Abstract
Fission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Galpha and git5 Gbeta are both required for glucose-triggered cAMP signaling. The git5 Gbeta is a unique member of the Gbeta family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gbeta folding and interaction with Ggamma subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Ggamma gene. Co-immunoprecipitation studies confirm the composition of this Gbetagamma dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Galpha or the git5 Gbeta. Overexpression of the gpa2 Galpha partially suppresses loss of either the git5 Gbeta or the git11 Ggamma, while mutational activation of the Galpha fully suppresses loss of either Gbeta or Ggamma. Deletion of gpa2 (Galpha), git5 (Gbeta), or git11 (Ggamma) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Galpha subunit remains partially active in the absence of the Gbetagamma dimer and that the git5 Gbeta subunit remains partially active in the absence of the git11 Ggamma subunit. The addition of the CAAX box from the git11 Ggamma to the carboxy-terminus of the git5 Gbeta partially suppresses the loss of the Ggamma. Thus the Ggamma in this system is presumably required for localization of the Gbetagamma dimer but not for folding of the Gbeta subunit. In mammalian cells, the essential roles of the Gbeta amino-terminal coiled-coil domains and Ggamma partners in Gbeta folding may therefore reflect a mechanism used by cells that express multiple forms of both Gbeta and Ggamma subunits to regulate the composition and activity of its G proteins.
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- Bähler J., Wu J. Q., Longtine M. S., Shah N. G., McKenzie A., 3rd, Steever A. B., Wach A., Philippsen P., Pringle J. R. Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast. 1998 Jul;14(10):943–951. doi: 10.1002/(SICI)1097-0061(199807)14:10<943::AID-YEA292>3.0.CO;2-Y. [DOI] [PubMed] [Google Scholar]
- Celenza J. L., Eng F. J., Carlson M. Molecular analysis of the SNF4 gene of Saccharomyces cerevisiae: evidence for physical association of the SNF4 protein with the SNF1 protein kinase. Mol Cell Biol. 1989 Nov;9(11):5045–5054. doi: 10.1128/mcb.9.11.5045. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chang E. C., Barr M., Wang Y., Jung V., Xu H. P., Wigler M. H. Cooperative interaction of S. pombe proteins required for mating and morphogenesis. Cell. 1994 Oct 7;79(1):131–141. doi: 10.1016/0092-8674(94)90406-5. [DOI] [PubMed] [Google Scholar]
- Colombo S., Ma P., Cauwenberg L., Winderickx J., Crauwels M., Teunissen A., Nauwelaers D., de Winde J. H., Gorwa M. F., Colavizza D. Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae. EMBO J. 1998 Jun 15;17(12):3326–3341. doi: 10.1093/emboj/17.12.3326. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dal Santo P., Blanchard B., Hoffman C. S. The Schizosaccharomyces pombe pyp1 protein tyrosine phosphatase negatively regulates nutrient monitoring pathways. J Cell Sci. 1996 Jul;109(Pt 7):1919–1925. doi: 10.1242/jcs.109.7.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Durfee T., Becherer K., Chen P. L., Yeh S. H., Yang Y., Kilburn A. E., Lee W. H., Elledge S. J. The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev. 1993 Apr;7(4):555–569. doi: 10.1101/gad.7.4.555. [DOI] [PubMed] [Google Scholar]
- Garritsen A., van Galen P. J., Simonds W. F. The N-terminal coiled-coil domain of beta is essential for gamma association: a model for G-protein beta gamma subunit interaction. Proc Natl Acad Sci U S A. 1993 Aug 15;90(16):7706–7710. doi: 10.1073/pnas.90.16.7706. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
- Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell. 1993 Nov 19;75(4):805–816. doi: 10.1016/0092-8674(93)90499-g. [DOI] [PubMed] [Google Scholar]
- Hirsch J. P., Cross F. R. Pheromone response in yeast. Bioessays. 1992 Jun;14(6):367–373. doi: 10.1002/bies.950140604. [DOI] [PubMed] [Google Scholar]
- 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]
- Hoffman C. S., Winston F. Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway. Genes Dev. 1991 Apr;5(4):561–571. doi: 10.1101/gad.5.4.561. [DOI] [PubMed] [Google Scholar]
- Hoffman C. S., Winston F. Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe. Genetics. 1990 Apr;124(4):807–816. doi: 10.1093/genetics/124.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Isshiki T., Mochizuki N., Maeda T., Yamamoto M. Characterization of a fission yeast gene, gpa2, that encodes a G alpha subunit involved in the monitoring of nutrition. Genes Dev. 1992 Dec;6(12B):2455–2462. doi: 10.1101/gad.6.12b.2455. [DOI] [PubMed] [Google Scholar]
