Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae

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Abstract
Plasmids capable of complementing lap1, lap2 and lap3 mutations [R. J. Trumbly and G. Bradley (1983) J. Bacteriol. 156, 36–48] were isolated from a yeast YEp13 library by screening for activity against the chromogenic aminopeptidase substrate L-leucine β-naphthylamide in intact yeast colonies. The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and ysclIV. Plasmids containing the gene encoding aminopeptidase yscII of Saccharomyces cerevisiae, APE2 (LAP1) were analyzed in detail. APE2 was determined by DNA blot analysis to be a single-copy gene located on chromosome XI. The cloned fragment was used to idenify a 2.7-kb mRNA. The cloned APE2 gene was sequenced and found to consist of an open reading frame of 2583 bp encoding a protein of 861 amino acids. The protein sequence contains two putative N-glycosylation sites. A significant amino acid similarity was detected between the APE2 gene product and members of the zinc-dependent metallopeptidase gene family. Chromosomal disruption of the APE2 gene completely abolished the distinct activity band previously identified as aminopeptidase yscII [H. H. Hirsch, P. Suárez-Rendueles. T. Achstetter and D. ri. Wolf (1988) Eur. J. Biochem 173, 589–598] in crude extrats subjected to non-denaturing polyacrylamide gel electrophoresis and subsequent aminopeptidase activity staining. No vital consequence of aminopeptidase yscII absence on cell growth could be detected.