Extracellular processing of carboxypeptidase Y secreted by a Saccharomyces cerevisiae ssl1 mutant strain

Abstract

Carboxypeptidase Y (CPY; EC 3. 4. 16. 1) encoded by the PRC1 gene is a yeast vacuolar protease. It enters the vacuole as a zymogen, proCPY, which is activated by vacuolar enzymes, proteinase A (PrA) and proteinase B (PrB). We previously showed that active CPY was efficiently secreted from the Saccharomyces cerevisiae ssl1 (supersecretion of lysozyme) mutant carrying a multicopy plasmid which contains the PRC1 gene fused to an inducible GAL10 promoter. In this study, we detected PrA and PrB activities in the culture supernatant of the ssl1 mutant harboring the CPY expression plasmid. The N-terminal amino acid sequence of extracellular CPY coincided with that of the original vacuolar one. Furthermore, studies using protease inhibitors suggested that CPY was secreted into the medium in the form of a precursor and was mainly activated by extracellular PrB. The ssl1 mutant secreted CPY, PrA and PrB into the medium even with a single copy of the PRC1 gene. On the other hand, a cytoplasmic marker enzyme, glucose-6-phosphate dehydrogenase, and a vacuolar membraneassociated enzyme, α-mannosidase, were not detected in the medium, whether the PRC1 gene was overexpressed or not. It is suggested that secretion of vacuolar proteases is caused by characteristics of the ssl1 mutation or overexpression of the PRC1 gene.