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. 1991 Jul;11(7):3537–3544. doi: 10.1128/mcb.11.7.3537

Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that encodes a putative GTP-binding protein involved in the control of cell polarity.

M Ziman 1, J M O'Brien 1, L A Ouellette 1, W R Church 1, D I Johnson 1
PMCID: PMC361094  PMID: 1904541

Abstract

The Saccharomyces cerevisiae CDC42 gene product, a member of the ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity. We have analyzed the effects of three CDC42 mutations (Gly to Val-12, Gln to Leu-61, and Asp to Ala-118) in the putative GTP-binding and hydrolysis domains and one mutation (Cys to Ser-188) in the putative isoprenylation site. The first three mutations resulted in either a dominant-lethal or dose-dependent dominant-lethal phenotype when present on plasmids in haploid cdc42-1ts or wild-type strains. Both wild-type and cdc42-1ts cells carrying plasmids (pGAL) with either the CDC42Val-12 or CDC42Leu-61 alleles under the control of a GAL promoter were arrested with a novel phenotype of large cells with elongated or multiple buds. Cells carrying pGAL-CDC42Ala-118 were arrested as large, round, unbudded cells reminiscent of cdc42-1ts arrested cells. The different phenotype of the CDC42Ala-118 mutant versus the CDC42Val-12 and CDC42Leu-61 mutants was unexpected since the phenotypes of all three analogous ras mutants were similar to each other. This suggests that aspects of the biochemical properties of the Cdc42 protein differ from those of the Ras protein. The cdc42Ser-188 mutant gene was incapable of complementing the cdc42-1ts mutation and was recessive to both wild-type and cdc42-1ts. In double-mutant alleles, the cdc42Ser-188 mutation was capable of suppressing the dominant lethality associated with the three putative GTP-binding and hydrolysis mutations, suggesting that isoprenylation is necessary for the activity of the wild-type and mutant proteins.

