• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Feb 1994; 14(2): 1438–1449.
PMCID: PMC358499

Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export.

Abstract

Eukaryotic proteins initially synthesized with a C-terminal CAAX motif (C is Cys, A is aliphatic, and X can be one of several amino acids) undergo a series of modifications involving isoprenylation of the Cys residue, proteolysis of AAX, and alpha-carboxyl methyl esterification of the newly formed isoprenyl cysteine. We have previously demonstrated that STE14 encodes the enzyme which mediates carboxyl methylation of the Saccharomyces cerevisiae CAAX proteins a-factor, RAS1, and RAS2. Here we report the nucleotide sequence of STE14, which indicates that STE14 encodes a protein of 239 amino acids, predicted to contain multiple membrane-spanning segments. Mapping data indicate that STE14 resides on chromosome IV, tightly linked to ADE8. By analysis of ste14 null alleles, we demonstrated that MATa ste14 mutants are unable to mate but are viable and exhibit no apparent growth defects. Additional analysis of ste14 ras 1 and ste14 ras2 double mutants, which grow normally, reinforces our previous conclusion that RAS function is not significantly influenced by its methylation status. We examine a-factor biogenesis in a ste14 null mutant by metabolic labeling and immunoprecipitation and demonstrate that although proteolytic processing and membrane localization of a-factor are normal, the ste14 null mutant exhibits a profound block in a-factor export. This observation suggests that the methyl group is likely to be a critical recognition determinant for the a-factor transporter, STE6, thus providing insight into the substrate specificity of STE6 and also supporting the hypothesis that carboxyl methylation can have a dramatic impact on protein-protein interactions.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.7M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Anderegg RJ, Betz R, Carr SA, Crabb JW, 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]
  • Ashby MN, King DS, Rine J. Endoproteolytic processing of a farnesylated peptide in vitro. Proc Natl Acad Sci U S A. 1992 May 15;89(10):4613–4617. [PMC free article] [PubMed]
  • Ashby MN, Errada PR, Boyartchuk VL, Rine J. Isolation and DNA sequence of the STE14 gene encoding farnesyl cysteine: carboxyl methyltransferase. Yeast. 1993 Aug;9(8):907–913. [PubMed]
  • Berkower C, Michaelis S. Mutational analysis of the yeast a-factor transporter STE6, a member of the ATP binding cassette (ABC) protein superfamily. EMBO J. 1991 Dec;10(12):3777–3785. [PMC free article] [PubMed]
  • Berkower C, Michaelis S. Effects of nucleotide binding fold mutations on STE6, a yeast ABC protein. Soc Gen Physiol Ser. 1993;48:129–146. [PubMed]
  • Carle GF, Olson MV. An electrophoretic karyotype for yeast. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3756–3760. [PMC free article] [PubMed]
  • Casadaban MJ, Martinez-Arias A, Shapira SK, Chou J. Beta-galactosidase gene fusions for analyzing gene expression in escherichia coli and yeast. Methods Enzymol. 1983;100:293–308. [PubMed]
  • Casey PJ. Biochemistry of protein prenylation. J Lipid Res. 1992 Dec;33(12):1731–1740. [PubMed]
  • Clarke S. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem. 1992;61:355–386. [PubMed]
  • Cox AD, Der CJ. Protein prenylation: more than just glue? Curr Opin Cell Biol. 1992 Dec;4(6):1008–1016. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Finegold AA, Johnson DI, Farnsworth CC, Gelb MH, Judd SR, Glomset JA, 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. [PMC free article] [PubMed]
  • Finegold AA, Schafer WR, 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. [PubMed]
  • Gerring SL, Connelly C, Hieter P. Positional mapping of genes by chromosome blotting and chromosome fragmentation. Methods Enzymol. 1991;194:57–77. [PubMed]
  • Goodman LE, Judd SR, Farnsworth CC, Powers S, Gelb MH, Glomset JA, 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. [PMC free article] [PubMed]
  • Gottesman MM, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem. 1993;62:385–427. [PubMed]
  • Hancock JF, Cadwallader K, Marshall CJ. Methylation and proteolysis are essential for efficient membrane binding of prenylated p21K-ras(B). EMBO J. 1991 Mar;10(3):641–646. [PMC free article] [PubMed]
  • He B, Chen P, Chen SY, Vancura KL, 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. [PMC free article] [PubMed]
  • Higgins CF. ABC transporters: from microorganisms to man. Annu Rev Cell Biol. 1992;8:67–113. [PubMed]
  • Hrycyna CA, 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. [PMC free article] [PubMed]
  • Hrycyna CA, 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]
  • Hrycyna CA, Sapperstein SK, 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. [PMC free article] [PubMed]
  • Ingrosso D, Fowler AV, Bleibaum J, Clarke S. Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. Common sequence motifs for protein, DNA, RNA, and small molecule S-adenosylmethionine-dependent methyltransferases. J Biol Chem. 1989 Nov 25;264(33):20131–20139. [PubMed]
  • Kronstad JW, Holly JA, MacKay VL. A yeast operator overlaps an upstream activation site. Cell. 1987 Jul 31;50(3):369–377. [PubMed]
  • Kuchler K, Sterne RE, 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. [PMC free article] [PubMed]
  • Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Marcus S, Caldwell GA, Miller D, Xue CB, Naider F, Becker JM. 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. [PMC free article] [PubMed]
  • Marr RS, Blair LC, 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]
  • McGrath JP, Varshavsky A. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature. 1989 Aug 3;340(6232):400–404. [PubMed]
  • Michaelis S. STE6, the yeast a-factor transporter. Semin Cell Biol. 1993 Feb;4(1):17–27. [PubMed]
  • Michaelis S, Herskowitz I. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol Cell Biol. 1988 Mar;8(3):1309–1318. [PMC free article] [PubMed]
  • Nasmyth KA, Tatchell K. The structure of transposable yeast mating type loci. Cell. 1980 Mar;19(3):753–764. [PubMed]
  • Nicolas A, Treco D, Schultes NP, Szostak JW. An initiation site for meiotic gene conversion in the yeast Saccharomyces cerevisiae. Nature. 1989 Mar 2;338(6210):35–39. [PubMed]
  • Ohya Y, Goebl M, Goodman LE, Petersen-Bjørn S, Friesen JD, 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]
  • Oshima T, Takano I. Mutants Showing Heterothallism from a Homothallic Strain of SACCHAROMYCES CEREVISIAE. Genetics. 1980 Apr;94(4):841–857. [PMC free article] [PubMed]
  • Riles L, Dutchik JE, Baktha A, McCauley BK, Thayer EC, Leckie MP, Braden VV, Depke JE, Olson MV. Physical maps of the six smallest chromosomes of Saccharomyces cerevisiae at a resolution of 2.6 kilobase pairs. Genetics. 1993 May;134(1):81–150. [PMC free article] [PubMed]
  • Robinson GW, Tsay YH, Kienzle BK, Smith-Monroy CA, Bishop RW. Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol Cell Biol. 1993 May;13(5):2706–2717. [PMC free article] [PubMed]
  • Rossi G, Yu JA, Newman AP, Ferro-Novick S. Dependence of Ypt1 and Sec4 membrane attachment on Bet2. Nature. 1991 May 9;351(6322):158–161. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schafer WR, Rine J. Protein prenylation: genes, enzymes, targets, and functions. Annu Rev Genet. 1992;26:209–237. [PubMed]
  • Schafer WR, Trueblood CE, Yang CC, Mayer MP, Rosenberg S, Poulter CD, Kim SH, 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. [PubMed]
  • Schultes NP, Szostak JW. Decreasing gradients of gene conversion on both sides of the initiation site for meiotic recombination at the ARG4 locus in yeast. Genetics. 1990 Dec;126(4):813–822. [PMC free article] [PubMed]
  • Sikorski RS, 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. [PMC free article] [PubMed]
  • Sinensky M, Lutz RJ. The prenylation of proteins. Bioessays. 1992 Jan;14(1):25–31. [PubMed]
  • Stephenson RC, 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]
  • Stephenson RC, Clarke S. Characterization of a rat liver protein carboxyl methyltransferase involved in the maturation of proteins with the -CXXX C-terminal sequence motif. J Biol Chem. 1992 Jul 5;267(19):13314–13319. [PubMed]
  • Struhl K. Molecular mechanisms of transcriptional regulation in yeast. Annu Rev Biochem. 1989;58:1051–1077. [PubMed]
  • Zaret KS, Sherman F. DNA sequence required for efficient transcription termination in yeast. Cell. 1982 Mar;28(3):563–573. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...