Biogenesis of the yeast lysosome (vacuole): biosynthesis and maturation of proteinase yscB.

The biosynthesis and processing of the vacuolar (lysosomal) proteinase yscB was followed in vivo and in vitro. In vitro transcription--translation of the cloned proteinase yscB gene results in a high mol. wt precursor protein (HMr-precursor) of Mr = 73,000. In vivo a precursor of identical mol. wt is found in sec6l mutant cells deficient in translocation of secretory protein precursors into the lumen of the endoplasmic reticulum (ER). In contrast to N-glycosylated intermediate mol. wt forms of proteinase yscB, the HMr-precursor of the enzyme is not N-glycosylated, indicating its appearance outside the ER. sec18 Mutant cells, wild type for translocation of proteins into the ER but blocked in the delivery step of secretory proteins to the Golgi apparatus, accumulate a lower mol. wt precursor (LMr-precursor) of proteinase yscB of Mr = 41,500. Processing of the HMr-precursor of proteinase yscB to the LMr-precursor requires proteolytic cleavage which is independent of the proteinase yscA gene product, a protein known to be involved in the processing event of the LMr-precursor of proteinase yscB to the mature enzyme of Mr = 33,000. A LMr-precursor of proteinase yscB of Mr = 42,000 accumulates in proteinase yscA-deficient mutant cells (allele pep4-3). This form is enzymatically active. Incubation in vitro of the LMr-precursor of proteinase yscB with mature proteinase yscA leads to further activation and to processing of the enzyme yielding the mature 33,000 Mr protein.(ABSTRACT TRUNCATED AT 250 WORDS)