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. 1991 Dec 15;88(24):11373–11377. doi: 10.1073/pnas.88.24.11373

RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins.

B He 1, P Chen 1, S Y Chen 1, K L Vancura 1, S Michaelis 1, S Powers 1
PMCID: PMC53137  PMID: 1763050

Abstract

In the yeast Saccharomyces cerevisiae, mutations in either of two unlinked genes, RAM1 or RAM2, abolish the farnesyltransferase activity responsible for prenylation of Ras proteins and the a-factor mating pheromone. Here we report that the function of RAM1 and RAM2 genes is required for the membrane localization of Ras proteins and a-factor. The RAM2 gene was sequenced and can encode a 38-kDa protein. We examined the functional interaction of RAM2 and RAM1 by expressing the genes in Escherichia coli. Extracts derived from an E. coli strain that coexpressed RAM1 and RAM2 efficiently farnesylated a-factor peptide and Ras protein substrates. In contrast, extracts derived from E. coli strains that expressed either RAM gene alone were devoid of activity; however, when the latter extracts were mixed, protein farnesyltransferase activity was reconstituted. These results indicate that the yeast farnesyl-protein transferase is comprised of Ram1 and Ram2 polypeptides. Although Ram1 is a component of the enzyme, disruption of the RAM1 gene in yeast was not lethal, indicating that the Ram1-Ram2 farnesyltransferase is not essential for viability. In contrast, disruption of RAM2 was lethal, suggesting that Ram2 has an essential function in addition to its role with Ram1 in protein farnesylation.

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Selected References

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

  1. Anderegg R. J., Betz R., Carr S. A., Crabb J. W., Duntze W. Structure of Saccharomyces cerevisiae mating hormone a-factor. Identification of S-farnesyl cysteine as a structural component. J Biol Chem. 1988 Dec 5;263(34):18236–18240. [PubMed] [Google Scholar]
  2. 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]
  3. 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]
  4. Casey P. J., Solski P. A., Der C. J., Buss J. E. p21ras is modified by a farnesyl isoprenoid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8323–8327. doi: 10.1073/pnas.86.21.8323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen W. J., Andres D. A., Goldstein J. L., Brown M. S. Cloning and expression of a cDNA encoding the alpha subunit of rat p21ras protein farnesyltransferase. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11368–11372. doi: 10.1073/pnas.88.24.11368. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen W. J., Andres D. A., Goldstein J. L., Russell D. W., Brown M. S. cDNA cloning and expression of the peptide-binding beta subunit of rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1. Cell. 1991 Jul 26;66(2):327–334. doi: 10.1016/0092-8674(91)90622-6. [DOI] [PubMed] [Google Scholar]
  7. Clarke S., Vogel J. P., Deschenes R. J., Stock J. Posttranslational modification of the Ha-ras oncogene protein: evidence for a third class of protein carboxyl methyltransferases. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4643–4647. doi: 10.1073/pnas.85.13.4643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DeFeo-Jones D., Tatchell K., Robinson L. C., Sigal I. S., Vass W. C., Lowy D. R., Scolnick E. M. Mammalian and yeast ras gene products: biological function in their heterologous systems. Science. 1985 Apr 12;228(4696):179–184. doi: 10.1126/science.3883495. [DOI] [PubMed] [Google Scholar]
  9. Der C. J., Cox A. D. Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity. Cancer Cells. 1991 Sep;3(9):331–340. [PubMed] [Google Scholar]
  10. 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]
  11. Farnsworth C. C., Kawata M., Yoshida Y., Takai Y., Gelb M. H., Glomset J. A. C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6196–6200. doi: 10.1073/pnas.88.14.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farnsworth C. C., Wolda S. L., Gelb M. H., Glomset J. A. Human lamin B contains a farnesylated cysteine residue. J Biol Chem. 1989 Dec 5;264(34):20422–20429. [PMC free article] [PubMed] [Google Scholar]
  13. Fedor-Chaiken M., Deschenes R. J., Broach J. R. SRV2, a gene required for RAS activation of adenylate cyclase in yeast. Cell. 1990 Apr 20;61(2):329–340. doi: 10.1016/0092-8674(90)90813-t. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Fujiyama A., Tamanoi F. RAS2 protein of Saccharomyces cerevisiae undergoes removal of methionine at N terminus and removal of three amino acids at C terminus. J Biol Chem. 1990 Feb 25;265(6):3362–3368. [PubMed] [Google Scholar]
  18. Gibbs J. B. Ras C-terminal processing enzymes--new drug targets? Cell. 1991 Apr 5;65(1):1–4. doi: 10.1016/0092-8674(91)90352-y. [DOI] [PubMed] [Google Scholar]
  19. Glomset J. A., Gelb M. H., Farnsworth C. C. Prenyl proteins in eukaryotic cells: a new type of membrane anchor. Trends Biochem Sci. 1990 Apr;15(4):139–142. doi: 10.1016/0968-0004(90)90213-u. [DOI] [PubMed] [Google Scholar]
  20. Goldstein J. L., Brown M. S. Regulation of the mevalonate pathway. Nature. 1990 Feb 1;343(6257):425–430. doi: 10.1038/343425a0. [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. 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]
  23. 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]
  24. Hrycyna C. A., Sapperstein S. K., Clarke S., Michaelis S. The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins. EMBO J. 1991 Jul;10(7):1699–1709. doi: 10.1002/j.1460-2075.1991.tb07694.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Kawata M., Farnsworth C. C., Yoshida Y., Gelb M. H., Glomset J. A., Takai Y. Posttranslationally processed structure of the human platelet protein smg p21B: evidence for geranylgeranylation and carboxyl methylation of the C-terminal cysteine. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8960–8964. doi: 10.1073/pnas.87.22.8960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Khosravi-Far R., Lutz R. J., Cox A. D., Conroy L., Bourne J. R., Sinensky M., Balch W. E., Buss J. E., Der C. J. Isoprenoid modification of rab proteins terminating in CC or CXC motifs. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6264–6268. doi: 10.1073/pnas.88.14.6264. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kinsella B. T., Maltese W. A. rab GTP-binding proteins implicated in vesicular transport are isoprenylated in vitro at cysteines within a novel carboxyl-terminal motif. J Biol Chem. 1991 May 5;266(13):8540–8544. [PubMed] [Google Scholar]
  29. Kohl N. E., Diehl R. E., Schaber M. D., Rands E., Soderman D. D., He B., Moores S. L., Pompliano D. L., Ferro-Novick S., Powers S. Structural homology among mammalian and Saccharomyces cerevisiae isoprenyl-protein transferases. J Biol Chem. 1991 Oct 5;266(28):18884–18888. [PubMed] [Google Scholar]
  30. Michaelis S., Herskowitz I. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol. 1988 Mar;8(3):1309–1318. doi: 10.1128/mcb.8.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakayama N., Arai K., Matsumoto K. Role of SGP2, a suppressor of a gpa1 mutation, in the mating-factor signaling pathway of Saccharomyces cerevisiae. Mol Cell Biol. 1988 Dec;8(12):5410–5416. doi: 10.1128/mcb.8.12.5410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ohya Y., Goebl M., Goodman L. E., Petersen-Bjørn S., Friesen J. D., Tamanoi F., Anraku Y. Yeast CAL1 is a structural and functional homologue to the DPR1 (RAM) gene involved in ras processing. J Biol Chem. 1991 Jul 5;266(19):12356–12360. [PubMed] [Google Scholar]
  33. 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]
  34. Reiss Y., Goldstein J. L., Seabra M. C., Casey P. J., Brown M. S. Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides. Cell. 1990 Jul 13;62(1):81–88. doi: 10.1016/0092-8674(90)90242-7. [DOI] [PubMed] [Google Scholar]
  35. Reiss Y., Seabra M. C., Armstrong S. A., Slaughter C. A., Goldstein J. L., Brown M. S. Nonidentical subunits of p21H-ras farnesyltransferase. Peptide binding and farnesyl pyrophosphate carrier functions. J Biol Chem. 1991 Jun 5;266(16):10672–10677. [PubMed] [Google Scholar]
  36. Reiss Y., Stradley S. J., Gierasch L. M., Brown M. S., Goldstein J. L. Sequence requirement for peptide recognition by rat brain p21ras protein farnesyltransferase. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):732–736. doi: 10.1073/pnas.88.3.732. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rine J., Kim S. H. A role for isoprenoid lipids in the localization and function of an oncoprotein. New Biol. 1990 Mar;2(3):219–226. [PubMed] [Google Scholar]
  38. Rossi G., Yu J. A., Newman A. P., Ferro-Novick S. Dependence of Ypt1 and Sec4 membrane attachment on Bet2. Nature. 1991 May 9;351(6322):158–161. doi: 10.1038/351158a0. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Seabra M. C., Reiss Y., Casey P. J., Brown M. S., Goldstein J. L. Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit. Cell. 1991 May 3;65(3):429–434. doi: 10.1016/0092-8674(91)90460-g. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Wilson K. L., Herskowitz I. STE16, a new gene required for pheromone production by a cells of Saccharomyces cerevisiae. Genetics. 1987 Mar;115(3):441–449. doi: 10.1093/genetics/115.3.441. [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]
  44. Yamane H. K., Farnsworth C. C., Xie H. Y., Howald W., Fung B. K., Clarke S., Gelb M. H., Glomset J. A. Brain G protein gamma subunits contain an all-trans-geranylgeranylcysteine methyl ester at their carboxyl termini. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5868–5872. doi: 10.1073/pnas.87.15.5868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yokoyama K., Goodwin G. W., Ghomashchi F., Glomset J. A., Gelb M. H. A protein geranylgeranyltransferase from bovine brain: implications for protein prenylation specificity. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5302–5306. doi: 10.1073/pnas.88.12.5302. [DOI] [PMC free article] [PubMed] [Google Scholar]

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