Biogenesis of the yeast vacuole (lysosome). Mutation in the active site of the vacuolar serine proteinase yscB abolishes proteolytic maturation of its 73-kDa precursor to the 41.5-kDa pro-enzyme and a newly detected 41-kDa peptide.
The codon of the catalytic serine in the active site of the vacuolar serine proteinase yscB (PrB) was changed to alanine, yielding the mutant gene prb1-Ala519. Following replacement of the wild-type PRB1 allele with prb1-Ala519, only a 73-kDa molecule was detected by immunoprecipitation with PrB-specific antiserum. The size of the mutant molecule corresponds to the unprocessed cytoplasmic precursor (pre-super-pro-PrB), as detected in sec61 mutants, when translocation into the endoplasmic reticulum is blocked. However, the mutant molecule is completely translocated into the secretory pathway, as indicated by protection from proteinase K digestion in spheroplast lysates in the absence of detergent. When N-glycosylation was inhibited in prb1-Ala519 mutant cells by tunicamycin, a smaller molecule of about 71 kDa appeared consistent with single N-glycosylation and signal-sequence cleavage of the translocated mutant PrB molecule in the endoplasmic reticulum. Thus, the active-site mutation prevents the wild-type processing of the N-glycosylated 73-kDa precursor of PrB to the 41.5 kDa pro-PrB in the endoplasmic reticulum. In order to characterize the processing of wild-type super-pro-PrB in more detail, we generated antibodies against the non-enzymatic superpeptide domain of the 73-kDa precursor expressed in Escherichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDS/PAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wild-type cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wild-type cell extracts. In contrast, no degradation of radioactively labeled 41-kDa superpeptide was observed within 60 min in mutant strains deficient in the vacuolar proteinase yscA (PrA), in which maturation of vacuolar pro-PrB to active PrB is blocked. Accordingly, superpeptide antigenic material was readily detected by immunoblotting cell extracts and enriched in vacuolar preparations of PrA deficient mutant cells. These results indicate that the superpeptide and pro-PrB travel to the vacuole, where the superpeptide is rapidly degraded upon pro-PrB activation to PrB. Using purified vacuoles, rapid degradation of the superpeptide was reconstituted in vitro by addition of either mature PrA or mature PrB. However, the PrA-triggered in vitro degradation of the superpeptide required PrB activity, as this process was inhibited in the presence of the PrB inhibitor chymostatin.(ABSTRACT TRUNCATED AT 400 WORDS)
Hirsch
H H
HH
Institut für Biochemie, Universität Stuttgart, Federal Republic of Germany.
Schiffer
H H
HH
Müller
H
H
Wolf
D H
DH
D001665:Binding Sites; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000201:enzymology; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006031:Glycosylation; D006868:Hydrolysis; D008247:Lysosomes / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D014415:Tunicamycin / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins
D004792:Enzyme Precursors; D012697:Serine Endopeptidases; D014415:Tunicamycin; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins