pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro.
Carboxypeptidase Y is a vacuolar enzyme from Saccharomyces cerevisiae. It enters the vacuole as a zymogen, procarboxypeptidase Y, which is immediately processed in a reaction involving two endoproteases, proteinase A and proteinase B. We have investigated the in vitro activation of purified procarboxypeptidase Y by purified proteinase A. This has identified two different processing intermediates; one active and one inactive. The intermediates define a 33 amino acid segment of the 91 amino acid propeptide as sufficient for maintaining the enzyme in an inactive state. The inactive intermediate was isolated from a processing reaction at neutral pH. In order to investigate the influence of vacuolar pH on processing in vivo, the autoactivation of proteinase A and its processing of procarboxypeptidase Y were studied in a vma2 prb1 mutant, which is deficient in vacuolar acidification and proteinase B activity. Efficient processing of procarboxypeptidase Y in the absence of proteinase B is dependent on acidic vacuolar pH, and the processing at neutral pH is slow and takes place in two steps similar to those identified in vitro.
Sørensen
S O
SO
Carlsberg Laboratory, Department of Yeast Genetics, Copenhagen, Valby, Denmark.
van den Hazel
H B
HB
Kielland-Brandt
M C
MC
Winther
J R
JR
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002268:Carboxypeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043402:Cathepsin A; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005800:Genes, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000201:enzymology
D004792:Enzyme Precursors; D029701:Saccharomyces cerevisiae Proteins; D002268:Carboxypeptidases; D043402:Cathepsin A; C499283:PRC1 protein, S cerevisiae; C022633:serine carboxypeptidase; D012697:Serine Endopeptidases; C023302:yeast proteinase B; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases