein signaling. Science. 1996 Feb 23;271(5252):1056–1058. doi: 10.1126/science.271.5252.1056. [DOI] [PubMed] [Google Scholar]
  84. Salama S. R., Hendricks K. B., Thorner J. G1 cyclin degradation: the PEST motif of yeast Cln2 is necessary, but not sufficient, for rapid protein turnover. Mol Cell Biol. 1994 Dec;14(12):7953–7966. doi: 10.1128/mcb.14.12.7953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  85. 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]
  86. Schandel K. A., Jenness D. D. Direct evidence for ligand-induced internalization of the yeast alpha-factor pheromone receptor. Mol Cell Biol. 1994 Nov;14(11):7245–7255. doi: 10.1128/mcb.14.11.7245. [DOI] [PMC free article] [PubMed] [Google Scholar]
  87. Scherer S., Davis R. W. Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4951–4955. doi: 10.1073/pnas.76.10.4951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  88. Schultz J., Ferguson B., Sprague G. F., Jr Signal transduction and growth control in yeast. Curr Opin Genet Dev. 1995 Feb;5(1):31–37. doi: 10.1016/s0959-437x(95)90050-0. [DOI] [PubMed] [Google Scholar]
  89. 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]
  90. Siderovski D. P., Hessel A., Chung S., Mak T. W., Tyers M. A new family of regulators of G-protein-coupled receptors? Curr Biol. 1996 Feb 1;6(2):211–212. doi: 10.1016/s0960-9822(02)00454-2. [DOI] [PubMed] [Google Scholar]
  91. 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]
  92. Stirling C. J., Rothblatt J., Hosobuchi M., Deshaies R., Schekman R. Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum. Mol Biol Cell. 1992 Feb;3(2):129–142. doi: 10.1091/mbc.3.2.129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  93. 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]
  94. 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]
  95. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Tan P. K., Davis N. G., Sprague G. F., Payne G. S. Clathrin facilitates the internalization of seven transmembrane segment receptors for mating pheromones in yeast. J Cell Biol. 1993 Dec;123(6 Pt 2):1707–1716. doi: 10.1083/jcb.123.6.1707. [DOI] [PMC free article] [PubMed] [Google Scholar]
  97. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  98. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  99. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  100. Van Dop C., Tsubokawa M., Bourne H. R., Ramachandran J. Amino acid sequence of retinal transducin at the site ADP-ribosylated by cholera toxin. J Biol Chem. 1984 Jan 25;259(2):696–698. [PubMed] [Google Scholar]
  101. 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]
  102. Whiteway M. S., Thomas D. Y. Site-directed mutations altering the CAAX box of Ste18, the yeast pheromone-response pathway G gamma subunit. Genetics. 1994 Aug;137(4):967–976. doi: 10.1093/genetics/137.4.967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  103. 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]
  104. 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]
  105. Whiteway M., Hougan L., Thomas D. Y. Expression of MF alpha 1 in MATa cells supersensitive to alpha-factor leads to self-arrest. Mol Gen Genet. 1988 Sep;214(1):85–88. doi: 10.1007/BF00340184. [DOI] [PubMed] [Google Scholar]
  106. 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]
  107. Wilson B. S., Komuro M., Farquhar M. G. Cellular variations in heterotrimeric G protein localization and expression in rat pituitary. Endocrinology. 1994 Jan;134(1):233–244. doi: 10.1210/endo.134.1.8275939. [DOI] [PubMed] [Google Scholar]
  108. Wu H. K., Heng H. H., Shi X. M., Forsdyke D. R., Tsui L. C., Mak T. W., Minden M. D., Siderovski D. P. Differential expression of a basic helix-loop-helix phosphoprotein gene, G0S8, in acute leukemia and localization to human chromosome 1q31. Leukemia. 1995 Aug;9(8):1291–1298. [PubMed] [Google Scholar]
  109. Xu G. F., O'Connell P., Viskochil D., Cawthon R., Robertson M., Culver M., Dunn D., Stevens J., Gesteland R., White R. The neurofibromatosis type 1 gene encodes a protein related to GAP. Cell. 1990 Aug 10;62(3):599–608. doi: 10.1016/0092-8674(90)90024-9. [DOI] [PubMed] [Google Scholar]
  110. 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]
  111. 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]
  112. Zheng Y., Cerione R., Bender A. Control of the yeast bud-site assembly GTPase Cdc42. Catalysis of guanine nucleotide exchange by Cdc24 and stimulation of GTPase activity by Bem3. J Biol Chem. 1994 Jan 28;269(4):2369–2372. [PubMed] [Google Scholar]
  113. Zheng Y., Hart M. J., Shinjo K., Evans T., Bender A., Cerione R. A. Biochemical comparisons of the Saccharomyces cerevisiae Bem2 and Bem3 proteins. Delineation of a limit Cdc42 GTPase-activating protein domain. J Biol Chem. 1993 Nov 25;268(33):24629–24634. [PubMed] [Google Scholar]
  114. Ziman M., Preuss D., Mulholland J., O'Brien J. M., Botstein D., Johnson D. I. Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol Biol Cell. 1993 Dec;4(12):1307–1316. doi: 10.1091/mbc.4.12.1307. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES