title: Endoproteolytic processing of a farnesylated peptide in vitro.
abstract: Numerous eukaryotic proteins containing a carboxyl-terminal CAAX motif (C, cysteine; A, aliphatic amino acid; X, any amino acid) require a three-step posttranslational processing for localization and function. The a mating factor of Saccharomyces cerevisiae is one such protein, requiring cysteine farnesylation, proteolysis of the terminal three amino acids, and carboxyl methylation for biological activity. We have used farnesylated a-factor peptides to examine the proteolytic step in the maturation of CAAX-containing proteins. Three distinct carboxyl-terminal protease activities were found in yeast cell extracts that could remove the terminal three residues of a-factor. Two of the proteolytic activities were in cytosolic fractions. One of these activities was a PEP4-dependent carboxypeptidase that was sensitive to phenylmethylsulfonyl fluoride. The other cytosolic activity was PEP4-independent, sensitive to 1,10-phenanthroline, and effectively inhibited by an unfarnesylated a-factor peptide. In contrast, a protease activity in membrane fractions was unaffected by phenylmethylsulfonyl fluoride, 1,10-phenanthroline, or unfarnesylated a-factor peptide. Incubation of membrane preparations from either yeast or rat liver with a radiolabeled farnesylated a-factor peptide released the terminal three amino acids intact as a tripeptide, indicating that this reaction occurred by an endoproteolytic mechanism and that the enzyme most likely possesses a broad substrate specificity. The yeast endoprotease was not significantly affected by a panel of protease inhibitors, suggesting that the enzyme is novel. Zinc ion was shown to inhibit the endoprotease (Ki less than 100 microM). The specific activities of the a-factor carboxyl-terminal membrane endoprotease and methyltransferase clearly indicated that the proteolytic reaction was not rate-limiting in these processing reactions in vitro.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley 94720.
authors.forename: M N
authors.initials: MN
authors.lastname: Ashby
authors.forename: D S
authors.initials: DS
authors.lastname: King
authors.forename: J
authors.initials: J
authors.lastname: Rine
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000201:enzymology; D003545:Cysteine / Q000031:analogs & derivatives* / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D000072235:Mating Factor; D008780:Methyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis; D010455:Peptides / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D009842:Oligopeptides; D010455:Peptides; D010675:Pheromones; D000072235:Mating Factor; C057785:S-farnesylcysteine; D008780:Methyltransferases; D010450:Endopeptidases; D003545:Cysteine