Phenotypic analysis of proteinase a mutants. Implications for autoactivation and the maturation pathway of the vacuolar hydrolases of Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry Vol. 268; no. 12; pp. 8990 - 8998
• Main Authors: Woolford, C.A, Noble, J.A, Garman, J.D, Tam, M.F, Innis, M.A, Jones, E.W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25.04.1993
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Aspartic Acid Endopeptidases - genetics; Aspartic Acid Endopeptidases - isolation & purification; Aspartic Acid Endopeptidases - metabolism; Base Sequence; Binding Sites; Biological and medical sciences; DNA, Fungal; Enzyme Activation; Enzymes and enzyme inhibitors; FENOTIPOS; Fundamental and applied biological sciences. Psychology; Genetic Complementation Test; Hydrolases; Hydrolases - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; MUTANT; MUTANTES; PHENOTYPE; PROTEASAS; PROTEASE; Restriction Mapping; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins; SECUENCIA NUCLEICA; SEQUENCE NUCLEIQUE; VACUOLA; VACUOLE; Vacuoles - enzymology; Fungi; Molecular processing; Enzymatic activity; Structure activity relation; Enzyme; Proteinase; Site directed mutagenesis; Ascomycetes; Activation; Saccharomyces cerevisiae; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)52969-0

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