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. 1997 Mar;17(3):1543–1551. doi: 10.1128/mcb.17.3.1543

Genes encoding farnesyl cysteine carboxyl methyltransferase in Schizosaccharomyces pombe and Xenopus laevis.

Y Imai 1, J Davey 1, M Kawagishi-Kobayashi 1, M Yamamoto 1
PMCID: PMC231880  PMID: 9032282

Abstract

The mam4 mutation of Schizosaccharomyces pombe causes mating deficiency in h- cells but not in h+ cells. h- cells defective in mam4 do not secrete active mating pheromone M-factor. We cloned mam4 by complementation. The mam4 gene encodes a protein of 236 amino acids, with several potential membrane-spanning domains, which is 44% identical with farnesyl cysteine carboxyl methyltransferase encoded by STE14 and required for the modification of a-factor in Saccharomyces cerevisiae. Analysis of membrane fractions revealed that mam4 is responsible for the methyltransferase activity in S. pombe. Cells defective in mam4 produced farnesylated but unmethylated cysteine and small peptides but no intact M-factor. These observations strongly suggest that the mam4 gene product is farnesyl cysteine carboxyl methyltransferase that modifies M-factor. Furthermore, transcomplementation of S. pombe mam4 allowed us to isolate an apparent homolog of mam4 from Xenopus laevis (Xmam4). In addition to its sequence similarity to S. pombe mam4, the product of Xmam4 was shown to have a farnesyl cysteine carboxyl methyltransferase activity in S. pombe cells. The isolation of a vertebrate gene encoding farnesyl cysteine carboxyl methyltransferase opens the way to in-depth studies of the role of methylation in a large body of proteins, including Ras superfamily proteins.