- Jin M., Fujita M., Culley B. M., Apolinario E., Yamamoto M., Maundrell K., Hoffman C. S. sck1, a high copy number suppressor of defects in the cAMP-dependent protein kinase pathway in fission yeast, encodes a protein homologous to the Saccharomyces cerevisiae SCH9 kinase. Genetics. 1995 Jun;140(2):457–467. doi: 10.1093/genetics/140.2.457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim D. U., Park S. K., Chung K. S., Choi M. U., Yoo H. S. The G protein beta subunit Gpb1 of Schizosaccharomyces pombe is a negative regulator of sexual development. Mol Gen Genet. 1996 Aug 27;252(1-2):20–32. doi: 10.1007/BF02173201. [DOI] [PubMed] [Google Scholar]
- 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]
- Landry S., Pettit M. T., Apolinario E., Hoffman C. S. The fission yeast git5 gene encodes a Gbeta subunit required for glucose-triggered adenylate cyclase activation. Genetics. 2000 Apr;154(4):1463–1471. doi: 10.1093/genetics/154.4.1463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Maeda T., Mochizuki N., Yamamoto M. Adenylyl cyclase is dispensable for vegetative cell growth in the fission yeast Schizosaccharomyces pombe. Proc Natl Acad Sci U S A. 1990 Oct;87(20):7814–7818. doi: 10.1073/pnas.87.20.7814. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McLeod M., Stein M., Beach D. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast. EMBO J. 1987 Mar;6(3):729–736. doi: 10.1002/j.1460-2075.1987.tb04814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakafuku M., Obara T., Kaibuchi K., Miyajima I., Miyajima A., Itoh H., Nakamura S., Arai K., Matsumoto K., Kaziro Y. Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: studies on its structure and possible functions. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1374–1378. doi: 10.1073/pnas.85.5.1374. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Neer E. J. Heterotrimeric G proteins: organizers of transmembrane signals. Cell. 1995 Jan 27;80(2):249–257. doi: 10.1016/0092-8674(95)90407-7. [DOI] [PubMed] [Google Scholar]
- Nocero M., Isshiki T., Yamamoto M., Hoffman C. S. Glucose repression of fbp1 transcription of Schizosaccharomyces pombe is partially regulated by adenylate cyclase activation by a G protein alpha subunit encoded by gpa2 (git8). Genetics. 1994 Sep;138(1):39–45. doi: 10.1093/genetics/138.1.39. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Obara T., Nakafuku M., Yamamoto M., Kaziro Y. Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5877–5881. doi: 10.1073/pnas.88.13.5877. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pellegrino S., Zhang S., Garritsen A., Simonds W. F. The coiled-coil region of the G protein beta subunit. Mutational analysis of Ggamma and effector interactions. J Biol Chem. 1997 Oct 3;272(40):25360–25366. doi: 10.1074/jbc.272.40.25360. [DOI] [PubMed] [Google Scholar]
- Sayle R. A., Milner-White E. J. RASMOL: biomolecular graphics for all. Trends Biochem Sci. 1995 Sep;20(9):374–374. doi: 10.1016/s0968-0004(00)89080-5. [DOI] [PubMed] [Google Scholar]
- 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]
- Southern J. A., Young D. F., Heaney F., Baumgärtner W. K., Randall R. E. Identification of an epitope on the P and V proteins of simian virus 5 that distinguishes between two isolates with different biological characteristics. J Gen Virol. 1991 Jul;72(Pt 7):1551–1557. doi: 10.1099/0022-1317-72-7-1551. [DOI] [PubMed] [Google Scholar]
- Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wach A., Brachat A., Alberti-Segui C., Rebischung C., Philippsen P. Heterologous HIS3 marker and GFP reporter modules for PCR-targeting in Saccharomyces cerevisiae. Yeast. 1997 Sep 15;13(11):1065–1075. doi: 10.1002/(SICI)1097-0061(19970915)13:11<1065::AID-YEA159>3.0.CO;2-K. [DOI] [PubMed] [Google Scholar]
- Watanabe Y., Lino Y., Furuhata K., Shimoda C., Yamamoto M. The S.pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 1988 Mar;7(3):761–767. doi: 10.1002/j.1460-2075.1988.tb02873.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Welton R. M., Hoffman C. S. Glucose monitoring in fission yeast via the Gpa2 galpha, the git5 Gbeta and the git3 putative glucose receptor. Genetics. 2000 Oct;156(2):513–521. doi: 10.1093/genetics/156.2.513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Xue Y., Batlle M., Hirsch J. P. GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p Galpha subunit and functions in a Ras-independent pathway. EMBO J. 1998 Apr 1;17(7):1996–2007. doi: 10.1093/emboj/17.7.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yun C. W., Tamaki H., Nakayama R., Yamamoto K., Kumagai H. Gpr1p, a putative G-protein coupled receptor, regulates glucose-dependent cellular cAMP level in yeast Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1998 Nov 9;252(1):29–33. doi: 10.1006/bbrc.1998.9600. [DOI] [PubMed] [Google Scholar]