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Selected References

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

  1. Adams A. E., Johnson D. I., Longnecker R. M., Sloat B. F., Pringle J. R. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J Cell Biol. 1990 Jul;111(1):131–142. doi: 10.1083/jcb.111.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Adams A. E., Pringle J. R. Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae. J Cell Biol. 1984 Mar;98(3):934–945. doi: 10.1083/jcb.98.3.934. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  4. Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [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. Capon D. J., Chen E. Y., Levinson A. D., Seeburg P. H., Goeddel D. V. Complete nucleotide sequences of the T24 human bladder carcinoma oncogene and its normal homologue. Nature. 1983 Mar 3;302(5903):33–37. doi: 10.1038/302033a0. [DOI] [PubMed] [Google Scholar]
  8. Church W. R., Messier T., Howard P. R., Amiral J., Meyer D., Mann K. G. A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding. J Biol Chem. 1988 May 5;263(13):6259–6267. [PubMed] [Google Scholar]
  9. Coleman K. G., Steensma H. Y., Kaback D. B., Pringle J. R. Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: isolation and characterization of the CDC24 gene and adjacent regions of the chromosome. Mol Cell Biol. 1986 Dec;6(12):4516–4525. doi: 10.1128/mcb.6.12.4516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Drubin D. G., Miller K. G., Botstein D. Yeast actin-binding proteins: evidence for a role in morphogenesis. J Cell Biol. 1988 Dec;107(6 Pt 2):2551–2561. doi: 10.1083/jcb.107.6.2551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Evans T., Brown M. L., Fraser E. D., Northup J. K. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem. 1986 May 25;261(15):7052–7059. [PubMed] [Google Scholar]
  12. Finegold A. A., Johnson D. I., Farnsworth C. C., Gelb M. H., Judd S. R., Glomset J. A., Tamanoi F. Protein geranylgeranyltransferase of Saccharomyces cerevisiae is specific for Cys-Xaa-Xaa-Leu motif proteins and requires the CDC43 gene product but not the DPR1 gene product. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4448–4452. doi: 10.1073/pnas.88.10.4448. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goodman L. E., Perou C. M., Fujiyama A., Tamanoi F. Structure and expression of yeast DPR1, a gene essential for the processing and intracellular localization of ras proteins. Yeast. 1988 Dec;4(4):271–281. doi: 10.1002/yea.320040405. [DOI] [PubMed] [Google Scholar]
  14. Haarer B. K., Lillie S. H., Adams A. E., Magdolen V., Bandlow W., Brown S. S. Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells. J Cell Biol. 1990 Jan;110(1):105–114. doi: 10.1083/jcb.110.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haarer B. K., Pringle J. R. Immunofluorescence localization of the Saccharomyces cerevisiae CDC12 gene product to the vicinity of the 10-nm filaments in the mother-bud neck. Mol Cell Biol. 1987 Oct;7(10):3678–3687. doi: 10.1128/mcb.7.10.3678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  17. Hart M. J., Polakis P. G., Evans T., Cerione R. A. The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system. J Biol Chem. 1990 Apr 15;265(11):5990–6001. [PubMed] [Google Scholar]
  18. Hill J. E., Myers A. M., Koerner T. J., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986 Sep;2(3):163–167. doi: 10.1002/yea.320020304. [DOI] [PubMed] [Google Scholar]
  19. Johnson D. I., O'Brien J. M., Jacobs C. W. Isolation and sequence analysis of CDC43, a gene involved in the control of cell polarity in Saccharomyces cerevisiae. Gene. 1990 May 31;90(1):93–98. doi: 10.1016/0378-1119(90)90443-u. [DOI] [PubMed] [Google Scholar]
  20. Johnson D. I., Pringle J. R. Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity. J Cell Biol. 1990 Jul;111(1):143–152. doi: 10.1083/jcb.111.1.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Johnston M., Davis R. W. Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Aug;4(8):1440–1448. doi: 10.1128/mcb.4.8.1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lillie S. H., Pringle J. R. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J Bacteriol. 1980 Sep;143(3):1384–1394. doi: 10.1128/jb.143.3.1384-1394.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Liu H. P., Bretscher A. Disruption of the single tropomyosin gene in yeast results in the disappearance of actin cables from the cytoskeleton. Cell. 1989 Apr 21;57(2):233–242. doi: 10.1016/0092-8674(89)90961-6. [DOI] [PubMed] [Google Scholar]
  25. Miyamoto S., Ohya Y., Ohsumi Y., Anraku Y. Nucleotide sequence of the CLS4 (CDC24) gene of Saccharomyces cerevisiae. Gene. 1987;54(1):125–132. doi: 10.1016/0378-1119(87)90354-4. [DOI] [PubMed] [Google Scholar]
  26. Munemitsu S., Innis M. A., Clark R., McCormick F., Ullrich A., Polakis P. Molecular cloning and expression of a G25K cDNA, the human homolog of the yeast cell cycle gene CDC42. Mol Cell Biol. 1990 Nov;10(11):5977–5982. doi: 10.1128/mcb.10.11.5977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  28. Novick P., Osmond B. C., Botstein D. Suppressors of yeast actin mutations. Genetics. 1989 Apr;121(4):659–674. doi: 10.1093/genetics/121.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ohya Y., Miyamoto S., Ohsumi Y., Anraku Y. Calcium-sensitive cls4 mutant of Saccharomyces cerevisiae with a defect in bud formation. J Bacteriol. 1986 Jan;165(1):28–33. doi: 10.1128/jb.165.1.28-33.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ohya Y., Ohsumi Y., Anraku Y. Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants. Mol Gen Genet. 1984;193(3):389–394. doi: 10.1007/BF00382073. [DOI] [PubMed] [Google Scholar]
  31. Ohya Y., Ohsumi Y., Anraku Y. Isolation and characterization of Ca2+-sensitive mutants of Saccharomyces cerevisiae. J Gen Microbiol. 1986 Apr;132(4):979–988. doi: 10.1099/00221287-132-4-979. [DOI] [PubMed] [Google Scholar]
  32. Polakis P. G., Snyderman R., Evans T. Characterization of G25K, a GTP-binding protein containing a novel putative nucleotide binding domain. Biochem Biophys Res Commun. 1989 Apr 14;160(1):25–32. doi: 10.1016/0006-291x(89)91615-x. [DOI] [PubMed] [Google Scholar]
  33. Pringle J. R., Preston R. A., Adams A. E., Stearns T., Drubin D. G., Haarer B. K., Jones E. W. Fluorescence microscopy methods for yeast. Methods Cell Biol. 1989;31:357–435. doi: 10.1016/s0091-679x(08)61620-9. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Schafer W. R., Trueblood C. E., Yang C. C., Mayer M. P., Rosenberg S., Poulter C. D., Kim S. H., Rine J. Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the ras protein. Science. 1990 Sep 7;249(4973):1133–1139. doi: 10.1126/science.2204115. [DOI] [PubMed] [Google Scholar]
  36. Schmitt H. D., Wagner P., Pfaff E., Gallwitz D. The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell. 1986 Nov 7;47(3):401–412. doi: 10.1016/0092-8674(86)90597-0. [DOI] [PubMed] [Google Scholar]
  37. Shinjo K., Koland J. G., Hart M. J., Narasimhan V., Johnson D. I., Evans T., Cerione R. A. Molecular cloning of the gene for the human placental GTP-binding protein Gp (G25K): identification of this GTP-binding protein as the human homolog of the yeast cell-division-cycle protein CDC42. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9853–9857. doi: 10.1073/pnas.87.24.9853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Sloat B. F., Adams A., Pringle J. R. Roles of the CDC24 gene product in cellular morphogenesis during the Saccharomyces cerevisiae cell cycle. J Cell Biol. 1981 Jun;89(3):395–405. doi: 10.1083/jcb.89.3.395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sloat B. F., Pringle J. R. A mutant of yeast defective in cellular morphogenesis. Science. 1978 Jun 9;200(4346):1171–1173. doi: 10.1126/science.349694. [DOI] [PubMed] [Google Scholar]
  41. Tamanoi F., Hsueh E. C., Goodman L. E., Cobitz A. R., Detrick R. J., Brown W. R., Fujiyama A. Posttranslational modification of ras proteins: detection of a modification prior to fatty acid acylation and cloning of a gene responsible for the modification. J Cell Biochem. 1988 Mar;36(3):261–273. doi: 10.1002/jcb.240360307. [DOI] [PubMed] [Google Scholar]
  42. Walworth N. C., Goud B., Kabcenell A. K., Novick P. J. Mutational analysis of SEC4 suggests a cyclical mechanism for the regulation of vesicular traffic. EMBO J. 1989 Jun;8(6):1685–1693. doi: 10.1002/j.1460-2075.1989.tb03560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamane H. K., Farnsworth C. C., Xie H. Y., Evans T., Howald W. N., Gelb M. H., Glomset J. A., Clarke S., Fung B. K. Membrane-binding domain of the small G protein G25K contains an S-(all-trans-geranylgeranyl)cysteine methyl ester at its carboxyl terminus. Proc Natl Acad Sci U S A. 1991 Jan 1;88(1):286–290. doi: 10.1073/pnas.88.1.286. [DOI] [PMC free article] [PubMed] [Google Scholar]

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