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Selected References

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  1. Akopyan T. N., Couedel Y., Orlowski M., Fournie-Zaluski M. C., Roques B. P. Proteolytic processing of farnesylated peptides: assay and partial purification from pig brain membranes of an endopeptidase which has the characteristics of E.C. 3.4.24.15. Biochem Biophys Res Commun. 1994 Jan 28;198(2):787–794. doi: 10.1006/bbrc.1994.1113. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Armstrong S. A., Seabra M. C., Südhof T. C., Goldstein J. L., Brown M. S. cDNA cloning and expression of the alpha and beta subunits of rat Rab geranylgeranyl transferase. J Biol Chem. 1993 Jun 5;268(16):12221–12229. [PubMed] [Google Scholar]
  4. Ashby M. N., Errada P. R., Boyartchuk V. L., Rine J. Isolation and DNA sequence of the STE14 gene encoding farnesyl cysteine: carboxyl methyltransferase. Yeast. 1993 Aug;9(8):907–913. doi: 10.1002/yea.320090810. [DOI] [PubMed] [Google Scholar]
  5. Beach D., Piper M., Nurse P. Construction of a Schizosaccharomyces pombe gene bank in a yeast bacterial shuttle vector and its use to isolate genes by complementation. Mol Gen Genet. 1982;187(2):326–329. doi: 10.1007/BF00331138. [DOI] [PubMed] [Google Scholar]
  6. Beach D., Rodgers L., Gould J. ran1+ controls the transition from mitotic division to meiosis in fission yeast. Curr Genet. 1985;10(4):297–311. doi: 10.1007/BF00365626. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Clarke S. Protein carboxyl methyltransferases: two distinct classes of enzymes. Annu Rev Biochem. 1985;54:479–506. doi: 10.1146/annurev.bi.54.070185.002403. [DOI] [PubMed] [Google Scholar]
  10. Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. doi: 10.1146/annurev.bi.61.070192.002035. [DOI] [PubMed] [Google Scholar]
  11. Danjoh I., Fujiyama A. Enzymic characterization of fission yeast farnesyl transferase: recognition of the -CAAL motif at the C-terminus. Eur J Biochem. 1996 Mar 15;236(3):847–851. doi: 10.1111/j.1432-1033.1996.00847.x. [DOI] [PubMed] [Google Scholar]
  12. Davey J. Mating pheromones of the fission yeast Schizosaccharomyces pombe: purification and structural characterization of M-factor and isolation and analysis of two genes encoding the pheromone. EMBO J. 1992 Mar;11(3):951–960. doi: 10.1002/j.1460-2075.1992.tb05134.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Edwards J. B., Delort J., Mallet J. Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning 5' ends of mRNAs and for constructing cDNA libraries by in vitro amplification. Nucleic Acids Res. 1991 Oct 11;19(19):5227–5232. doi: 10.1093/nar/19.19.5227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Egel R., Egel-Mitani M. Premeiotic DNA synthesis in fission yeast. Exp Cell Res. 1974 Sep;88(1):127–134. doi: 10.1016/0014-4827(74)90626-0. [DOI] [PubMed] [Google Scholar]
  15. Egel R. Genes involved in mating type expression of fission yeast. Mol Gen Genet. 1973 May 28;122(4):339–343. doi: 10.1007/BF00269434. [DOI] [PubMed] [Google Scholar]
  16. Egel R., Willer M., Kjaerulff S., Davey J., Nielsen O. Assessment of pheromone production and response in fission yeast by a halo test of induced sporulation. Yeast. 1994 Oct;10(10):1347–1354. doi: 10.1002/yea.320101012. [DOI] [PubMed] [Google Scholar]
  17. Fukui Y., Kaziro Y., Yamamoto M. Mating pheromone-like diffusible factor released by Schizosaccharomyces pombe. EMBO J. 1986 Aug;5(8):1991–1993. doi: 10.1002/j.1460-2075.1986.tb04454.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fukui Y., Kozasa T., Kaziro Y., Takeda T., Yamamoto M. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell. 1986 Jan 31;44(2):329–336. doi: 10.1016/0092-8674(86)90767-1. [DOI] [PubMed] [Google Scholar]
  19. Gibbs J. B., Marshall M. S. The ras oncogene--an important regulatory element in lower eucaryotic organisms. Microbiol Rev. 1989 Jun;53(2):171–185. doi: 10.1128/mr.53.2.171-185.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Grimm C., Kohli J., Murray J., Maundrell K. Genetic engineering of Schizosaccharomyces pombe: a system for gene disruption and replacement using the ura4 gene as a selectable marker. Mol Gen Genet. 1988 Dec;215(1):81–86. doi: 10.1007/BF00331307. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Hrycyna C. A., Clarke S. Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Oct;10(10):5071–5076. doi: 10.1128/mcb.10.10.5071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hrycyna C. A., Clarke S. Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro. J Biol Chem. 1992 May 25;267(15):10457–10464. [PubMed] [Google Scholar]
  25. 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]
  26. Imai Y., Miyake S., Hughes D. A., Yamamoto M. Identification of a GTPase-activating protein homolog in Schizosaccharomyces pombe. Mol Cell Biol. 1991 Jun;11(6):3088–3094. doi: 10.1128/mcb.11.6.3088. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Imai Y., Yamamoto M. The fission yeast mating pheromone P-factor: its molecular structure, gene structure, and ability to induce gene expression and G1 arrest in the mating partner. Genes Dev. 1994 Feb 1;8(3):328–338. doi: 10.1101/gad.8.3.328. [DOI] [PubMed] [Google Scholar]
  28. Kitamura K., Shimoda C. The Schizosaccharomyces pombe mam2 gene encodes a putative pheromone receptor which has a significant homology with the Saccharomyces cerevisiae Ste2 protein. EMBO J. 1991 Dec;10(12):3743–3751. doi: 10.1002/j.1460-2075.1991.tb04943.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kjaerulff S., Davey J., Nielsen O. Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: identification of a third gene, mfm3. Mol Cell Biol. 1994 Jun;14(6):3895–3905. doi: 10.1128/mcb.14.6.3895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. 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]
  31. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  34. Leiser M., Efrat S., Fleischer N. Evidence that Rap1 carboxylmethylation is involved in regulated insulin secretion. Endocrinology. 1995 Jun;136(6):2521–2530. doi: 10.1210/endo.136.6.7750474. [DOI] [PubMed] [Google Scholar]
