Skip to main content
Genetics logoLink to Genetics
. 2001 Dec;159(4):1435–1448. doi: 10.1093/genetics/159.4.1435

Overactivation of the protein kinase C-signaling pathway suppresses the defects of cells lacking the Rho3/Rho4-GAP Rgd1p in Saccharomyces cerevisiae.

G de Bettignies 1, D Thoraval 1, C Morel 1, M F Peypouquet 1, M Crouzet 1
PMCID: PMC1461911  PMID: 11779787

Abstract

The nonessential RGD1 gene encodes a Rho-GTPase activating protein for the Rho3 and Rho4 proteins in Saccharomyces cerevisiae. Previous studies have revealed genetic interactions between RGD1 and the SLG1 and MID2 genes, encoding two putative sensors for cell integrity signaling, and VRP1 encoding an actin and myosin interacting protein involved in polarized growth. To better understand the role of Rgd1p, we isolated multicopy suppressor genes of the cell lethality of the double mutant rgd1Delta mid2Delta. RHO1 and RHO2 encoding two small GTPases, MKK1 encoding one of the MAP-kinase kinases in the protein kinase C (PKC) pathway, and MTL1, a MID2-homolog, were shown to suppress the rgd1Delta defects strengthening the functional links between RGD1 and the cell integrity pathway. Study of the transcriptional activity of Rlm1p, which is under the control of Mpk1p, the last kinase of the PKC pathway, and follow-up of the PST1 transcription, which is positively regulated by Rlm1p, indicate that the lack of RGD1 function diminishes the PKC pathway activity. We hypothesize that the rgd1Delta inactivation, at least through the hyperactivation of the small GTPases Rho3p and Rho4p, alters the secretory pathway and/or the actin cytoskeleton and decreases activity of the PKC pathway.

Full Text

The Full Text of this article is available as a PDF (456.0 KB).

