We have isolated a gene from Neurospora crassa that appears to encode a
pepstatin-sensitive
protease found both in membranes and in soluble contents of vacuoles. The gene contains two introns and encodes a 396-residue
protein with a molecular mass of 42,900 Da. Because of the similarity of the
protein to
proteinase A in Saccharomyces cerevisiae the gene has been named pep-4. Strains with mutations in the pep-4 gene were generated in vivo by the gene RIPing procedure described by Selker and Garrett (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874). The mutant strains were deficient in
pepstatin-sensitive
protease activity and did not appear to produce a major 42-kDa
polypeptide in the vacuole. The mutant strains grew at the same rate as the wild type and had no other observable phenotype. When compared with inactivation of the PEP4 gene of S. cerevisiae, inactivation of the pep-4 gene in N. crassa produced a phenotype that was different in several ways. In N. crassa the mutant strains did not exhibit reduced sporulation or reduced viability after
nitrogen starvation, and they had elevated levels of
proteinase B and
carboxypeptidase activities. The pep-4 gene appears to encode the N. crassa, homolog of
proteinase A, but the maturation of vacuolar
hydrolases appeared to be less dependent on this
protease than has been observed in S. cerevisiae.