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. 1994 Jul 1;13(13):3050–3064. doi: 10.1002/j.1460-2075.1994.tb06604.x

Molecular characterization of SIG1, a Saccharomyces cerevisiae gene involved in negative regulation of G-protein-mediated signal transduction.

E Leberer 1, D Dignard 1, D Harcus 1, M Whiteway 1, D Y Thomas 1
PMCID: PMC395195  PMID: 8039500

Abstract

Two recessive mutations in the Saccharomyces cerevisiae SIG1 (suppressor of inhibitory G-protein) gene have been identified by their ability to suppress the signalling defect of dominant-negative variants of the mating response G-protein beta-subunit. The mutations and deletion of SIG1 enhance the sensitivity of the cells to pheromone and stimulate the basal transcription of a mating specific gene, FUS1, suggesting that Sig1p plays a negatively regulatory role in G beta gamma-mediated signal transduction. An additional function of Sig1p in vegetatively growing cells is suggested by the finding that the mutations and deletion of SIG1 cause temperature-sensitive growth defects. The SIG1 gene encodes a protein with a molecular weight of 65 kDa that contains at the amino-terminus two zinc finger-like sequence motifs. Epistasis experiments localize the action of Sig1p within the pheromone signalling pathway at a position at or shortly after the G-protein. We propose that Sig1p represents a novel negative regulator of G beta gamma-mediated signal transduction.