Selected References

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

  1. Adamo J. E., Rossi G., Brennwald P. The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity. Mol Biol Cell. 1999 Dec;10(12):4121–4133. doi: 10.1091/mbc.10.12.4121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aves S. J., Durkacz B. W., Carr A., Nurse P. Cloning, sequencing and transcriptional control of the Schizosaccharomyces pombe cdc10 'start' gene. EMBO J. 1985 Feb;4(2):457–463. doi: 10.1002/j.1460-2075.1985.tb03651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barthe C., de Bettignies G., Louvet O., Peypouquet M. F., Morel C., Doignon F., Crouzet M. First characterization of the gene RGD1 in the yeast Saccharomyces cerevisiae. C R Acad Sci III. 1998 Jun;321(6):453–462. doi: 10.1016/s0764-4469(98)80776-2. [DOI] [PubMed] [Google Scholar]
  4. Berben G., Dumont J., Gilliquet V., Bolle P. A., Hilger F. The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast. 1991 Jul;7(5):475–477. doi: 10.1002/yea.320070506. [DOI] [PubMed] [Google Scholar]
  5. Bialek-Wyrzykowska U., Bauer B. E., Wagner W., Kohlwein S. D., Schweyen R. J., Ragnini A. Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance. Mol Microbiol. 2000 Mar;35(6):1295–1311. doi: 10.1046/j.1365-2958.2000.01782.x. [DOI] [PubMed] [Google Scholar]
  6. Broach J. R., Strathern J. N., Hicks J. B. Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene. Gene. 1979 Dec;8(1):121–133. doi: 10.1016/0378-1119(79)90012-x. [DOI] [PubMed] [Google Scholar]
  7. Costigan C., Gehrung S., Snyder M. A synthetic lethal screen identifies SLK1, a novel protein kinase homolog implicated in yeast cell morphogenesis and cell growth. Mol Cell Biol. 1992 Mar;12(3):1162–1178. doi: 10.1128/mcb.12.3.1162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Daignan-Fornier B., Nguyen C. C., Reisdorf P., Lemeignan B., Bolotin-Fukuhara M. MBR1 and MBR3, two related yeast genes that can suppress the growth defect of hap2, hap3 and hap4 mutants. Mol Gen Genet. 1994 Jun 3;243(5):575–583. doi: 10.1007/BF00284206. [DOI] [PubMed] [Google Scholar]
  9. Deere D., Shen J., Vesey G., Bell P., Bissinger P., Veal D. Flow cytometry and cell sorting for yeast viability assessment and cell selection. Yeast. 1998 Jan 30;14(2):147–160. doi: 10.1002/(SICI)1097-0061(19980130)14:2<147::AID-YEA207>3.0.CO;2-L. [DOI] [PubMed] [Google Scholar]
  10. Doignon F., Biteau N., Crouzet M., Aigle M. The complete sequence of a 19,482 bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae. Yeast. 1993 Feb;9(2):189–199. doi: 10.1002/yea.320090210. [DOI] [PubMed] [Google Scholar]
  11. Doignon F., Weinachter C., Roumanie O., Crouzet M. The yeast Rgd1p is a GTPase activating protein of the Rho3 and rho4 proteins. FEBS Lett. 1999 Oct 15;459(3):458–462. doi: 10.1016/s0014-5793(99)01293-4. [DOI] [PubMed] [Google Scholar]
  12. Drgonová J., Drgon T., Roh D. H., Cabib E. The GTP-binding protein Rho1p is required for cell cycle progression and polarization of the yeast cell. J Cell Biol. 1999 Jul 26;146(2):373–387. doi: 10.1083/jcb.146.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Drgonová J., Drgon T., Tanaka K., Kollár R., Chen G. C., Ford R. A., Chan C. S., Takai Y., Cabib E. Rho1p, a yeast protein at the interface between cell polarization and morphogenesis. Science. 1996 Apr 12;272(5259):277–279. doi: 10.1126/science.272.5259.277. [DOI] [PubMed] [Google Scholar]
  14. Drubin D. G., Nelson W. J. Origins of cell polarity. Cell. 1996 Feb 9;84(3):335–344. doi: 10.1016/s0092-8674(00)81278-7. [DOI] [PubMed] [Google Scholar]
  15. Errede B., Cade R. M., Yashar B. M., Kamada Y., Levin D. E., Irie K., Matsumoto K. Dynamics and organization of MAP kinase signal pathways. Mol Reprod Dev. 1995 Dec;42(4):477–485. doi: 10.1002/mrd.1080420416. [DOI] [PubMed] [Google Scholar]
