Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Mar;15(3):1333–1342. doi: 10.1128/mcb.15.3.1333

Mutations in the SHR5 gene of Saccharomyces cerevisiae suppress Ras function and block membrane attachment and palmitoylation of Ras proteins.

V Jung 1, L Chen 1, S L Hofmann 1, M Wigler 1, S Powers 1
PMCID: PMC230357  PMID: 7532279

Abstract

We have identified a gene, SHR5, in a screen for extragenic suppressors of the hyperactive RAS2Val-19 mutation in the budding yeast Saccharomyces cerevisiae. SHR5 was cloned, sequenced, and found to encode a 23-kDa protein not significantly homologous to other proteins in the current data bases. Genetic evidence arguing that Shr5 operates at the level of Ras is presented. We tested whether SHR5, like previously isolated suppressors of hyperactivated RAS2, acts by affecting the membrane attachment and/or posttranslational modification of Ras proteins. We found that less Ras protein is attached to the membrane in shr5 mutants than in wild-type cells and that the Ras proteins are markedly underpalmitoylated, suggesting that Shr5 is involved in palmitoylation of Ras proteins. However, shr5null mutants exhibit normal palmitoyltransferase activity measured in vitro. Further, shr5null mutations attenuate Ras function in cells containing mutant Ras2 proteins that are not palmitoylated or farnesylated. We conclude that SHR5 encodes a protein that participates in the membrane localization of Ras but also interacts in vivo with completely unprocessed and cytosolic Ras proteins.

Full Text

The Full Text of this article is available as a PDF (621.4 KB).

