PURIFICATION AND CHARACTERIZATION OF A NOVEL METALLOENDOPEPTIDASE FROM SACCHAROMYCES-CEREVISIAE

We previously identified an activity in the soluble fraction of the ye ast Saccharomyces cerevisiae that is a candidate for catalyzing the pr oteolytic maturation of farnesylated-CXXX precursor polypeptides. We d escribe here a 1259-fold purification of this activity by chromatograp hy on DEAE-cellulose, hydroxylapatite, phenyl-Sepharose, and Sephacryl S-200. Sodium dodecyl sulfate gel electrophoresis of this preparation demonstrated a single 68-kDa polypeptide chain. The experimentally de termined N-terminal amino acid sequence was identical at all 20 positi ons with residues 28-47 of the deduced sequence of the S. cerevisiae Y CL57w gene product. This analysis suggests that the YCL57w gene encode s this enzyme and that the initial translation product may contain a l eader peptide. Its complete deduced amino acid sequence shows signific ant homology to a number of zinc metallopeptidases and is most closely related to rat metalloendopeptidase 24.15 (E.C. 3.4.24.15), an enzyme that preferentially cleaves after hydrophobic residues. Using the pur ified yeast enzyme, we show a unique cleavage site in the peptides bra dykinin and beta-neoendorphin four residues from the C-terminus on the carboxyl side of a hydrophobic amino acid. The cleavage pattern for n eurotensin revealed a major site three residues from the C-terminus al so on the carboxyl side of a hydrophobic residue and a minor site four residues from the C-terminus of the peptide. This specificity is simi lar to that of rat endopeptidase 24.15 and may explain why the farnesy lated peptide employed in our studies is a good substrate for the yeas t enzyme. However, subcellular localization experiments revealed that this activity does not appear to be cytosolic; its distribution follow s that of Golgi and vacuolar marker enzymes. These results suggest tha t this enzyme may not be involved in the physiological processing of c ytosolic-CXXX containing proteins but may be important in other types of protein or peptide metabolism.