BIOGENESIS OF THE YEAST VACUOLE (LYSOSOME) - THE USE OF ACTIVE-SITE MUTANTS OF PROTEINASE YSCA TO DETERMINE THE NECESSITY OF THE ENZYME FOR VACUOLAR PROTEINASE MATURATION AND PROTEINASE YSCB STABILITY

The activation process of vacuolar proteinases in the yeast Saccharomyces cerevisiae via precursor maturation is initiated by the PRA1/PEP4 gene product, proteinase yscA. Chromosomal deletion of the PRA1/PEP4 locus leads to accumulation of inactive pro-proteinases in the vacuole. Nine active-site mutations of proteinase yscA have been constructed in vitro. All these mutations lead to the expression of proteinase yscA species in vivo that are inactive against the in vitro substrate hemoglobin and the in vivo substrates pro-proteinase yscB and pro-carboxypeptidase yscY. However, three active-site mutations in proteinase yscA sustained the precursor maturation of proteinase yscB and carboxypeptidase yscY after exchange of the genomic wild-type allele with the respective proteinase yscA mutant alleles. In contrast to yeast strains deleted in proteinase yscA, the respective mutants carry out all cellular functions that rely on a proteolytically active vacuole. This wild-type behaviour of proteinase yscA mutant cells is dependent on the presence of active proteinase yscB. Proteinase yscA and proteinase yscB are equally able to fulfil essential cellular functions. For instance, either proteinase is able to maintain viability under starvation. However, mature proteinase yscB is not stable in the absence of proteinase yscA. The wild-type-like conformation of proteolytically inactive mutant proteinase yscA proteins stabilizes mature proteinase yscB and thus enables continuous maturation of pro-proteinase yscB by active proteinase yscB. After inhibition of the proteolytic activity of proteinase yscB in these proteinase yscA mutants with phenylmethylsulfonyl fluoride or deletion of the PRB1 gene, maturation of all zymogens investigated in the vacuole, including the proteinase yscA mutant proteins, is blocked. The proteolytic activities of the vacuole in such a strain can be regained, however, by introduction of a wild-type proteinase yscA gene allowing subsequent autocatalytic maturation of wild-type pro-proteinase yscA. This indicates that an initial self-activation process of proteinase yscA is necessary for the activation of vacuolar zymogens.