Selected References

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

  1. Ballester R., Marchuk D., Boguski M., Saulino A., Letcher R., Wigler M., Collins F. The NF1 locus encodes a protein functionally related to mammalian GAP and yeast IRA proteins. Cell. 1990 Nov 16;63(4):851–859. doi: 10.1016/0092-8674(90)90151-4. [DOI] [PubMed] [Google Scholar]
  2. Ballester R., Michaeli T., Ferguson K., Xu H. P., McCormick F., Wigler M. Genetic analysis of mammalian GAP expressed in yeast. Cell. 1989 Nov 17;59(4):681–686. doi: 10.1016/0092-8674(89)90014-7. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Broek D., Samiy N., Fasano O., Fujiyama A., Tamanoi F., Northup J., Wigler M. Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins. Cell. 1985 Jul;41(3):763–769. doi: 10.1016/s0092-8674(85)80057-x. [DOI] [PubMed] [Google Scholar]
  5. Broek D., Toda T., Michaeli T., Levin L., Birchmeier C., Zoller M., Powers S., Wigler M. The S. cerevisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell. 1987 Mar 13;48(5):789–799. doi: 10.1016/0092-8674(87)90076-6. [DOI] [PubMed] [Google Scholar]
  6. Buss J. E., Sefton B. M. Direct identification of palmitic acid as the lipid attached to p21ras. Mol Cell Biol. 1986 Jan;6(1):116–122. doi: 10.1128/mcb.6.1.116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cadwallader K. A., Paterson H., Macdonald S. G., Hancock J. F. N-terminally myristoylated Ras proteins require palmitoylation or a polybasic domain for plasma membrane localization. Mol Cell Biol. 1994 Jul;14(7):4722–4730. doi: 10.1128/mcb.14.7.4722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Camonis J. H., Kalékine M., Gondré B., Garreau H., Boy-Marcotte E., Jacquet M. Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cerevisiae. EMBO J. 1986 Feb;5(2):375–380. doi: 10.1002/j.1460-2075.1986.tb04222.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chant J., Herskowitz I. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 1991 Jun 28;65(7):1203–1212. doi: 10.1016/0092-8674(91)90015-q. [DOI] [PubMed] [Google Scholar]
  10. Cox A. D., Hisaka M. M., Buss J. E., Der C. J. Specific isoprenoid modification is required for function of normal, but not oncogenic, Ras protein. Mol Cell Biol. 1992 Jun;12(6):2606–2615. doi: 10.1128/mcb.12.6.2606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Deschenes R. J., Broach J. R. Fatty acylation is important but not essential for Saccharomyces cerevisiae RAS function. Mol Cell Biol. 1987 Jul;7(7):2344–2351. doi: 10.1128/mcb.7.7.2344. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Deschenes R. J., Resh M. D., Broach J. R. Acylation and prenylation of proteins. Curr Opin Cell Biol. 1990 Dec;2(6):1108–1113. doi: 10.1016/0955-0674(90)90164-a. [DOI] [PubMed] [Google Scholar]
  13. Evans T., Hart M. J., Cerione R. A. The Ras superfamilies: regulatory proteins and post-translational modifications. Curr Opin Cell Biol. 1991 Apr;3(2):185–191. doi: 10.1016/0955-0674(91)90137-n. [DOI] [PubMed] [Google Scholar]
  14. Field J., Nikawa J., Broek D., MacDonald B., Rodgers L., Wilson I. A., Lerner R. A., Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. doi: 10.1128/mcb.8.5.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Field J., Vojtek A., Ballester R., Bolger G., Colicelli J., Ferguson K., Gerst J., Kataoka T., Michaeli T., Powers S. Cloning and characterization of CAP, the S. cerevisiae gene encoding the 70 kd adenylyl cyclase-associated protein. Cell. 1990 Apr 20;61(2):319–327. doi: 10.1016/0092-8674(90)90812-s. [DOI] [PubMed] [Google Scholar]
  16. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  17. Finegold A. A., Schafer W. R., Rine J., Whiteway M., Tamanoi F. Common modifications of trimeric G proteins and ras protein: involvement of polyisoprenylation. Science. 1990 Jul 13;249(4965):165–169. doi: 10.1126/science.1695391. [DOI] [PubMed] [Google Scholar]
  18. Fujiyama A., Matsumoto K., Tamanoi F. A novel yeast mutant defective in the processing of ras proteins: assessment of the effect of the mutation on processing steps. EMBO J. 1987 Jan;6(1):223–228. doi: 10.1002/j.1460-2075.1987.tb04742.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gietz R. D., Sugino A. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988 Dec 30;74(2):527–534. doi: 10.1016/0378-1119(88)90185-0. [DOI] [PubMed] [Google Scholar]
