VACUOLAR AND EXTRACELLULAR MATURATION OF SACCHAROMYCES-CEREVISIAE PROTEINASE-A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cer evisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specif ic proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is o verexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determinatio n of extracellular, as well as vacuolar pseudoPrA showed that it conta ins nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordan ce with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 tran sformant of a prb1 mutant was grown in the presence of the aspartyl pr otease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 le ads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vac uolar processing reactions. Amino acid sequencing of secreted proPrA c onfirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.