TY - JOUR

T1 - pH-dependent processing of yeast procarboxypeptidase Y by proteinase A in vivo and in vitro

AU - Sørensen, S O

AU - van den Hazel, H B

AU - Kielland-Brandt, Morten

AU - Winther, Jakob R.

PY - 1994

Y1 - 1994

N2 - 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.

AB - 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.

KW - Amino Acid Sequence

KW - Aspartic Acid Endopeptidases

KW - Binding Sites

KW - Carboxypeptidases

KW - Cathepsin A

KW - Enzyme Activation

KW - Enzyme Precursors

KW - Genes, Fungal

KW - Hydrogen-Ion Concentration

KW - Molecular Sequence Data

KW - Mutation

KW - Protein Processing, Post-Translational

KW - Saccharomyces cerevisiae

KW - Saccharomyces cerevisiae Proteins

KW - Serine Endopeptidases

KW - Vacuoles

M3 - Journal article

C2 - 8119286

VL - 220

SP - 19

EP - 27

JO - FEBS Journal

JF - FEBS Journal

SN - 1742-464X

IS - 1

ER -