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Endoproteolytic Processing of a Farnesylated Peptide in vitro

Matthew N. Ashby, David S. King and Jasper Rine
Proceedings of the National Academy of Sciences of the United States of America
Vol. 89, No. 10 (May 15, 1992), pp. 4613-4617
Stable URL: http://www.jstor.org/stable/2359379
Page Count: 5
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Since scans are not currently available to screen readers, please contact JSTOR User Support for access. We'll provide a PDF copy for your screen reader.
Endoproteolytic Processing of a Farnesylated Peptide in vitro
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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 < 100 μ M). 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.

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