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Selected References

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

  1. Bauer P. H., Müller S., Puzicha M., Pippig S., Obermaier B., Helmreich E. J., Lohse M. J. Phosducin is a protein kinase A-regulated G-protein regulator. Nature. 1992 Jul 2;358(6381):73–76. doi: 10.1038/358073a0. [DOI] [PubMed] [Google Scholar]
  2. Berg J. M. Zinc fingers and other metal-binding domains. Elements for interactions between macromolecules. J Biol Chem. 1990 Apr 25;265(12):6513–6516. [PubMed] [Google Scholar]
  3. 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]
  4. Broach J. R. Construction of high copy yeast vectors using 2-microns circle sequences. Methods Enzymol. 1983;101:307–325. doi: 10.1016/0076-6879(83)01024-1. [DOI] [PubMed] [Google Scholar]
  5. Cairns B. R., Ramer S. W., Kornberg R. D. Order of action of components in the yeast pheromone response pathway revealed with a dominant allele of the STE11 kinase and the multiple phosphorylation of the STE7 kinase. Genes Dev. 1992 Jul;6(7):1305–1318. doi: 10.1101/gad.6.7.1305. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Chang F., Herskowitz I. Identification of a gene necessary for cell cycle arrest by a negative growth factor of yeast: FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell. 1990 Nov 30;63(5):999–1011. doi: 10.1016/0092-8674(90)90503-7. [DOI] [PubMed] [Google Scholar]
  8. Chvatchko Y., Howald I., Riezman H. Two yeast mutants defective in endocytosis are defective in pheromone response. Cell. 1986 Aug 1;46(3):355–364. doi: 10.1016/0092-8674(86)90656-2. [DOI] [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. 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]
  11. Collart M. A., Struhl K. CDC39, an essential nuclear protein that negatively regulates transcription and differentially affects the constitutive and inducible HIS3 promoters. EMBO J. 1993 Jan;12(1):177–186. doi: 10.1002/j.1460-2075.1993.tb05643.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davis J. L., Kunisawa R., Thorner J. A presumptive helicase (MOT1 gene product) affects gene expression and is required for viability in the yeast Saccharomyces cerevisiae. Mol Cell Biol. 1992 Apr;12(4):1879–1892. doi: 10.1128/mcb.12.4.1879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Elion E. A., Satterberg B., Kranz J. E. FUS3 phosphorylates multiple components of the mating signal transduction cascade: evidence for STE12 and FAR1. Mol Biol Cell. 1993 May;4(5):495–510. doi: 10.1091/mbc.4.5.495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Errede B., Gartner A., Zhou Z., Nasmyth K., Ammerer G. MAP kinase-related FUS3 from S. cerevisiae is activated by STE7 in vitro. Nature. 1993 Mar 18;362(6417):261–264. doi: 10.1038/362261a0. [DOI] [PubMed] [Google Scholar]
  18. Errede B., Levin D. E. A conserved kinase cascade for MAP kinase activation in yeast. Curr Opin Cell Biol. 1993 Apr;5(2):254–260. doi: 10.1016/0955-0674(93)90112-4. [DOI] [PubMed] [Google Scholar]
  19. Froehner S. C. Expression of RNA transcripts for the postsynaptic 43 kDa protein in innervated and denervated rat skeletal muscle. FEBS Lett. 1989 Jun 5;249(2):229–233. doi: 10.1016/0014-5793(89)80629-5. [DOI] [PubMed] [Google Scholar]
  20. Gartner A., Nasmyth K., Ammerer G. Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes Dev. 1992 Jul;6(7):1280–1292. doi: 10.1101/gad.6.7.1280. [DOI] [PubMed] [Google Scholar]
  21. Hagen D. C., McCaffrey G., Sprague G. F., Jr Pheromone response elements are necessary and sufficient for basal and pheromone-induced transcription of the FUS1 gene of Saccharomyces cerevisiae. Mol Cell Biol. 1991 Jun;11(6):2952–2961. doi: 10.1128/mcb.11.6.2952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. Irie K., Nomoto S., Miyajima I., Matsumoto K. SGV1 encodes a CDC28/cdc2-related kinase required for a G alpha subunit-mediated adaptive response to pheromone in S. cerevisiae. Cell. 1991 May 31;65(5):785–795. doi: 10.1016/0092-8674(91)90386-d. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Jenness D. D., Spatrick P. Down regulation of the alpha-factor pheromone receptor in S. cerevisiae. Cell. 1986 Aug 1;46(3):345–353. doi: 10.1016/0092-8674(86)90655-0. [DOI] [PubMed] [Google Scholar]
  27. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Leberer E., Dignard D., Harcus D., Hougan L., Whiteway M., Thomas D. Y. Cloning of Saccharomyces cerevisiae STE5 as a suppressor of a Ste20 protein kinase mutant: structural and functional similarity of Ste5 to Far1. Mol Gen Genet. 1993 Nov;241(3-4):241–254. doi: 10.1007/BF00284675. [DOI] [PubMed] [Google Scholar]
  34. Leberer E., Dignard D., Harcus D., Thomas D. Y., Whiteway M. The protein kinase homologue Ste20p is required to link the yeast pheromone response G-protein beta gamma subunits to downstream signalling components. EMBO J. 1992 Dec;11(13):4815–4824. doi: 10.1002/j.1460-2075.1992.tb05587.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Lee K. S., Irie K., Gotoh Y., Watanabe Y., Araki H., Nishida E., Matsumoto K., Levin D. E. A yeast mitogen-activated protein kinase homolog (Mpk1p) mediates signalling by protein kinase C. Mol Cell Biol. 1993 May;13(5):3067–3075. doi: 10.1128/mcb.13.5.3067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee R. H., Ting T. D., Lieberman B. S., Tobias D. E., Lolley R. N., Ho Y. K. Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin. J Biol Chem. 1992 Dec 15;267(35):25104–25112. [PubMed] [Google Scholar]
  38. Link A. J., Olson M. V. Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution. Genetics. 1991 Apr;127(4):681–698. doi: 10.1093/genetics/127.4.681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. Mukai Y., Harashima S., Oshima Y. Function of the ste signal transduction pathway for mating pheromones sustains MAT alpha 1 transcription in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Apr;13(4):2050–2060. doi: 10.1128/mcb.13.4.2050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Neiman A. M., Chang F., Komachi K., Herskowitz I. CDC36 and CDC39 are negative elements in the signal transduction pathway of yeast. Cell Regul. 1990 Apr;1(5):391–401. doi: 10.1091/mbc.1.5.391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Perkins D. D. Biochemical Mutants in the Smut Fungus Ustilago Maydis. Genetics. 1949 Sep;34(5):607–626. doi: 10.1093/genetics/34.5.607. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Perlman R., Yablonski D., Simchen G., Levitzki A. Cloning of the STE5 gene of Saccharomyces cerevisiae as a suppressor of the mating defect of cdc25 temperature-sensitive mutants. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5474–5478. doi: 10.1073/pnas.90.12.5474. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. Rose M. D., Novick P., Thomas J. H., Botstein D., Fink G. R. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene. 1987;60(2-3):237–243. doi: 10.1016/0378-1119(87)90232-0. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  53. 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]
  54. Sherman F., Wakem P. Mapping yeast genes. Methods Enzymol. 1991;194:38–57. doi: 10.1016/0076-6879(91)94006-x. [DOI] [PubMed] [Google Scholar]
  55. 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]
  56. Sprague G. F., Jr Assay of yeast mating reaction. Methods Enzymol. 1991;194:77–93. doi: 10.1016/0076-6879(91)94008-z. [DOI] [PubMed] [Google Scholar]
  57. Stevenson B. J., Rhodes N., Errede B., Sprague G. F., Jr Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev. 1992 Jul;6(7):1293–1304. doi: 10.1101/gad.6.7.1293. [DOI] [PubMed] [Google Scholar]
  58. 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]
  59. 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]
  60. 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]
  61. 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]
  62. Woolford C. A., Dixon C. K., Manolson M. F., Wright R., Jones E. W. Isolation and characterization of PEP5, a gene essential for vacuolar biogenesis in Saccharomyces cerevisiae. Genetics. 1990 Aug;125(4):739–752. doi: 10.1093/genetics/125.4.739. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Zhou Z., Gartner A., Cade R., Ammerer G., Errede B. Pheromone-induced signal transduction in Saccharomyces cerevisiae requires the sequential function of three protein kinases. Mol Cell Biol. 1993 Apr;13(4):2069–2080. doi: 10.1128/mcb.13.4.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. de Barros Lopes M., Ho J. Y., Reed S. I. Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway. Mol Cell Biol. 1990 Jun;10(6):2966–2972. doi: 10.1128/mcb.10.6.2966. [DOI] [PMC free article] [PubMed] [Google Scholar]

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