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 i mmediately processed in a reaction involving two endoproteases, protei nase A and proteinase B. We have investigated the in vitro activation of purified procarboxypeptidase Y by purified proteinase A. This has i dentified two different processing intermediates; one active and one i nactive. The intermediates define a 33 amino acid segment of the 91 am ino acid propeptide as sufficient for maintaining the enzyme in an ina ctive state. The inactive intermediate was isolated from a processing reaction at neutral pH. In order to investigate the influence of vacuo lar pH on processing in vivo, the autoactivation of proteinase A and i ts processing of procarboxypeptidase Y were studied in a vma2 prbl mut ant, which is deficient in vacuolar acidification and proteinase B act ivity. Efficient processing of procarboxypeptidase Y in the absence of proteinase B is dependent on acidic vacuolar pH, and the processing a t neutral pH is slow and takes place in two steps similar to those ide ntified in vitro.