Processing pathway for protease B of Saccharomyces cerevisiae.

Abstract

Abstract. The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparaginelinked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.T nF. lysosome-like vacuole of the yeast Saccharomyces cerevisiae contains several hydrolytic activities, including protease A (PrA),~ protease B (PrB), carboxypeptidase Y (CpY), a 600-kD aminopeptidase, a repressible alkaline phosphatase, and at least one RNase (39,65). In all cases studied to date, these hydrolases are synthesized as inactive glycoprotein precursors (reviewed in reference 28) that require the PEP4 gene product, protease A, for activation (2, 28, 66). Vacuolar hydrolases may be thought of as internally secreted proteins, as they traverse the ER and Golgi apparatus en route to the vacuole. Mutations that block transport from the ER or Golgi apparatus affect internally and externally secreted proteins equally (31, 57).Some preliminary work on the structure of PrB and its precursors has been reported. In kinetic experiments using a 20-min pulse of [35S]methionine, Mechler et al. (43) identiffed a 42-kD precursor of PrB that is subsequently processed to the mature form. The relative molecular mass of the precursor was 3 kD smaller in tunicamycin-treated cells, but the relative molecular mass of the mature protease was unaffected (43). This kinetic precursor had the same relative molecular mass as the zymogen form of protease B observed to accumulate in a pep4 mutant (44). Mature protease B has been reported to be a glycoprotein that contains "no more than 0.5%" carbohydrate (61). A different group reported C. M. Moehle's present address is Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20892. PrB to be a 33-kD glycoprotein with 8-9% neutral sugars and 1.5 % amino sugars (32). The neutral/amino sugar ratio was suggestive of an Asn-linked carbohydrate moiety. Abbreviations used in this paperWe recently reported the cloning and sequencing of PRB1 and also the purification and amino-terminal sequence of PrB (46,47). Unexpectedly, analysis of the...

Resolution of organelle docking and fusion kinetics in a cell-free assay

“…We now report that a second protease, Prb1p, is rate limiting for ALP maturation. Because Pep4p is required for Prb1p activation, pep4 mutant strains lack both Pep4p and Prb1p activities (4,14,18). Vacuoles isolated from any of several pep4 strains contain pro-ALP but lack significant ALP activity.…”

Purification and biochemical characterization of the ATH1 gene product, vacuolar acid trehalase, from Saccharomyces cerevisiae

“…The reported PEP4 dependence of ATH activity [11] suggests the presence of a propeptide that is cleaved in the vacuole by PrA or a PrA-dependent hydrolase such as PrB, This is consistent with the fact that the 85 kDa ATH is substantially smaller than the potential 117 kDa protein encoded by the A TH1 open reading frame. A 30 kDa peptide would be large but is approximately the size of the N-terminal propeptide of the vacuolar hydrolase proteinase B [30]. Because our antiserum to Athlp only detects ATH in western blots and not by immunoprecipitation, we are not able to carry out a kinetic analysis to directly demonstrate processing of the enzyme.…”

Substrate specificity of proteinase yscA from Saccharomyces cerevisiae

“…The cleavages necessary for "correct" N-terminal processing ofproteinase yscB, carboxypeptidase yscY and proteinase yscA itself, occur between amino acids with low cleavage probability by proteinase yscA: Glu3-Ala2-Thr x -Glu~-Phe 2-Asp 3-in proteinase yscB (21), -Arg3-ValE-Asn l -Lys~-Ile2-Lys 3-in carboxypeptidase yscY (30) and -Phe-3-ThrE-Glu -I Glyt-GlyE-His 3-in proteinase yscA itself (2,32). Maturation of the precursor of proteinase yscB additionally involves sequential removal of C-terminal fragments, one of which is catalyzed by proteinase yscA (20). The exact position of the cleaved bond has not yet been established.…”

“…The exact position of the cleaved bond has not yet been established. Hydrolase maturations are multi-step limited proteolytic reactions, and proteinase yscA is most likely involved in cleavages leading to slightly higher molecular weight intermediates (19,20). Such intermediates are catalytically active and might be physiologically important, although the proteinases obtained from cells at the end of growth and after extensive autoactivation of the cell lysate during enzyme purification have lower molecular weights.…”