  21. Goodman L. E., Judd S. R., Farnsworth C. C., Powers S., Gelb M. H., Glomset J. A., Tamanoi F. Mutants of Saccharomyces cerevisiae defective in the farnesylation of Ras proteins. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9665–9669. doi: 10.1073/pnas.87.24.9665. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Green N., Alexander H., Olson A., Alexander S., Shinnick T. M., Sutcliffe J. G., Lerner R. A. Immunogenic structure of the influenza virus hemagglutinin. Cell. 1982 Mar;28(3):477–487. doi: 10.1016/0092-8674(82)90202-1. [DOI] [PubMed] [Google Scholar]
  23. Gutierrez L., Magee A. I. Characterization of an acyltransferase acting on p21N-ras protein in a cell-free system. Biochim Biophys Acta. 1991 Jun 24;1078(2):147–154. doi: 10.1016/0167-4838(91)99003-b. [DOI] [PubMed] [Google Scholar]
  24. Gutierrez L., Magee A. I., Marshall C. J., Hancock J. F. Post-translational processing of p21ras is two-step and involves carboxyl-methylation and carboxy-terminal proteolysis. EMBO J. 1989 Apr;8(4):1093–1098. doi: 10.1002/j.1460-2075.1989.tb03478.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Hancock J. F., Magee A. I., Childs J. E., Marshall C. J. All ras proteins are polyisoprenylated but only some are palmitoylated. Cell. 1989 Jun 30;57(7):1167–1177. doi: 10.1016/0092-8674(89)90054-8. [DOI] [PubMed] [Google Scholar]
  26. He B., Chen P., Chen S. Y., Vancura K. L., Michaelis S., Powers S. RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11373–11377. doi: 10.1073/pnas.88.24.11373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jones J. S., Prakash L. Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers. Yeast. 1990 Sep-Oct;6(5):363–366. doi: 10.1002/yea.320060502. [DOI] [PubMed] [Google Scholar]
  28. Kataoka T., Broek D., Wigler M. DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell. 1985 Dec;43(2 Pt 1):493–505. doi: 10.1016/0092-8674(85)90179-5. [DOI] [PubMed] [Google Scholar]
  29. Kataoka T., Powers S., Cameron S., Fasano O., Goldfarb M., Broach J., Wigler M. Functional homology of mammalian and yeast RAS genes. Cell. 1985 Jan;40(1):19–26. doi: 10.1016/0092-8674(85)90304-6. [DOI] [PubMed] [Google Scholar]
  30. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M. Genetic analysis of yeast RAS1 and RAS2 genes. Cell. 1984 Jun;37(2):437–445. doi: 10.1016/0092-8674(84)90374-x. [DOI] [PubMed] [Google Scholar]
  31. Kato K., Cox A. D., Hisaka M. M., Graham S. M., Buss J. E., Der C. J. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci U S A. 1992 Jul 15;89(14):6403–6407. doi: 10.1073/pnas.89.14.6403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kato K., Der C. J., Buss J. E. Prenoids and palmitate: lipids that control the biological activity of Ras proteins. Semin Cancer Biol. 1992 Aug;3(4):179–188. [PubMed] [Google Scholar]
  33. Kuroda Y., Suzuki N., Kataoka T. The effect of posttranslational modifications on the interaction of Ras2 with adenylyl cyclase. Science. 1993 Jan 29;259(5095):683–686. doi: 10.1126/science.8430318. [DOI] [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Lowy D. R., Willumsen B. M. Protein modification: new clue to Ras lipid glue. Nature. 1989 Oct 5;341(6241):384–385. doi: 10.1038/341384a0. [DOI] [PubMed] [Google Scholar]
  36. Magee A. I., Gutierrez L., McKay I. A., Marshall C. J., Hall A. Dynamic fatty acylation of p21N-ras. EMBO J. 1987 Nov;6(11):3353–3357. doi: 10.1002/j.1460-2075.1987.tb02656.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marshall C. J. Protein prenylation: a mediator of protein-protein interactions. Science. 1993 Mar 26;259(5103):1865–1866. doi: 10.1126/science.8456312. [DOI] [PubMed] [Google Scholar]
  38. Mitchell D. A., Farh L., Marshall T. K., Deschenes R. J. A polybasic domain allows nonprenylated Ras proteins to function in Saccharomyces cerevisiae. J Biol Chem. 1994 Aug 26;269(34):21540–21546. [PubMed] [Google Scholar]
  39. Niman H. L., Houghten R. A., Walker L. E., Reisfeld R. A., Wilson I. A., Hogle J. M., Lerner R. A. Generation of protein-reactive antibodies by short peptides is an event of high frequency: implications for the structural basis of immune recognition. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4949–4953. doi: 10.1073/pnas.80.16.4949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Powers S., Kataoka T., Fasano O., Goldfarb M., Strathern J., Broach J., Wigler M. Genes in S. cerevisiae encoding proteins with domains homologous to the mammalian ras proteins. Cell. 1984 Mar;36(3):607–612. doi: 10.1016/0092-8674(84)90340-4. [DOI] [PubMed] [Google Scholar]
