Biogenesis of the yeast vacuole (lysosome)

Abstract

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 prhl-Ala519. Following replacement of the wildtype PRBl allele withprhl-AZu519, 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 prbl-A11519 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 Eschrrichia coli. We find that, in addition to pro-PrB, a distinct protein (superpeptide) with a mobility of about 41 kDa in SDSjPAGE is generated in the endoplasmic reticulum. Pulse-chase experiments indicate rapid degradation of the 41-kDa superpeptide in wildtype cells. Correspondingly, the superpeptide was virtually undetectable by immunoblotting wildtype 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. In a more detailed kinetic analysis, rapid PrA-triggered degradation of superpeptide was observed only coincidently with the maturation of the PrA-processed 40-kDa intermediate pro-PrB form to the mature PrB of 33 kDa. Slow degradation of the superpeptide only by incubation was correlated with unmasking of the cryptic pro-PrB activity. These results suggest that the complex maturation events of the vacuolar serine PrB precursors involve several timely and spatially controlled (auto-)proteolytic proces...

A newly characterized vacuolar serine carboxypeptidase, Atg42/Ybr139w, is required for normal vacuole function and the terminal steps of autophagy in the yeastSaccharomyces cerevisiae

“…, 2014 ). Prb1 undergoes a self-catalyzed N-terminal cleavage event in the ER followed by glycosylation, resulting in a 40-kDa species (proPrb1) being delivered to the vacuole ( Nebes and Jones, 1991 ; Hirsch et al. , 1992 ; Van Den Hazel et al.…”

Calnexin Is Essential for Survival under Nitrogen Starvation and Stationary Phase in Schizosaccharomyces pombe

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The proteolytic systems and heterologous proteins degradation in the methylotrophic yeastPichia pastoris

“…The removal of the N-terminal propeptide is presumably autocatalytic, as it does not occur in strains producing an active-site mutant form of PrB. This step occurs before delivery to the Golgi complex since it is not dependent on a functional Sec l 8p (Moehle et al, 1989;Hirsch et al, 1992). The large initial PrB precursor contains five acceptor sites for Asn-linked glycosylation.…”