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Mutational analysis of theβ-subunit of yeast geranylgeranyl transferase I

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Abstract

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes theβ subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.

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References

  • Adams AEM, Johnson DI, Longnecker RM, Sloat BF, Pringle JR (1990)CDC42 andCDC43, two additional genes involved in budding and the establishment of cell polarity in the yeastSaccharomyces cerevisiae. J Cell Biol 111: 131–142

    Google Scholar 

  • Adamson P, Marshall CJ, Hall A, Tilbrook PA (1992) Post-translational modifications of p21rho proteins. J Biol Chem 267: 20033–20038

    Google Scholar 

  • Armstrong SA, Hannah VC, Goldstein JL, Brown MS (1995) CAAX geranylgeranyl transferase transfers farnesyl as efficiently as geranylgeranyl to RhoB. J Biol Chem 270: 7864–7868

    Google Scholar 

  • Boguski MS, Murray AW, Powers S (1992) Novel repetitive sequence motifs in theα andβ subunits of prenyl-protein transferases and homology of theα subunit to theMAD2 gene product of yeast. New Biologist 4: 408–411

    Google Scholar 

  • Caplin BE, Hettich LA, Marshall MS (1994) Substrate characterization of theSaccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase. Biochim Biophys Acta 1205: 39–48

    Google Scholar 

  • Chen W, Andres DA, Goldstein JL, Brown MS (1991a) Cloning and expression of a cDNA encoding theα subunit of rat p21ras protein farnesyltransferase. Proc Natl Acad Sci USA 88: 11368–11372

    Google Scholar 

  • Chen W, Andres DA, Goldstein JL, Russell DW, Brown MS (1991b) cDNA cloning and expression of the peptide-bindingβ subunit of rat p21ras farnesyltransferase, the counterpart of yeastDPRI/RAMI. Cell 66: 327–334

    Google Scholar 

  • Clarke S (1992) Protein isoprenylation and methylation at carboxylterminal cysteine residues. Annu Rev Biochem 61: 355–386

    Google Scholar 

  • Diaz M, Sanchez Y, Bennett T, Sun CR, Godoy C, Tamanoi F, Duran A, Perez P (1993) TheSchizosaccharomyces pombe cwg2 − gene codes for the beta subunit of a geranylgeranyltransferase type I required for beta-glucan synthesis. EMBO J 12: 5245–5254

    Google Scholar 

  • Dolence JM, Poulter CD (1995) A mechanism for posttranslational modification of proteins by yeast protein farnesyltransferase. Proc. Natl Acad Sci USA 92: 5008–5011

    Google Scholar 

  • Finegold AA, Johnson DI, Farnsworth CC, Gelb MH, Judd SR, Glomset JA, Tamanoi F (1991) Geranylgeranyl: protein transferase ofSaccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires theCDC43 gene product, but not theDPRI gene product. Proc Natl Acad Sci USA 88: 4448–4452

    Google Scholar 

  • Fujimura K, Tanaka K, Nakano A, Toh-e A (1994) TheSaccharomyces cerevisiae MS14 gene encodes the yeast counterpart of component A of Rab geranylgeranyltransferase. J Biol Chem 269: 9205–9212

    Google Scholar 

  • Goodman LE, Perou CM, Fujiyama A, Tamanoi F (1988) Structure and expression of yeastDPR1, a gene essential for the processing and intracellular localization of Ras proteins. Yeast 4: 271–281

    Google Scholar 

  • He B, Chen P, Chen S, Vancura KL, Michaelis S, Powers S (1991)RAM2, an essential gene of yeast, andRAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc Natl Acad Sci USA 88: 11373–11377

    Google Scholar 

  • Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153: 163–168

    Google Scholar 

  • Johnson DI, Pringle JR (1990) Molecular characterization ofCDC42, aSaccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol 111: 143–152

    Google Scholar 

  • Johnson DI, O'Brien JM, Jacobs CW (1991) Isolation and sequence analysis ofCDC43, a gene involved in the control of cell polarity inSaccharomyces cerevisiae. Gene 98: 149–150

    Google Scholar 

  • Kalman VK, Erdman RA, Maltese WA, Robishaw JD (1995) Regions outside of the CAAX motif influence the specificity of prenylation of G protein gamma subunits. J Biol Chem 270: 14835–14841

