title: Synthesis and processing of in vitro and in vivo precursors of the vacuolar yeast enzyme carboxypeptidase Y.
abstract: The biosynthesis of carboxypeptidase Y, which is located in the lysosome-like vacuole of Saccharomyces cerevisiae, has been studied in vitro in a cell-free translation system from wheat germ and in vivo in intact spheroplasts. When a wheat germ system was programmed with yeast RNA, a translation product was immunoprecipitated by anti-carboxypeptidase Y antibodies, which had a slightly smaller molecular weight (Mr = 59,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the authentic glycoprotein (Mr = 60,000). In the presence of dog pancreatic microsomal membranes, an additional cross-reacting translation product of Mr = 68,000 was formed, which in contrast to the 59,000-dalton form was not susceptible to digestion by externally added proteinases, suggesting its segregation within the microsomal vesicles. The observed increase in molecular weight may be consistent with a core glycosylation of the translocated protein. During a pulse-chase labeling of spheroplasts, the antibody initially precipitated a form of carboxypeptidase Y, which co-migrated on sodium dodecyl sulfate gels with the 68,000-dalton in vitro translation product. Following a chase of 60 min, this early labeled immunoreactive protein was completely converted into the authentic enzyme (Mr = 60,000) and is therefore coincident with the in vivo precursor of carboxypeptidase Y previously described (Hasilik, A., and Tanner, W. (1978) Eur. J. Biochem. 85, 599-608). These data suggest that a vacuolar yeast enzyme is synthesized via a cotranslational segregation of its nascent polypeptide chain within the endoplasmic reticulum giving rise to a proenzyme, which is further processed in vivo into the vacuole-located mature enzyme.
mesh_terms: D002268:Carboxypeptidases / Q000235:genetics*; D043402:Cathepsin A; D004792:Enzyme Precursors / Q000235:genetics*; D009940:Organoids / Q000201:enzymology*; D010944:Plants / Q000378:metabolism; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014908:Triticum / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D004792:Enzyme Precursors; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase