Phenotypic analysis of proteinase A mutants. Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae.
We have isolated a number of mutants deficient in activity of the vacuolar hydrolase proteinase A (PrA). The mutations were sequenced and although they all map in the PEP4 gene, which encodes the precursor to PrA, three distinguishable phenotypes have surfaced. The properties of the pep4-7 missense mutant suggested that the activation of the precursor to proteinase A is due to an autocatalytic cleavage. PrA active site mutations were constructed and resulted in accumulation of PrA antigen in the inactive precursor form. Although protease B (PrB), another vacuolar hydrolase, is not required for the production of active PrA, the active form of PrA that accumulates in a strain lacking PrB is larger than that found in a strain containing active PrB. We have purified this larger form of PrA and determined that it bears 7 additional amino acids at its NH2 terminus. It has become apparent from all the studies performed on the maturation pathway of the vacuolar hydrolases that there is a great deal of redundancy built into the system.
Woolford
C A
CA
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Noble
J A
JA
Garman
J D
JD
Tam
M F
MF
Innis
M A
MA
Jones
E W
EW
D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal; D004789:Enzyme Activation; D005816:Genetic Complementation Test; D006867:Hydrolases / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014617:Vacuoles / Q000201:enzymology*
D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D006867:Hydrolases; C022325:aspartic proteinase A; C499267:PEP4 protein, S cerevisiae; D016282:Aspartic Acid Endopeptidases