    Google Scholar 

  • Kinsella BT, Erdman RA, Maltese WA (1991) Post-translational modification of Ha-ras p21 by farnesyl versus geranylgeranyl isoprenoids is determined by the COOH-terminal amino acid. Proc Natl Acad Sci USA 88: 8934–8938

    Google Scholar 

  • Kohl NE, Diehl RE, Schaber MD, Rands E, Soderman DD, He B, Moores SL, Pompliano DL, Ferro-Novick S, Powers S, Thomas KA, Gibbs JB (1991) Structural homology among mammalian andSaccharomyces cerevisiae isoprenyl-protein transferases. J Biol Chem 266: 18884–18888

    Google Scholar 

  • Madaule P, Axel R, Myers AM (1987) Characterization of two members of therho gene family from the yeastSaccharomyces cerevisiae. Proc Natl Acad Sci USA 84: 779–783

    Google Scholar 

  • Mayer ML, Caplin BE, Marshall MS (1992)CDC43 andRAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase. J Biol Chem 267: 20589–20593

    Google Scholar 

  • Mitsuzawa H, Esson K, Tamanoi F (1995) Mutant farnesyltransferase beta subunit ofSaccharomyces cerevisiae that can substitute for geranylgeranyltransferase type I beta subunit. Proc Natl Acad Sci USA 92: 1704–1708

    Google Scholar 

  • Moomaw J, Casey PJ (1992) Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements. J Biol Chem 267: 17438–17443

    Google Scholar 

  • Moores SL, Schaber MD, Mosser SD, Rands E, O'Hara MB, Garsky VM, Marshall MS, Pompliano DL, Gibbs JB (1991) Sequence dependence of protein isoprenylation. J Biol Chem 266: 14603–14610

    Google Scholar 

  • Ohya Y, Ohsumi Y, Anraku Y (1984) Genetic study of the role of calcium ions in the cell division cycle ofSaccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants. Mol Gen Genet 193: 389–394

    Google Scholar 

  • Ohya Y, Goebl M, Goodman LE, Petersen-Bjørn S, Friesen JD, Tamanoi F, Anraku Y (1991) YeastCAL1 is a structural and functional homologue to theDPR1 (RAM) gene involved in ras processing. J Biol Chem 266: 12356–12360

    Google Scholar 

  • Ohya Y, Qadota H, Anraku Y, Pringle JR, Botstein D (1993) Suppression of yeast geranylgeranyl transferase I detect by alternative prenylation of two target GTPases, Rholp and Cdc42p. Mol Biol Cell 4: 1017–1025

    Google Scholar 

  • Omer CA, Gibbs JB (1994) Protein prenylation in eukaryotic micro-organisms: genetics, biology and biochemistry. Mol Microbiol 11: 219–225

    Google Scholar 

  • Orr-Weaver TL, Szostak JW, Rothstein RJ (1983) Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 101: 228–245

    Google Scholar 

  • Qudota H, Ishi I, Fujiyama A, Ohya Y, Anraku Y (1992)RHO gene products, putative small GTP-binding proteins, are important for activation of theCAL1/CDC43 gene product, a protein geranyl-geranyl-transferase inSaccharomyces cerevisiae. Yeast 8: 735–741

    Google Scholar 

  • Qadota H, Anraku Y, Botstein D, Ohya Y (1994) Conditional lethality of a yeast strain expressing humanRHoA in place ofRHO1. Proc Natl Acad Sci USA 91: 9317–9321

    Google Scholar 

  • Reiss Y, Goldstein JL, Seabra MC, Casey PJ, Brown MS (1990) Inhibition of purified p21ras farnesyl: protein transferase by Cys-AAX tetrapeptides. Cell 62: 81–88

    Google Scholar 

  • Reiss Y, Seabra MC, Armstrong SA, Slaughter CA, Goldstein JL, Brown MS (1991a) Nonidentical subunits of p21H-ras farnesyltransferase. J Biol Chem 266: 10672–10677

    Google Scholar 

  • Reiss Y, Stradley SJ, Gierasch LM, Brown MS, Goldstein JL (1991b) Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase. Proc Natl Acad Sci USA 88: 732–736

    Google Scholar 

  • Rose MD, Winson (1990) Methods in yeast genetics. Cold Spring Harbor Laboratory Press, Plainview, New York