  16. Evangelista M., Blundell K., Longtine M. S., Chow C. J., Adames N., Pringle J. R., Peter M., Boone C. Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science. 1997 Apr 4;276(5309):118–122. doi: 10.1126/science.276.5309.118. [DOI] [PubMed] [Google Scholar]
  17. Garcia-Ranea J. A., Valencia A. Distribution and functional diversification of the ras superfamily in Saccharomyces cerevisiae. FEBS Lett. 1998 Sep 4;434(3):219–225. doi: 10.1016/s0014-5793(98)00967-3. [DOI] [PubMed] [Google Scholar]
  18. Gray J. V., Ogas J. P., Kamada Y., Stone M., Levin D. E., Herskowitz I. A role for the Pkc1 MAP kinase pathway of Saccharomyces cerevisiae in bud emergence and identification of a putative upstream regulator. EMBO J. 1997 Aug 15;16(16):4924–4937. doi: 10.1093/emboj/16.16.4924. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heinisch J. J., Lorberg A., Schmitz H. P., Jacoby J. J. The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol Microbiol. 1999 May;32(4):671–680. doi: 10.1046/j.1365-2958.1999.01375.x. [DOI] [PubMed] [Google Scholar]
  20. Iida H., Nakamura H., Ono T., Okumura M. S., Anraku Y. MID1, a novel Saccharomyces cerevisiae gene encoding a plasma membrane protein, is required for Ca2+ influx and mating. Mol Cell Biol. 1994 Dec;14(12):8259–8271. doi: 10.1128/mcb.14.12.8259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Imai J., Toh-e A., Matsui Y. Genetic analysis of the Saccharomyces cerevisiae RHO3 gene, encoding a rho-type small GTPase, provides evidence for a role in bud formation. Genetics. 1996 Feb;142(2):359–369. doi: 10.1093/genetics/142.2.359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Imamura H., Tanaka K., Hihara T., Umikawa M., Kamei T., Takahashi K., Sasaki T., Takai Y. Bni1p and Bnr1p: downstream targets of the Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisiae. EMBO J. 1997 May 15;16(10):2745–2755. doi: 10.1093/emboj/16.10.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Irie K., Takase M., Lee K. S., Levin D. E., Araki H., Matsumoto K., Oshima Y. MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C. Mol Cell Biol. 1993 May;13(5):3076–3083. doi: 10.1128/mcb.13.5.3076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kamada Y., Jung U. S., Piotrowski J., Levin D. E. The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response. Genes Dev. 1995 Jul 1;9(13):1559–1571. doi: 10.1101/gad.9.13.1559. [DOI] [PubMed] [Google Scholar]
  25. Kamada Y., Qadota H., Python C. P., Anraku Y., Ohya Y., Levin D. E. Activation of yeast protein kinase C by Rho1 GTPase. J Biol Chem. 1996 Apr 19;271(16):9193–9196. doi: 10.1074/jbc.271.16.9193. [DOI] [PubMed] [Google Scholar]
  26. Kohno H., Tanaka K., Mino A., Umikawa M., Imamura H., Fujiwara T., Fujita Y., Hotta K., Qadota H., Watanabe T. Bni1p implicated in cytoskeletal control is a putative target of Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J. 1996 Nov 15;15(22):6060–6068. [PMC free article] [PubMed] [Google Scholar]
  27. 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]
  28. Lee K. S., Levin D. E. Dominant mutations in a gene encoding a putative protein kinase (BCK1) bypass the requirement for a Saccharomyces cerevisiae protein kinase C homolog. Mol Cell Biol. 1992 Jan;12(1):172–182. doi: 10.1128/mcb.12.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Levin D. E., Fields F. O., Kunisawa R., Bishop J. M., Thorner J. A candidate protein kinase C gene, PKC1, is required for the S. cerevisiae cell cycle. Cell. 1990 Jul 27;62(2):213–224. doi: 10.1016/0092-8674(90)90360-q. [DOI] [PubMed] [Google Scholar]
