Molecular cloning of soluble aminopeptidases from Saccharomyces cerevisiae. Sequence analysis of aminopeptidase yscII, a putative zinc-metallopeptidase.
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 beta-naphthylamide in intact yeast colonies. The genomic inserts were shown to contain the structural genes for aminopeptidases yscII, yscIII and yscIV. 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 identify 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 abolishes the distinct activity band previously identified as aminopeptidase yscII [H.H. Hirsch, P. Suárez-Rendueles, T. Achstetter and D.H. Wolf (1988) Eur. J. Biochem. 173, 589-598] in crude extracts 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.
García-Alvarez
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Departamento de Biología Funcional, Facultad de Medicina, Universidad de Oviedo, Spain.
Cueva
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Suárez-Rendueles
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D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular / Q000379:methods; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D005800:Genes, Fungal; D016681:Genome, Fungal; D015698:Genomic Library; D006801:Humans; D007527:Isoenzymes / Q000235:genetics*; D008666:Metalloendopeptidases / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010957:Plasmids; D051381:Rats; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D000626:Aminopeptidases; C021108:yscII protein, S cerevisiae; D008666:Metalloendopeptidases