  41. Powers S., Michaelis S., Broek D., Santa Anna S., Field J., Herskowitz I., Wigler M. RAM, a gene of yeast required for a functional modification of RAS proteins and for production of mating pheromone a-factor. Cell. 1986 Nov 7;47(3):413–422. doi: 10.1016/0092-8674(86)90598-2. [DOI] [PubMed] [Google Scholar]
  42. Powers S., O'Neill K., Wigler M. Dominant yeast and mammalian RAS mutants that interfere with the CDC25-dependent activation of wild-type RAS in Saccharomyces cerevisiae. Mol Cell Biol. 1989 Feb;9(2):390–395. doi: 10.1128/mcb.9.2.390. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Powers S. Protein prenylation: a modification that sticks. Curr Biol. 1991 Apr;1(2):114–116. doi: 10.1016/0960-9822(91)90295-8. [DOI] [PubMed] [Google Scholar]
  44. Rothstein R. J. One-step gene disruption in yeast. Methods Enzymol. 1983;101:202–211. doi: 10.1016/0076-6879(83)01015-0. [DOI] [PubMed] [Google Scholar]
  45. Ruggieri R., Bender A., Matsui Y., Powers S., Takai Y., Pringle J. R., Matsumoto K. RSR1, a ras-like gene homologous to Krev-1 (smg21A/rap1A): role in the development of cell polarity and interactions with the Ras pathway in Saccharomyces cerevisiae. Mol Cell Biol. 1992 Feb;12(2):758–766. doi: 10.1128/mcb.12.2.758. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. 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]
  47. Sass P., Field J., Nikawa J., Toda T., Wigler M. Cloning and characterization of the high-affinity cAMP phosphodiesterase of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1986 Dec;83(24):9303–9307. doi: 10.1073/pnas.83.24.9303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schafer W. R., Rine J. Protein prenylation: genes, enzymes, targets, and functions. Annu Rev Genet. 1992;26:209–237. doi: 10.1146/annurev.ge.26.120192.001233. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Tanaka K., Nakafuku M., Satoh T., Marshall M. S., Gibbs J. B., Matsumoto K., Kaziro Y., Toh-e A. S. cerevisiae genes IRA1 and IRA2 encode proteins that may be functionally equivalent to mammalian ras GTPase activating protein. Cell. 1990 Mar 9;60(5):803–807. doi: 10.1016/0092-8674(90)90094-u. [DOI] [PubMed] [Google Scholar]
  52. Toda T., Cameron S., Sass P., Wigler M. SCH9, a gene of Saccharomyces cerevisiae that encodes a protein distinct from, but functionally and structurally related to, cAMP-dependent protein kinase catalytic subunits. Genes Dev. 1988 May;2(5):517–527. doi: 10.1101/gad.2.5.517. [DOI] [PubMed] [Google Scholar]
  53. Toda T., Cameron S., Sass P., Zoller M., Scott J. D., McMullen B., Hurwitz M., Krebs E. G., Wigler M. Cloning and characterization of BCY1, a locus encoding a regulatory subunit of the cyclic AMP-dependent protein kinase in Saccharomyces cerevisiae. Mol Cell Biol. 1987 Apr;7(4):1371–1377. doi: 10.1128/mcb.7.4.1371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Toda T., Cameron S., Sass P., Zoller M., Wigler M. Three different genes in S. cerevisiae encode the catalytic subunits of the cAMP-dependent protein kinase. Cell. 1987 Jul 17;50(2):277–287. doi: 10.1016/0092-8674(87)90223-6. [DOI] [PubMed] [Google Scholar]
  55. Toda T., Uno I., Ishikawa T., Powers S., Kataoka T., Broek D., Cameron S., Broach J., Matsumoto K., Wigler M. In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell. 1985 Jan;40(1):27–36. doi: 10.1016/0092-8674(85)90305-8. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. Wigler M., Field J., Powers S., Broek D., Toda T., Cameron S., Nikawa J., Michaeli T., Colicelli J., Ferguson K. Studies of RAS function in the yeast Saccharomyces cerevisiae. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 2):649–655. doi: 10.1101/sqb.1988.053.01.074. [DOI] [PubMed] [Google Scholar]
  58. Willumsen B. M., Christensen A., Hubbert N. L., Papageorge A. G., Lowy D. R. The p21 ras C-terminus is required for transformation and membrane association. Nature. 1984 Aug 16;310(5978):583–586. doi: 10.1038/310583a0. [DOI] [PubMed] [Google Scholar]
  59. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES