Complementing Yeast rho1 Mutation Groups with Distinct Functional Defects

Abstract

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1Aand rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-β-glucan synthase, respectively. Heteroallelic diploid strains withrho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementingrho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-β-glucan synthase activity. Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1Aand rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-β-glucan synthase, respectively. Heteroallelic diploid strains withrho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementingrho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-β-glucan synthase activity. 1,3-β-glucan synthase mitogen-activated protein kinase guanosine 5′-O-(3-thiotriphosphate) After establishment of cell polarity, morphogenesis of plant and fungal cells is determined by organization of the intracellular cytoskeleton and construction of the extracellular cell wall. A Rho-type small GTP-binding protein (Rho1p) in the budding yeastSaccharomyces cerevisiae has been shown to play a pivotal role in cell morphogenesis by regulating its effector proteins. Rho1p binds and activates Fks1p and Fks2p, two closely related catalytic subunits of 1,3-β-glucan synthase (GS),1 thereby directly controlling cell wall synthesis (1Drgonová J. Drgon T. Tanaka K. Kollár R. Chen G.-C. Ford R.A. Chan C.S.M. Takai Y. Cabib E. Science. 1996; 272: 277-279Crossref PubMed Scopus (301) Google Scholar, 2Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google Scholar). Rho1p also binds and activates Pkc1p, a yeast homolog of mammalian protein kinase C. Through the mitogen-activated protein kinase (MAPK) cascade, Pkc1p regulates organization of the actin cytoskeleton and transcription of several genes involved in cell wall integrity (3Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.-L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (181) Google Scholar, 4Nonaka H. Tanaka K. Hirano H. Fujiwara T. Kohno H. Umikawa M. Mino A. Takai Y. EMBO J. 1995; 14: 5931-5938Crossref PubMed Scopus (302) Google Scholar, 5Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 6Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar). Other Rho1p-interacting proteins include Bni1p, Skn7p, and Sec3p (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar, 8Evangelista M. Blundell K. Longtine M.S. Chow C.J. Adames N. Pringle J.R. Peter M. Boone C. Science. 1997; 276: 118-122Crossref PubMed Scopus (527) Google Scholar, 9Alberts A.S. Bouquin N. Johnston L.H. Treisman R. J. Biol. Chem. 1998; 273: 8616-8622Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 10Guo W. Tamanoi F. Novick P. Nat. Cell Biol. 2001; 3: 353-360Crossref PubMed Scopus (252) Google Scholar). Gene disruption analyses revealed that among the five Rho1p effector proteins, Fks1/2p and Pkc1p are the most important in yeast cell growth. Δfks1Δfks2 and Δpkc1 are both lethal in complete medium (11Levin D.E. Fields F.O. Kunisawa R. Bishop J.M. Thorner J. Cell. 1990; 62: 213-224Abstract Full Text PDF PubMed Scopus (310) Google Scholar, 12Mazur P. Morin N. Baginsky W. El-Sherbeini M. Clemas J.A. Nielsen J.B. Foor F. Mol. Cell. Biol. 1995; 15: 5671-5681Crossref PubMed Google Scholar), whereas Δsec3 shows slow growth in synthetic medium (13Haarer B.K. Corbett A. Kweon Y. Petzold A.S. Silver P. Brown S.S. Genetics. 1996; 144: 495-510Crossref PubMed Google Scholar), and Δbni1 and Δskn7 display normal growth (7Kohno H. Tanaka K. Mino A. Umikawa M. Imamura H. Fujiwara T. Fujita Y. Hotta K. Qadota H. Watanabe T. Ohya Y. Takai Y. EMBO J. 1996; 15: 6060-6068Crossref PubMed Scopus (241) Google Scholar, 15Morgan B.A. Bouquin N. Merrill G.F. Johnston L.H. EMBO J. 1995; 14: 5679-5689Crossref PubMed Scopus (87) Google Scholar). Thus, Rho1p controls cell morphogenesis by regulating the activities of two essential effector proteins important for cell wall synthesis and actin cytoskeleton organization. Several conditional lethal mutations (high temperature-sensitive mutations) in the RHO1 gene (rho1-2,rho1-3, rho1-4, rho1-5) have been isolated in our laboratory and characterized for elucidation of Rho1p function. Biochemical analyses of the rho1 mutants greatly contributed to the understanding of the essential pathways downstream of Rho1p (2Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google Scholar, 5Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 6Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar). However, the rho1 mutants did not always exhibit a single unique phenotype. Helliwell et al. (6Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar) reported