Biogenesis of the yeast vacuole (lysosome) Active site mutation in the vacuolar aspartate proteinase yscA blocks maturation of vacuolar proteinases

Abstract The activation process of vacuolar (lysosomal) proteinases in the yeast Saccharomyces cerevisiae is initiated by the PRAI ( PEP4 ) gene product, proteinase yscA. To elucidate the activation process of proteinase yscA the catalytically active amino acid Asp 294 of the enzyme was exchanged with Ala 294 using site directed mutagenesis. The resulting proteinase yscA-Ala 294 showed no proteolytic activity against the substrate hemoglobin. The mutant protein did not undergo processing in vivo. This phenomenon is in good agreement with the hypothesis that maturation of proteinase yscA is due to self-processing or pro-proteinase yscA. Furthermore, proteinase yscA-Ala 294 is not able to convert the zymogens pro-proteinase yscB and pro-carboxypeptidase yscY to the mature enzymes.