  35. Li R., Murray A. W. Feedback control of mitosis in budding yeast. Cell. 1991 Aug 9;66(3):519–531. doi: 10.1016/0092-8674(81)90015-5. [DOI] [PubMed] [Google Scholar]
  36. Ma Y. T., Chaudhuri A., Rando R. R. Substrate specificity of the isoprenylated protein endoprotease. Biochemistry. 1992 Dec 1;31(47):11772–11777. doi: 10.1021/bi00162a014. [DOI] [PubMed] [Google Scholar]
  37. Marcus S., Caldwell G. A., Miller D., Xue C. B., Naider F., Becker J. M. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol Cell Biol. 1991 Jul;11(7):3603–3612. doi: 10.1128/mcb.11.7.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marr R. S., Blair L. C., Thorner J. Saccharomyces cerevisiae STE14 gene is required for COOH-terminal methylation of a-factor mating pheromone. J Biol Chem. 1990 Nov 25;265(33):20057–20060. [PubMed] [Google Scholar]
  39. Maundrell K. Thiamine-repressible expression vectors pREP and pRIP for fission yeast. Gene. 1993 Jan 15;123(1):127–130. doi: 10.1016/0378-1119(93)90551-d. [DOI] [PubMed] [Google Scholar]
  40. Mayer M. L., Caplin B. E., Marshall M. S. CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase. J Biol Chem. 1992 Oct 15;267(29):20589–20593. [PubMed] [Google Scholar]
  41. McLeod M., Stein M., Beach D. The product of the mei3+ gene, expressed under control of the mating-type locus, induces meiosis and sporulation in fission yeast. EMBO J. 1987 Mar;6(3):729–736. doi: 10.1002/j.1460-2075.1987.tb04814.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Nadin-Davis S. A., Nasim A., Beach D. Involvement of ras in sexual differentiation but not in growth control in fission yeast. EMBO J. 1986 Nov;5(11):2963–2971. doi: 10.1002/j.1460-2075.1986.tb04593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Nielsen O., Davey J. Pheromone communication in the fission yeast Schizosaccharomyces pombe. Semin Cell Biol. 1995 Apr;6(2):95–104. doi: 10.1016/1043-4682(95)90006-3. [DOI] [PubMed] [Google Scholar]
  44. Obara T., Nakafuku M., Yamamoto M., Kaziro Y. Isolation and characterization of a gene encoding a G-protein alpha subunit from Schizosaccharomyces pombe: involvement in mating and sporulation pathways. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5877–5881. doi: 10.1073/pnas.88.13.5877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. 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]
  46. Okazaki K., Okazaki N., Kume K., Jinno S., Tanaka K., Okayama H. High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe. Nucleic Acids Res. 1990 Nov 25;18(22):6485–6489. doi: 10.1093/nar/18.22.6485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Philips M. R., Pillinger M. H., Staud R., Volker C., Rosenfeld M. G., Weissmann G., Stock J. B. Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils. Science. 1993 Feb 12;259(5097):977–980. doi: 10.1126/science.8438158. [DOI] [PubMed] [Google Scholar]
  48. Pérez-Sala D., Tan E. W., Cañada F. J., Rando R. R. Methylation and demethylation reactions of guanine nucleotide-binding proteins of retinal rod outer segments. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3043–3046. doi: 10.1073/pnas.88.8.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. 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]
  50. 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]
  51. Sapperstein S., Berkower C., Michaelis S. Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export. Mol Cell Biol. 1994 Feb;14(2):1438–1449. doi: 10.1128/mcb.14.2.1438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. 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]
  53. 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]
  54. Sinensky M., Lutz R. J. The prenylation of proteins. Bioessays. 1992 Jan;14(1):25–31. doi: 10.1002/bies.950140106. [DOI] [PubMed] [Google Scholar]
  55. Sipiczki M., Ferenczy L. Protoplast fusion of Schizosaccharomyces pombe Auxotrophic mutants of identical mating-type. Mol Gen Genet. 1977 Feb 28;151(1):77–81. doi: 10.1007/BF00446915. [DOI] [PubMed] [Google Scholar]
  56. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  57. Springer M. S., Goy M. F., Adler J. Protein methylation in behavioural control mechanisms and in signal transduction. Nature. 1979 Jul 26;280(5720):279–284. doi: 10.1038/280279a0. [DOI] [PubMed] [Google Scholar]
  58. Stephenson R. C., Clarke S. Identification of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate. J Biol Chem. 1990 Sep 25;265(27):16248–16254. [PubMed] [Google Scholar]
  59. Tanaka K., Davey J., Imai Y., Yamamoto M. Schizosaccharomyces pombe map3+ encodes the putative M-factor receptor. Mol Cell Biol. 1993 Jan;13(1):80–88. doi: 10.1128/mcb.13.1.80. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Volker C., Lane P., Kwee C., Johnson M., Stock J. A single activity carboxyl methylates both farnesyl and geranylgeranyl cysteine residues. FEBS Lett. 1991 Dec 16;295(1-3):189–194. doi: 10.1016/0014-5793(91)81415-5. [DOI] [PubMed] [Google Scholar]
  61. Willer M., Hoffmann L., Styrkársdóttir U., Egel R., Davey J., Nielsen O. Two-step activation of meiosis by the mat1 locus in Schizosaccharomyces pombe. Mol Cell Biol. 1995 Sep;15(9):4964–4970. doi: 10.1128/mcb.15.9.4964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Xue C. B., Ewenson A., Becker J. M., Naider F. Solution phase synthesis of Saccharomyces cerevisiae a-mating factor and its analogs. Int J Pept Protein Res. 1990 Oct;36(4):362–373. doi: 10.1111/j.1399-3011.1990.tb01295.x. [DOI] [PubMed] [Google Scholar]
  63. Yabana N., Yamamoto M. Schizosaccharomyces pombe map1+ encodes a MADS-box-family protein required for cell-type-specific gene expression. Mol Cell Biol. 1996 Jul;16(7):3420–3428. doi: 10.1128/mcb.16.7.3420. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Yamamoto M. The molecular control mechanisms of meiosis in fission yeast. Trends Biochem Sci. 1996 Jan;21(1):18–22. [PubMed] [Google Scholar]
  65. Zhang F. L., Diehl R. E., Kohl N. E., Gibbs J. B., Giros B., Casey P. J., Omer C. A. cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I. J Biol Chem. 1994 Feb 4;269(5):3175–3180. [PubMed] [Google Scholar]

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