Extracellular processing of carboxypeptidase Y secreted by a Saccharomycescerevisiae ssl1 mutant strain

Carhoxypeptidase Y (CPY; EC 3. 4. 16. 1) encoded by the PRC1 gene is a yeas t vacuolar protease. It enters the vacuole as a zymogen, proCPY, which is a ctivated by vacuolar enzymes, proteinase A (PrA) and proteinase B (PrB). We previously showed that active CPY was efficiently secreted from the Saccha romyces cerevisiae ssl1 (supersecretion of lysozyme) mutant carrying a mult icopy plasmid which contains the PRC1 gene fused to an inducible GAL10 prom oter. In this study, we detected PrA and PrB activities in the culture supe rnatant of the ssl1 mutant harboring the CPY expression plasmid. The N-term inal amino acid sequence of extracellular CPY coincided with that of the or iginal vacuolar one. Furthermore, studies using protease inhibitors suggest ed 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 a nd PrB into the medium even with a single copy of the PRC1 gene. On the oth er hand, a cytoplasmic marker enzyme, glucose-6-phosphate dehydrogenase, an d a vacuolar membrane-associated enzyme, alpha-mannosidase, were not detect ed in the medium, whether the PRC1 gene was overexpressed or not. It is sug gested that secretion of vacuolar proteases is caused by characteristics of the sail mutation or overexpression of the PRC1 gene.