| Title: | Endoproteolytic processing of a farnesylated peptide in vitro |
| Author(s): | Ashby MN; King DS; Rine J; |
| Address: | "Department of Molecular and Cell Biology, University of California, Berkeley 94720" |
| ISSN/ISBN: | 0027-8424 (Print) 1091-6490 (Electronic) 0027-8424 (Linking) |
| 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" |
| Keywords: | Amino Acid Sequence Cell Membrane/enzymology Cysteine/*analogs & derivatives/metabolism Cytosol/enzymology Endopeptidases/isolation & purification/*metabolism Kinetics Mating Factor Methyltransferases/isolation & purification/*metabolism Molecular Sequenc; |
| Notes: | "MedlineAshby, M N King, D S Rine, J eng 1F32CA09091-01/CA/NCI NIH HHS/ GM35827/GM/NIGMS NIH HHS/ Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. 1992/05/15 Proc Natl Acad Sci U S A. 1992 May 15; 89(10):4613-7. doi: 10.1073/pnas.89.10.4613" |