  30. Li Y., Moir R. D., Sethy-Coraci I. K., Warner J. R., Willis I. M. Repression of ribosome and tRNA synthesis in secretion-defective cells is signaled by a novel branch of the cell integrity pathway. Mol Cell Biol. 2000 Jun;20(11):3843–3851. doi: 10.1128/mcb.20.11.3843-3851.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Manning B. D., Padmanabha R., Snyder M. The Rho-GEF Rom2p localizes to sites of polarized cell growth and participates in cytoskeletal functions in Saccharomyces cerevisiae. Mol Biol Cell. 1997 Oct;8(10):1829–1844. doi: 10.1091/mbc.8.10.1829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Marcoux N., Bourbonnais Y., Charest P. M., Pallotta D. Overexpression of MID2 suppresses the profilin-deficient phenotype of yeast cells. Mol Microbiol. 1998 Jul;29(2):515–526. doi: 10.1046/j.1365-2958.1998.00944.x. [DOI] [PubMed] [Google Scholar]
  34. Marcoux N., Cloutier S., Zakrzewska E., Charest P. M., Bourbonnais Y., Pallotta D. Suppression of the profilin-deficient phenotype by the RHO2 signaling pathway in Saccharomyces cerevisiae. Genetics. 2000 Oct;156(2):579–592. doi: 10.1093/genetics/156.2.579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Martín H., Castellanos M. C., Cenamor R., Sánchez M., Molina M., Nombela C. Molecular and functional characterization of a mutant allele of the mitogen-activated protein-kinase gene SLT2(MPK1) rescued from yeast autolytic mutants. Curr Genet. 1996 May;29(6):516–522. doi: 10.1007/BF02426955. [DOI] [PubMed] [Google Scholar]
  36. Martín H., Rodríguez-Pachón J. M., Ruiz C., Nombela C., Molina M. Regulatory mechanisms for modulation of signaling through the cell integrity Slt2-mediated pathway in Saccharomyces cerevisiae. J Biol Chem. 2000 Jan 14;275(2):1511–1519. doi: 10.1074/jbc.275.2.1511. [DOI] [PubMed] [Google Scholar]
  37. Matsui Y., Toh-E A. Yeast RHO3 and RHO4 ras superfamily genes are necessary for bud growth, and their defect is suppressed by a high dose of bud formation genes CDC42 and BEM1. Mol Cell Biol. 1992 Dec;12(12):5690–5699. doi: 10.1128/mcb.12.12.5690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsui Y., Toh-e A. Isolation and characterization of two novel ras superfamily genes in Saccharomyces cerevisiae. Gene. 1992 May 1;114(1):43–49. doi: 10.1016/0378-1119(92)90705-t. [DOI] [PubMed] [Google Scholar]
  39. Nierras C. R., Warner J. R. Protein kinase C enables the regulatory circuit that connects membrane synthesis to ribosome synthesis in Saccharomyces cerevisiae. J Biol Chem. 1999 May 7;274(19):13235–13241. doi: 10.1074/jbc.274.19.13235. [DOI] [PubMed] [Google Scholar]
  40. Nonaka H., Tanaka K., Hirano H., Fujiwara T., Kohno H., Umikawa M., Mino A., Takai Y. A downstream target of RHO1 small GTP-binding protein is PKC1, a homolog of protein kinase C, which leads to activation of the MAP kinase cascade in Saccharomyces cerevisiae. EMBO J. 1995 Dec 1;14(23):5931–5938. doi: 10.1002/j.1460-2075.1995.tb00281.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ono T., Suzuki T., Anraku Y., Iida H. The MID2 gene encodes a putative integral membrane protein with a Ca(2+)-binding domain and shows mating pheromone-stimulated expression in Saccharomyces cerevisiae. Gene. 1994 Dec 30;151(1-2):203–208. doi: 10.1016/0378-1119(94)90657-2. [DOI] [PubMed] [Google Scholar]
  42. Ozaki K., Tanaka K., Imamura H., Hihara T., Kameyama T., Nonaka H., Hirano H., Matsuura Y., Takai Y. Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae. EMBO J. 1996 May 1;15(9):2196–2207. [PMC free article] [PubMed] [Google Scholar]
  43. Philip B., Levin D. E. Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol Cell Biol. 2001 Jan;21(1):271–280. doi: 10.1128/MCB.21.1.271-280.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Pruyne D., Bretscher A. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J Cell Sci. 2000 Feb;113(Pt 3):365–375. doi: 10.1242/jcs.113.3.365. [DOI] [PubMed] [Google Scholar]
