Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.
Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.
Nebes
V L
VL
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Jones
E W
EW
D001483:Base Sequence; D002384:Catalysis; D004271:DNA, Fungal; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D011498:Protein Precursors; D012697:Serine Endopeptidases; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins