Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1990 Oct;10(10):5071–5076. doi: 10.1128/mcb.10.10.5071

Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae.

C A Hrycyna 1, S Clarke 1
PMCID: PMC361172  PMID: 2204804

Abstract

Membrane extracts of sterile Saccharomyces cerevisiae strains containing the a-specific ste14 mutation lack a farnesyl cysteine C-terminal carboxyl methyltransferase activity that is present in wild-type a and alpha cells. Other a-specific sterile strains with ste6 and ste16 mutations also have wild-type levels of the farnesyl cysteine carboxyl methyltransferase activity. This enzyme activity, detected by using a synthetic peptide sequence based on the C-terminus of a ras protein, may be responsible not only for the essential methylation of the farnesyl cysteine residue of a mating factor, but also for the methylation of yeast RAS1 and RAS2 proteins and possibly other polypeptides with similar C-terminal structures. We demonstrate that the farnesylation of the cysteine residue in the peptide is required for the methyltransferase activity, suggesting that methyl esterification follows the lipidation reaction in the cell. To show that the loss of methyltransferase activity is a direct result of the ste14 mutation, we transformed ste14 mutant cells with a plasmid complementing the mating defect of this strain and found that active enzyme was produced. Finally, we demonstrated that a similar transformation of cells possessing the wild-type STE14 gene resulted in sixfold overproduction of the enzyme. Although more complicated possibilities cannot be ruled out, these results suggest that STE14 is a candidate for the structural gene for a methyltransferase involved in the formation of isoprenylated cysteine alpha-methyl ester C-terminal structures.

Full text

PDF
5071

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. Betz R., Crabb J. W., Meyer H. E., Wittig R., Duntze W. Amino acid sequences of a-factor mating peptides from Saccharomyces cerevisiae. J Biol Chem. 1987 Jan 15;262(2):546–548. [PubMed] [Google Scholar]
  3. 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]
  4. Chelsky D., Olson J. F., Koshland D. E., Jr Cell cycle-dependent methyl esterification of lamin B. J Biol Chem. 1987 Mar 25;262(9):4303–4309. [PubMed] [Google Scholar]
  5. Chelsky D., Sobotka C., O'Neill C. L. Lamin B methylation and assembly into the nuclear envelope. J Biol Chem. 1989 May 5;264(13):7637–7643. [PubMed] [Google Scholar]
  6. Chen Z. Q., Ulsh L. S., DuBois G., Shih T. Y. Posttranslational processing of p21 ras proteins involves palmitylation of the C-terminal tetrapeptide containing cysteine-186. J Virol. 1985 Nov;56(2):607–612. doi: 10.1128/jvi.56.2.607-612.1985. [DOI] [PMC free article] [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. 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]
  9. Deschenes R. J., Stimmel J. B., Clarke S., Stock J., Broach J. R. RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus. J Biol Chem. 1989 Jul 15;264(20):11865–11873. [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Fung B. K., Yamane H. K., Ota I. M., Clarke S. The gamma subunit of brain G-proteins is methyl esterified at a C-terminal cysteine. FEBS Lett. 1990 Jan 29;260(2):313–317. doi: 10.1016/0014-5793(90)80132-3. [DOI] [PubMed] [Google Scholar]
  14. Georgatos S. D., Maroulakou I., Blobel G. Lamin A, lamin B, and lamin B receptor analogues in yeast. J Cell Biol. 1989 Jun;108(6):2069–2082. doi: 10.1083/jcb.108.6.2069. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. Herskowitz I. A regulatory hierarchy for cell specialization in yeast. Nature. 1989 Dec 14;342(6251):749–757. doi: 10.1038/342749a0. [DOI] [PubMed] [Google Scholar]
  19. Holtz D., Tanaka R. A., Hartwig J., McKeon F. The CaaX motif of lamin A functions in conjunction with the nuclear localization signal to target assembly to the nuclear envelope. Cell. 1989 Dec 22;59(6):969–977. doi: 10.1016/0092-8674(89)90753-8. [DOI] [PubMed] [Google Scholar]
  20. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kuchler K., Sterne R. E., Thorner J. Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO J. 1989 Dec 20;8(13):3973–3984. doi: 10.1002/j.1460-2075.1989.tb08580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McGrath J. P., Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. doi: 10.1038/340400a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Ong O. C., Ota I. M., Clarke S., Fung B. K. The membrane binding domain of rod cGMP phosphodiesterase is posttranslationally modified by methyl esterification at a C-terminal cysteine. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9238–9242. doi: 10.1073/pnas.86.23.9238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ota I. M., Clarke S. Enzymatic methylation of 23-29-kDa bovine retinal rod outer segment membrane proteins. Evidence for methyl ester formation at carboxyl-terminal cysteinyl residues. J Biol Chem. 1989 Aug 5;264(22):12879–12884. [PubMed] [Google Scholar]
  26. 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]
  27. Sakagami Y., Yoshida M., Isogai A., Suzuki A. Peptidal Sex Hormones Inducing Conjugation Tube Formation in Compatible Mating-Type Cells of Tremella mesenterica. Science. 1981 Jun 26;212(4502):1525–1527. doi: 10.1126/science.212.4502.1525. [DOI] [PubMed] [Google Scholar]
  28. Schafer W. R., Kim R., Sterne R., Thorner J., Kim S. H., Rine J. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science. 1989 Jul 28;245(4916):379–385. doi: 10.1126/science.2569235. [DOI] [PubMed] [Google Scholar]
  29. Serrano R. In vivo glucose activation of the yeast plasma membrane ATPase. FEBS Lett. 1983 May 30;156(1):11–14. doi: 10.1016/0014-5793(83)80237-3. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Vorburger K., Kitten G. T., Nigg E. A. Modification of nuclear lamin proteins by a mevalonic acid derivative occurs in reticulocyte lysates and requires the cysteine residue of the C-terminal CXXM motif. EMBO J. 1989 Dec 20;8(13):4007–4013. doi: 10.1002/j.1460-2075.1989.tb08583.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. Wiedow O., Schröder J. M., Gregory H., Young J. A., Christophers E. Elafin: an elastase-specific inhibitor of human skin. Purification, characterization, and complete amino acid sequence. J Biol Chem. 1990 Sep 5;265(25):14791–14795. [PubMed] [Google Scholar]
  34. 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]
  35. Wolda S. L., Glomset J. A. Evidence for modification of lamin B by a product of mevalonic acid. J Biol Chem. 1988 May 5;263(13):5997–6000. [PubMed] [Google Scholar]

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

RESOURCES