Vacuolar ATPase mutants of Neurospora crassa

The vacuolar ATPase is the major proton pump of the endomembrane system. We wanted to develop methods to look at the phenotype of Neurospora crassa strains with deficient levels of vacuolar ATPase. Three strategies were employed, selection of mutants which are resistant to trifluoperazine (TFP), inactivation of the gene encoding the catalytic subunit (vma-1), and construction of strains with heterologous replacement of the vma-1 promoter.;To determine the phenotype of cells lacking the enzyme we inactivated the vma-1 gene. Preliminary experiments indicated that the vma-1 gene was essential. Therefore we developed a method to simultaneously inactivate the gene while complementing it with a functional copy. We call this method repeat induced point mutation (RIP) & Rescue. Two strains were mated, both contained an ectopic copy of the gene. Progeny which inherited a functional ectopic copy of the gene were selected. Sequencing showed the endogenous vma-1 gene of some of the progeny had been inactivated by RIP. Progeny from strains with an inactive endogenous vma-1 gene were inviable unless a functional ectopic copy of the gene cosegregated, indicating the vacuolar ATPase is essential in N. crassa.;In a final set of experiments the ectopic functional copy of the vma-1 gene was replaced with an altered version. This version had the qa-2 promoter fused to the vma-1 coding region. This generated strains in which the only functional copy of the vma-1 gene was the chimeric qa-vma-1 gene. These strains had no observable differences from the wild-type strain and had normal vacuolar ATPase activity suggesting that the vma-1 gene was constitutively expressed.;Resistance to trifluoperazine was used to select for strains with reduced vacuolar ATPase activity. Analysis of these mutant strains indicated lower levels of enzyme but no detectable kinetic alterations. One strain appear to be mutated in the 5...