that actin morphologies differ among therho1 mutants: rho1-3 and rho1-4display normal polarized actin patches, whereas rho1-2 andrho1-5 possess delocalized actin patches. In this study, we investigated what kind of phenotypic differences exist among therho1 mutants and why. We found that two groups ofrho1 mutations show “intragenic complementation.” Based on characterizations of the complementing rho1 mutations in terms of activation of each Rho1p effector protein, the mechanism of intragenic complementation is discussed in conjunction with multifunctional properties of Rho1p. Standard procedures were used for DNA manipulations and Escherichia coli transformation (16Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The E. coli strain SCS1 (Stratagene, San Diego, CA) was used for propagation of the plasmids used in this study (Table I). TheS. cerevisiae strains used are listed in TableII. All strains except Δpkc1/stt1 are isogenic derivatives of YPH499 and YPH500. Yeast transformation was carried out using the lithium acetate method (17Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Genetic manipulations for yeast were carried out as described (18Kaiser C.S. Michaelis S. Mitchell A. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). Yeast cells were grown either in rich medium (YPD; 1% Bacto-yeast extract (Difco), 2% Bacto-peptone (Difco), and 2% glucose (Wako Chemicals, Osaka, Japan)) or in synthetic growth medium (0.67% yeast nitrogen base (Difco) and 2% glucose) supplemented appropriately.Table IPlasmids used in this workNameParent plasmidMarkersRef.pRS314TRP1,CEN39Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpRS316URA3, CEN39Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarpYO701pRS314TRP1, RHO1promoter, RHO1 terminator, CEN36Qadota H. Anraku Y. Botstein D. Ohya Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9317-9321Crossref PubMed Scopus (42) Google ScholarpYO743 (YIpHade3S)HIS336Qadota H. Anraku Y. Botstein D. Ohya Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9317-9321Crossref PubMed Scopus (42) Google ScholarpYO774 (YCpUG-RHO1)pRS316URA3, GAL1 promoter,RHO1, CMK1 terminator,CEN36Qadota H. Anraku Y. Botstein D. Ohya Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9317-9321Crossref PubMed Scopus (42) Google Scholar Open table in a new tab Table IIStrains used in this workStrainRelevant genotypeRef.YPH499MAT a ade2 his3 leu2 lys2 trp1 ura339Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarYPH500MATα ade2 his3 leu2 lys2 trp1 ura339Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google ScholarYOC701MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 RHO1/rho1Δ∷HIS32Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC706MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 (pYO774)2Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC729MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷LYS2 ade3∷rho1-3∷HIS32Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC751MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-1∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC752MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-2∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC754MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-4∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC755MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-5∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC756MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rhoC112R∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC757MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-6∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC758MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-7∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC759MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-8∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC760MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-9∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC761MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-10∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC762MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-11∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC763MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rhoA∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC764MATα ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷RHO1∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC771MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-1∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC772MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-2∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC774MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-4∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC775MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-5∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC776MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rhoC112R∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC777MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-6∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC778MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-7∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC779MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-8∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC780MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-9∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC781MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-10∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC782MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-11∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC783MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rhoA∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC784MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷RHO1∷LEU22Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google ScholarYOC1081MAT a ade2 his3 leu2 lys2 trp1 ura3 fks1Δ∷HIS3 fks2Δ∷LYS2 ade3∷fks1-1125∷TRP12-aM. Abe, M. Minemura, T. Utsugi, K.-M. Sekiya, A. Hirata, H. Qadota, K. Morishita, T. Watanabe, and Y. Ohya, unpublished data.YOC1087MAT a ade2 his3 leu2 lys2 trp1 ura3 fks1Δ∷HIS3 fks2Δ∷LYS2 ade3∷fks1-1154∷TRP12-aM. Abe, M. Minemura, T. Utsugi, K.-M. Sekiya, A. Hirata, H. Qadota, K. Morishita, T. Watanabe, and Y. Ohya, unpublished data.YOC1943MAT a ade2 his3 leu2 lys2 trp1 ura335Yoshida S. Ikeda E. Uno I. Mitsuzawa H. Mol. Gen. Genet. 1992; 231: 337-344Crossref PubMed Scopus (71) Google ScholarYOC2005MAT a ade2 his3 leu2 lys2 trp1 ura3 stt1Δ∷HIS335Yoshida S. Ikeda E. Uno I. Mitsuzawa H. Mol. Gen. Genet. 1992; 231: 337-344Crossref PubMed Scopus (71) Google ScholarYOC2435MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-3∷LEU2This studyYOC2867MAT a ade2 his3 leu2 lys2 trp1 ura3 rho1Δ∷HIS3 ade3∷rho1-3∷HIS3This studyYOC2868MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷RHO1∷LEU2/ade3∷RHO1∷LEU2This studyYOC2869MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-2∷LEU2/ade3∷rho1-2∷LEU2This studyYOC2870MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-4∷LEU2/ade3∷rho1-4∷LEU2This studyYOC2871MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-2∷LEU2/ade3∷rho1-4∷LEU2This studyYOC2872MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-5∷LEU2/ade3∷rho1-5∷LEU2This studyYOC2873MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-10∷LEU2/ade3∷rho1-10∷LEU2This studyYOC2874MAT a /α ade2/ade2 his3/his3 leu2/leu2 lys2/lys2 trp1/trp1 ura3/ura3 rho1Δ∷HIS3/rho1Δ∷HIS3 ade3∷rho1-5∷LEU2/ade3∷rho1–10∷LEU2This study2-a M. Abe, M. Minemura, T. Utsugi, K.-M. Sekiya, A. Hirata, H. Qadota, K. Morishita, T. Watanabe, and Y. Ohya, unpublished data. Open table in a new tab To isolate temperature-sensitive rho1 mutants, we introduced random mutations into the entire region of RHO1 by the error-prone polymerase chain reaction method (19Cadwell R.C. Joyce G.F. PCR Methods Applications. 1992; 2: 28-32Crossref PubMed Scopus (819) Google Scholar). The RHO1 open reading frame was amplified by AmpliTaq polymerase with 5′-ATT AAC CCT CAC TAA AGA GAT CTC TAT AAC AAG ACA CAC TT-3′ and 5′-GGG GGA ATT CAT GTC ACA ACA AGT TG-3′. The amplified polymerase chain reaction fragment was cloned into the EcoRI-BglII site of pYO701. The resulting plasmids were transformed into the YOC706 strain containing pYO774 (YCpUG-RHO1) with the RHO1 gene deleted. Transformants were streaked onto YPD plates and incubated at 23 and 37 °C for screening temperature-sensitive mutants. The resultant candidates were selected on plates containing 5-fluoroorotic acid to eliminate pYO774. After rescue of the mutagenized plasmids, the plasmids were re-transformed into YOC706 and checked for the phenotypes. By scanning common mutations in 39 independently isolatedrho1 mutants, we identified 13 single mutations that may result in a temperature-sensitive phenotype. We then made 13 single mutations by site-directed mutagenesis and examined temperature sensitivity in their growth. Finally, 12 temperature-sensitiverho1 mutations were identified. Integration of the mutant rho1 genes into the chromosomalADE3 locus was performed as follows. Plasmids carrying the temperature-sensitive rho1 mutations were digested withBamHI and SalI, and the resulting fragments containing rho1 were cloned into plasmid pYO743. The resulting plasmids were digested with SacII, and the mutantrho1 genes were integrated into the ADE3 locus (20Ohya Y. Botstein D. Genetics. 1994; 138: PubMed Google Scholar) of the diploid strain the resultant strains, rho1 mutants were by (18Kaiser C.S. Michaelis S. Mitchell A. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1994Google Scholar). were grown at °C in medium in a in at A All the procedures were carried out at The cells were with and by for each with in of containing and After at for the was and to The was by at for in with The resultant was with containing and and with a The of the was to a described S.B. N. T. T. M. Y. C. Arisawa M. Y. Watanabe T. J. 1995; 231: PubMed Scopus Google Scholar). described S.B. N. T. T. M. Y. C. Arisawa M. Y. Watanabe T. J. 1995; 231: PubMed Scopus Google Scholar), the of were to at °C in YPD medium and to 37 °C for Cell were as described H. J.M. C. C. M. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). were onto to and with either or Yeast cells were incubated on a YPD at °C for and to 37 °C The was then with as described by et al. (3Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.-L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (181) Google Scholar). containing cells whereas even with was carried out as described J.R. R.A. T. B.K. Methods Cell Biol. 1989; PubMed Scopus Google Scholar). were with a with were using a and All were using To the of Rho1p, we systematically temperature-sensitiverho1 mutants. mutations were introduced into gene using error-prone polymerase chain reaction shown in we identified 12 temperature-sensitive rho1 of mutants revealed that of the were in mammalian the mutation of rho1-2 and were in the switch and switch in the proteins. are also the effector M.S. J.B. Mol. Cell. Biol. PubMed Scopus Google Scholar). are to proteins to in to J.B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, E.F. U. R.S. W. A. EMBO J. 1990; PubMed Scopus Google Scholar, J. J. D.E. Science. PubMed Scopus Google Scholar). By strains carrying 12 rho1 we diploid strains and checked their temperature sensitivity All diploid strains for the temperature-sensitive to grow at the restrictive diploid cells were able to grow at the restrictive heteroallelic at 37 and did is intragenic complementation. The of growth at the restrictive temperature for of the revealed two intragenic complementation groups of therho1 The rho1A of and mutants, whereas the and mutants. In mutant of the rho1A intragenic complementation was The of the mutants were not in either that Rho1p has at two essential of is in the rho1A mutants, and the in and Open table in a new tab The was each ofrho1 mutant cells grown at °C and was either at the temperature or to 37 We found that of therho1 mutant strains the strain both The is not to the to activate the a Δpkc1 mutant the strain (2Qadota H. Python C.P. Inoue S.B. Arisawa M. Anraku Y. Zheng Y. Watanabe T. Levin D.E. Ohya Y. Science. 1996; 272: 279-281Crossref PubMed Scopus (389) Google Scholar). is that the rho1B mutants mutants both to temperature-sensitive activities in A may of the mutant proteins at the restrictive temperature J. Drgon T. Cabib E. J. Cell Biol. PubMed Scopus Google Scholar). To this we isolated the the rho1 mutants grown at °C and their activities at both and We found that of the rho1B mutants temperature sensitivity for the in whereas the rho1A mutants did not We also in the of homoallelic and and heteroallelic diploid both the activities in the and diploid cells were the as in the corresponding mutants In heteroallelic the in was with that diploid of the To activation of the in the complementing rho1 mutant we to the in therho1 mutants. Pkc1p activates the of kinase and (MAPK) Levin D.E. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar, C. S. M. Mol. Cell. Biol. 1992; 12: PubMed Scopus Google Scholar, K. M. Levin D.E. H. K. Y. Mol. Cell. Biol. PubMed Scopus Google Scholar, K. Y. Watanabe Y. H. E. K. Levin D.E. Mol. Cell. Biol. PubMed Scopus Google Scholar). of is to by the growth temperature 23 to °C Y. J. Levin D.E. 1995; PubMed Scopus Google Scholar, P. C. C. EMBO J. 1996; 2: Scopus Google Scholar). was performed a using that or of the revealed essential the strain and of the rho1 mutants. However, was in rho1A cells to a in the the rho1B in that mutants in the rho1A are in activation of the We also examined the of in homoallelic and and heteroallelic diploid cells diploid cells the of as the cells of the corresponding In heteroallelic diploid cells of with cells of the rho1A mutants Biochemical analyses of the complementing demonstrated that mutations in and rho1B groups result in Pkc1p activation and respectively. To phenotypic differences exist the rho1A and cytological and phenotypic of the complementing rho1 mutant groups were is that mutants with defects in the display both actin and cell (3Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.