Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.
We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.
Cooper
A
A
Department of Biology, McGill University, Quebec, Canada.
Bussey
H
H
D001483:Base Sequence; D001667:Binding, Competitive; D002268:Carboxypeptidases / Q000235:genetics* / Q000378:metabolism; D043402:Cathepsin A; D002498:Centrifugation; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D055646:Killer Factors, Yeast; D000072235:Mating Factor; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009183:Mycotoxins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D011233:Precipitin Tests; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism / Q000502:physiology; D012100:Reproduction, Asexual; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D005656:Fungal Proteins; D055646:Killer Factors, Yeast; D008565:Membrane Proteins; D009183:Mycotoxins; D010455:Peptides; D011498:Protein Precursors; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D000072235:Mating Factor; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase