Analysis of vacuolar ATPase mutants of Neurospora crassa

The major proton pump found throughout the endomembrane system of eukaryotic cells is the vacuolar ATPase or V-ATPase. In this study, our goals were to determine the composition of the V0 sector and to gain insights into the functional role of some of the V-ATPase subunits within the Neurospora crassa enzyme.; To study the possible regulation of the V-ATPase by two loosely associated peripheral subunits, C and H, we inactivated genes encoding these subunits. Analysis of these two strains revealed that these subunits were necessary for V-ATPase activity.; To determine the composition of the V0 sector and to determine the phenotype of cells lacking V0 sector subunits we inactivated vph-1, vma-3, vma-11 and vma-16. Preliminary attempts at generating V0 sector mutants proved extremely difficult. We then developed a method to increase the frequency of mutant strain generation. This procedure readily yielded vma-11 mutant strains and eventually a single vma-16 mutant strain. This procedure failed to yield vma-3 mutant strains.; vma-11 mutant strains displayed variable growth phenotypes different from all other vma mutants isolated. We were therefore unsure if any of the vma-11 RIP strains represented the true null phenotype. To be sure, we generated a strain in which the vma-11 gene was replaced with the BAR gene. This strain displayed a phenotype like the other Deltavma strains but had the ability to grow at neutral pH and above. These data suggest that the c' subunit may not be absolutely essentially for V-ATPase activity. We also used homologous recombination to generate a strain in which the vma-3 gene was replaced with the inositol gene. This strain was morphologically indistinguishable from the other vma defective strains. However, the Deltavma-3 strain is sterile.; Using bioinformatics, we determined the vma-11 protein product to be a fungal specific V-ATPase component. Analyses of the vph-1 and STV1 protein sequences in fungal systems reveal them to be paralogs. Interestingly, this gene duplication event is restricted to the budding yeasts. We have inactivated the vph-1 gene in N. crassa and find that unlike in S. cerevisiae there is no STV1 ortholog and we observe the typical Deltavma phenotype.