-L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (181) Google Scholar, 5Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 6Helliwell S.B. Schmidt A. Ohya Y. Hall M.N. Curr. Biol. 1998; 8: 1211-1214Abstract Full Text Full Text PDF PubMed Google Scholar). actin and cell were in and temperature-sensitive mutants. was examined in rho1 and mutant cells incubated for to 37 °C and then with shown in cells of and the mutants as as of the normal of actin at the cell the and the mutants delocalized actin at the restrictive the rho1A and cell We also examined actin in homoallelic and and heteroallelic and diploid cells at the restrictive temperature homoallelic diploid cells and the defects in actin as the corresponding In the heteroallelic diploid cells and normal actin To directly the cell of rho1 and mutants, we carried out a in of cells is (3Paravicini G. Cooper M. Friedli L. Smith D.J. Carpentier J.-L. Klig L.S. Payton M.A. Mol. Cell. Biol. 1992; 12: 4896-4905Crossref PubMed Scopus (181) Google Scholar). the mutant strains of Δpkc1/stt1 were reported to result in cell were Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, S. Ikeda E. Uno I. Mitsuzawa H. Mol. Gen. Genet. 1992; 231: 337-344Crossref PubMed Scopus (71) Google Scholar, H. Anraku Y. Botstein D. Ohya Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9317-9321Crossref PubMed Scopus (42) Google Scholar), whereas as the strain and the rho1-3 mutant Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar) were shows that the rho1A and mutant cells in cell whereas the rho1B mutant cells did this into we that the rho1A and rho1B mutants exhibit to of mutants of Pkc1p and respectively. complementation is among mutations in a multifunctional the yeast protein is a multifunctional protein in and a mutation in this protein of Genetics. PubMed Google Scholar). intragenic complementation are with this Rho1p may not as into as the the that for intragenic that of subunits yeast Rho1p is to in as a The of the intragenic complementation analyses to rho1 mutants into and and and the intragenic complementation study of mutations that related to several Y. Botstein D. Science. 1994; PubMed Scopus Google Scholar). The that Rho1p regulates two essential Fks1/2p and Pkc1p, that the of this protein may also by groups of that each We examined each of the essential Rho1p was in the rho1A or rho1B of two essential downstream activities of Rho1p demonstrated that the ability to activate the and that the rho1B has defects in The of each mutant to activate each downstream was with phenotypic differences of the the rho1A mutants phenotypic to whereas mutants phenotypic Finally, of homoallelic and heteroallelic with rho1 mutations revealed complementation of the effector by mutations in and rho1B we most that the intragenic complementation of the rho1 mutations is to complementation of effector activities downstream of Rho1p study the showing the mechanism of intragenic complementation in mutations in the RHO1 of the the of complementing rho1 mutants to activate Pkc1p or is that the mutant proteins with Pkc1p or we that to showing a in Pkc1p Pkc1p and the of Rho1p by a However, also to the rho1A did not the not that mutation Pkc1p activation to the rho1B activation the with the the of Rho1p as the strain not We that the mutant protein or the in effector that is for their The of the of the protein has been K. S. M. T. M. S. K. T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Based on the the protein and yeast Rho1p, to Rho1p mutation onto the of the to the complementation were not in a region on the protein that the activation of effector proteins by the of Rho1p not However, we found that and were on the of the protein, with their to the of acid not the protein are to directly involved in the of effector proteins. are or the switch and switch that are reported to important for of the Rho-type with its effectors K. S. M. T. M. S. K. T. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (169) Google Scholar). and mutations the activation of Pkc1p and and involved in the of effector study a to understanding the multifunctional properties of Rho1p at the acid study, to systematically a ofrho1 mutants that have at or and to their ability to activate the effector proteins. or in the also We that on the and of a multifunctional protein Rho1p to a new of the in We are to for reading the and of the Laboratory of of of for