title: Maturation of isoprenylated proteins in Saccharomyces cerevisiae. Multiple activities catalyze the cleavage of the three carboxyl-terminal amino acids from farnesylated substrates in vitro.
abstract: Eukaryotic polypeptides containing COOH-terminal-CXXX sequences can be posttranslationally modified by isoprenylation of the cysteine residue via a thioether linkage, proteolytic removal of the three terminal amino acids, and alpha-carboxyl methylation of the cysteine residue. Through the development of an indirect coupled assay, we have identified three in vitro activities in the yeast Saccharomyces cerevisiae that can catalyze the proteolytic cleavage of the three COOH-terminal amino acids of the synthetic peptide substrate N-acetyl-KSKTK[S-farnesyl-Cys]VIM. One of these is the vacuolar protease carboxypeptidase Y. Using a mutant strain deficient in this enzyme, we find evidence for an additional soluble activity as well as for a membrane-associated activity. These latter activities are candidates for roles in the physiological processing of isoprenylated protein precursors. They are both insensitive to inhibitors of serine and aspartyl proteinases but are sensitive to sulfhydryl reagents and 0.5 mM ZnCl2. The soluble activity appears to be a metalloenzyme, inhibitable by 2 mM o-phenanthroline but not by 1 mM N-ethylmaleimide, whereas the membrane-associated enzyme is inhibitable by 1 mM N-ethylmaleimide but not 2 mM o-phenanthroline. We show that the membrane-bound protease is not an activity of the membrane-bound methyltransferase, because protease activity is observed in membrane preparations that lack the STE14-encoded methyltransferase. The soluble activity appears to be a novel carboxypeptidase of approximately 110 kDa that catalyzes a processive removal of amino acids from the COOH terminus from both the farnesylated and non-farnesylated substrate, but not from three other unrelated peptides. Finally, we find no evidence for non-vacuolar membrane or soluble activities that catalyze the ester hydrolysis of N-acetyl-S-farnesyl-L-cysteine methyl ester.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, Los Angeles 90024-1569.
authors.forename: C A
authors.initials: CA
authors.lastname: Hrycyna
authors.forename: S
authors.initials: S
authors.lastname: Clarke
mesh_terms: D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002384:Catalysis; D043402:Cathepsin A; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D006868:Hydrolysis; D008780:Methyltransferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D005656:Fungal Proteins; D011480:Protease Inhibitors; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D008780:Methyltransferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C064911:p21(ras) farnesyl-protein transferase; D002268:Carboxypeptidases; D010450:Endopeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase