Biogenesis of the yeast vacuole (lysosome). The precursor forms of the soluble hydrolase carboxypeptidase yscS are associated with the vacuolar membrane.

Abstract

We have studied the structure, biosynthesis, intracellular routing, and vacuolar localization of carboxypeptidase ysCS in the yeast Saccharomyces cerevisiae. Nondenaturing polyacrylamide gel electrophoresis revealed two forms of carboxypeptidase yscS with different electrophoretic mobility. Antibodies specific for carboxypeptidase yscS recognized two glycoproteins of 77- and 74-kDa apparent molecular mass which differ by one N-linked carbohydrate residue. Both observations suggest that carboxypeptidase yscS exists in two catalytically active forms. The enzyme was found to be synthesized as two active high molecular mass precursor forms which are converted to the mature forms with a half-time of 20 min. The mature forms of carboxypeptidase yscS appeared soluble in the vacuolar lumen, while the precursor proteins accumulated tightly associated with the vacuolar membrane. The single hydrophobic domain present at the N terminus is believed to be responsible for the membrane association of the precursor molecules. Double mutants defective in proteinase yscA and proteinase yscB synthesize solely the carboxypeptidase yscS precursor forms. Correct proteolytic cleavage of the precursor forms was performed using purified proteinase yscB in vitro. Sec61, sec18, and sec7 mutants, conditionally defective in the secretory pathway, accumulate carboxypeptidase yscS precursor protein. Thus the carboxypeptidase yscS precursor molecules are delivered to the vacuole in a membrane bound form via the secretory pathway. After assembly into the vacuolar membrane, proteinase yscB presumably cleaves the precursor molecules to release soluble carboxypeptidase yscS forms into the lumen of the vacuole. The proposed mechanism is different from the delivery mechanism found for the other soluble vacuolar hydrolases in yeast.