  45. Rajavel M., Philip B., Buehrer B. M., Errede B., Levin D. E. Mid2 is a putative sensor for cell integrity signaling in Saccharomyces cerevisiae. Mol Cell Biol. 1999 Jun;19(6):3969–3976. doi: 10.1128/mcb.19.6.3969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Robinson N. G., Guo L., Imai J., Toh-E A., Matsui Y., Tamanoi F. Rho3 of Saccharomyces cerevisiae, which regulates the actin cytoskeleton and exocytosis, is a GTPase which interacts with Myo2 and Exo70. Mol Cell Biol. 1999 May;19(5):3580–3587. doi: 10.1128/mcb.19.5.3580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Robzyk K., Kassir Y. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nucleic Acids Res. 1992 Jul 25;20(14):3790–3790. doi: 10.1093/nar/20.14.3790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rose A. H. Growth and handling of yeasts. Methods Cell Biol. 1975;12:1–16. doi: 10.1016/s0091-679x(08)60948-6. [DOI] [PubMed] [Google Scholar]
  49. Roumanie O., Peypouquet M. F., Bonneu M., Thoraval D., Doignon F., Crouzet M. Evidence for the genetic interaction between the actin-binding protein Vrp1 and the RhoGAP Rgd1 mediated through Rho3p and Rho4p in Saccharomyces cerevisiae. Mol Microbiol. 2000 Jun;36(6):1403–1414. doi: 10.1046/j.1365-2958.2000.01958.x. [DOI] [PubMed] [Google Scholar]
  50. Tanaka K., Takai Y. Control of reorganization of the actin cytoskeleton by Rho family small GTP-binding proteins in yeast. Curr Opin Cell Biol. 1998 Feb;10(1):112–116. doi: 10.1016/s0955-0674(98)80093-8. [DOI] [PubMed] [Google Scholar]
  51. Torres L., Martín H., García-Saez M. I., Arroyo J., Molina M., Sánchez M., Nombela C. A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants. Mol Microbiol. 1991 Nov;5(11):2845–2854. doi: 10.1111/j.1365-2958.1991.tb01993.x. [DOI] [PubMed] [Google Scholar]
  52. Verna J., Lodder A., Lee K., Vagts A., Ballester R. A family of genes required for maintenance of cell wall integrity and for the stress response in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1997 Dec 9;94(25):13804–13809. doi: 10.1073/pnas.94.25.13804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Watanabe Y., Irie K., Matsumoto K. Yeast RLM1 encodes a serum response factor-like protein that may function downstream of the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol. 1995 Oct;15(10):5740–5749. doi: 10.1128/mcb.15.10.5740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Watanabe Y., Takaesu G., Hagiwara M., Irie K., Matsumoto K. Characterization of a serum response factor-like protein in Saccharomyces cerevisiae, Rlm1, which has transcriptional activity regulated by the Mpk1 (Slt2) mitogen-activated protein kinase pathway. Mol Cell Biol. 1997 May;17(5):2615–2623. doi: 10.1128/mcb.17.5.2615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. White J. H., Barker D. G., Nurse P., Johnston L. H. Periodic transcription as a means of regulating gene expression during the cell cycle: contrasting modes of expression of DNA ligase genes in budding and fission yeast. EMBO J. 1986 Jul;5(7):1705–1709. doi: 10.1002/j.1460-2075.1986.tb04414.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Winston F., Dollard C., Ricupero-Hovasse S. L. Construction of a set of convenient Saccharomyces cerevisiae strains that are isogenic to S288C. Yeast. 1995 Jan;11(1):53–55. doi: 10.1002/yea.320110107. [DOI] [PubMed] [Google Scholar]
  57. Yamochi W., Tanaka K., Nonaka H., Maeda A., Musha T., Takai Y. Growth site localization of Rho1 small GTP-binding protein and its involvement in bud formation in Saccharomyces cerevisiae. J Cell Biol. 1994 Jun;125(5):1077–1093. doi: 10.1083/jcb.125.5.1077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. de Bettignies G., Barthe C., Morel C., Peypouquet M. F., Doignon F., Crouzet M. RGD1 genetically interacts with MID2 and SLG1, encoding two putative sensors for cell integrity signalling in Saccharomyces cerevisiae. Yeast. 1999 Dec;15(16):1719–1731. doi: 10.1002/(SICI)1097-0061(199912)15:16<1719::AID-YEA499>3.0.CO;2-F. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES