Analysis of strains with mutations in six genes encoding subunits of the V-ATPase -: Eukaryotes differ in the composition of the V0 sector of the enzyme

To address questions about the structure of the vacuolar ATPase, we have generated mutant strains of Neurospora crassa defective in six subunits, C, H, a, c, c', and c". Except for strains lacking subunit c', the mutant strains were indistinguishable from each other in most phenotypic characteristics. They did not accumulate arginine in the vacuoles, grew poorly at pH 5.8 with altered morphology, and failed to grow at alkaline pH. Consistent with findings from Saccharomyces cerevisiae, the data indicate that subunits C and H are essential for generation of a functional enzyme. Unlike S. cerevisiae, N. crassa has a single isoform of the a subunit. Analysis of other fungal genomes indicates that only the budding yeasts have a two-gene family for subunit a. It has been unclear whether subunit c', a small proteolipid, is a component of all V-ATPases. Our data suggest that this subunit is present in all fungi, but not in other organisms. Mutation or deletion of the N. crassa gene encoding subunit c' did not completely eliminate V-ATPase function. Unlike other V-ATPase null strains, they grew, although slowly, at alkaline pH, were able to form conidia (asexual spores), and were inhibited by concanamycin, a specific inhibitor of the V-ATPase. The phenotypic character in which strains differed was the ability to go through the sexual cycle to generate mature spores and viable mutant progeny. Strains lacking the integral membrane subunits a, c, c', and c" had more severe defects than strains lacking subunits C or H.