Disruption of vma-1, the Gene Encoding the Catalytic Subunit of the Vacuolar H+-ATPase, Causes Severe Morphological Changes in Neurospora crassa

Emma Jean Bowman,Ryan Kendle,Barry J Bowman

doi:10.1074/jbc.275.1.167

Emma Jean Bowman, Ryan Kendle + Show 1 more

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https://doi.org/10.1074/jbc.275.1.167

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Abstract

By using the process of Repeat-induced Point mutation (Selker, E. U., and Garrett, P. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 6870-6874), we inactivated vma-1, the gene encoding subunit A of the V-ATPase of Neurospora crassa. Two vma-1 mutant strains were characterized. One was mutated at multiple sites, did not make a protein product, and produced spores that only rarely germinated. The other had four point mutations, made a protein product, and produced viable spores. Neither strain had detectable V-ATPase activity. The vma-1 mutant strains did not grow in medium buffered to pH 7.0 or above or in medium supplemented with the cation Zn(2+). They were completely resistant to inhibition by concanamycin C, supporting our hypothesis that the V-ATPase is the in vivo target of this antibiotic. Inactivation of the vma-1 gene had a pronounced effect on morphology and development of the organism. In the mutants tip growth was inhibited, and multiple branching was induced. The vma-1 mutant strains could not differentiate conidia or perithecia. They could grow slowly as mycelia and could donate nuclei in a sexual cross. A mutation in the plasma membrane ATPase, which suppressed the sensitivity of wild type N. crassa to concanamycin, also proved effective in suppressing the sensitivity of a vma-1 null mutant to basic pH but did not correct the morphological defects.

Highlights

Vacuolar Hϩ-ATPases (V-ATPases)1 are large, complex enzymes found on multiple members of the endomembrane system of eukaryotic cells and on the plasma membranes of many specialized cells [1, 2]
More recently the VMA1 gene was disrupted in Ashbya gossypii, a filamentous fungus related to S. cerevisiae
N. crassa is a haploid organism, which readily takes up DNA by transformation and incorporates it into the genome either by homologous recombination or more frequently by recombination at Strains, genotypes, and phenotypes are defined in Table I and the text

Summary

Introduction

Vacuolar Hϩ-ATPases (V-ATPases) are large, complex enzymes found on multiple members of the endomembrane system of eukaryotic cells and on the plasma membranes of many specialized cells [1, 2]. Studies of V-ATPases in fungi have primarily focused on the enzyme in vacuolar membranes These organelles are important as storage reservoirs for basic amino acids and polyphosphate, as sites for protein degradation and turnover, and as sequestration sites of potentially toxic ions, especially Ca2ϩ [3,4,5]. Not strictly petϪ as originally thought [15], the deletion strains do not grow on non-fermentable substrates under usual conditions and need inositol in the medium [11] Vacuoles in such mutants are not acidified [9, 10], and cytosolic Ca2ϩ levels are elevated [11]. We wanted to determine if loss of the vacuolar ATPase caused the metabolic phenotypes observed in yeast VMA mutants, e.g. failure to grow on high concentrations of a non-fermentable carbon source.

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