    Google Scholar 

  • Rossi G, Jiang Y, Newman AP, Ferro-Novick S (1991) Dependence of Ypt1 and Sec4 membrane attachment on Bet2. Nature 351: 158–161

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Schafer WR, Rine J (1992) Protein prenylation: genes, enzymes, targets and functions. Annu Rev Genet 26: 209–237

    Google Scholar 

  • Schiest RH, Gietz RD (1989) High efficiency transformation of intact yeast cells using single-stranded nucleic acids as a carrier. Curr Genet 16: 339–346

    Google Scholar 

  • Seabra MC, Reiss Y, Casey PJ, Brown MS, Goldstein JL (1991) Protein farnesyltransferase and geranylgeranyltransferase share a commonα subunit. Cell 65: 429–434

    Google Scholar 

  • Seabra MC, Brown MS, Slaughter CA, Südhof TC, Goldstein JL (1992) Purification of component A of Rab geranylgeranyl transferase: possible identity with the choroideremia gene product. Cell 70: 1049–1057

    Google Scholar 

  • Seabra MC, Brown MS, Goldstein JL (1993) Retinal degeneration in choroideremia: deficiency of Rab geranylgeranyl transferase. Science 259: 377–381

    Google Scholar 

  • Sikorski RS, Hieter P (1989) A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA inSaccharomyces cerevisiae. Genetics 122: 19–27

    Google Scholar 

  • Trueblood CE, Ohya Y, Rine J (1993) Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I inSaccharomyces cerevisiae. Mol Cell Biol 13: 4260–4275

    Google Scholar 

  • Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. Methods Enzymol 153: 3–11

    Google Scholar 

  • Yokoyama K, Gelb MH (1993) Purification of a mammalian protein geranylgeranyltransferase. Formation and catalytic properties of an enzyme-geranylgeranyl pyrophosphate complex. J Biol Chem 268: 4055–4060

    Google Scholar 

  • Yokoyama K, Goodwin GW, Ghomashchi F, Glomset JA, Gelb MH (1991) A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity. Proc Natl Acad Sci USA 88: 5302–5306

    Google Scholar 

  • Yokoyama K, McGeady P, Gelb MH (1995) Mammalian protein geranylgeranyltransferase-I: substrate specificity, kinetic mechanism, metal requirements, and affinity labeling. Biochemistry 34: 1344–1354.

    Google Scholar 

  • Zhang FL, Diehl RE, Kohl NE, Gibbs JB, Giros B, Casey PJ, Omer CA (1994) cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I. J Biol Chem 269: 3175–3180

    Google Scholar 

  • Ziman M, Preuss D, Mulholland J, O'Brien JM, Protstein D, Johnson DI (1993) Subcellular localization of Cdc42p, aSaccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity. Mol Biol Cell 4: 1307–1316

    Google Scholar 

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Author notes

  1. John R. Pringle

    Present address: Department of Biology, The University of North Carolina, 27599-3280, Chapel Hill, North Carolina, USA

Authors and Affiliations

  1. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Bunkyo-ku, 113, Tokyo, Japan

    Yoshikazu Ohya, Hiroshi Qadota & Yasuhiro Anraku

  2. Department of Genetics, Stanford University School of Medicine, 94305-5120, Stanford, CA, USA

    Yoshikazu Ohya

  3. Division of Hematology and Oncology and Walther Oncology Center, Indiana University School of Medicine, 975 W Walnut Street, Rm, 501, 46202-5121, Indianapolis, IN, USA

    Brian E. Caplin & Mark S. Marshall

  4. Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 975 W Walnut Street, Rm. 501, 46202-5121, Indianapolis, IN, USA

    Brian E. Caplin & Mark S. Marshall

  5. Department of Biology, The University of Michigan, 48109-1048, Ann Arbor, MI, USA

    Michael F. Tibbetts & John R. Pringle

Authors
  1. Yoshikazu Ohya
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Communicated by C. P. Hollenberg

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Ohya, Y., Caplin, B.E., Qadota, H. et al. Mutational analysis of theβ-subunit of yeast geranylgeranyl transferase I. Molec. Gen. Genet. 252, 1–10 (1996). https://doi.org/10.1007/BF02173199

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  • DOI: https://doi.org/10.1007/BF02173199

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