Endoproteolytic processing of a farnesylated peptide in vitro

Proc Natl Acad Sci U S A. 1992 May 15;89(10):4613-7. doi: 10.1073/pnas.89.10.4613.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Cell Membrane / enzymology
  • Cysteine / analogs & derivatives*
  • Cysteine / metabolism
  • Cytosol / enzymology
  • Endopeptidases / isolation & purification
  • Endopeptidases / metabolism*
  • Kinetics
  • Mating Factor
  • Methyltransferases / isolation & purification
  • Methyltransferases / metabolism*
  • Molecular Sequence Data
  • Oligopeptides / chemical synthesis
  • Peptides / metabolism*
  • Pheromones / metabolism
  • Protein Processing, Post-Translational
  • Saccharomyces cerevisiae / enzymology*
  • Substrate Specificity

Substances

  • Oligopeptides
  • Peptides
  • Pheromones
  • Mating Factor
  • S-farnesylcysteine
  • Methyltransferases
  • Endopeptidases
  • Cysteine