Disruption of vma-1, the gene encoding the catalytic subunit of the vacuolar H(+)-ATPase, causes severe morphological changes in Neurospora crassa.
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.
Bowman
E J
EJ
Department of Biology, University of California, Santa Cruz, California 95064, USA. rbowman@biology.ucsc.edu
Kendle
R
R
Bowman
B J
BJ
D003433:Crosses, Genetic; D005800:Genes, Fungal; D005838:Genotype; D009154:Mutation; D009492:Neurospora crassa / Q000166:cytology* / Q000235:genetics* / Q000254:growth & development; D010641:Phenotype; D017354:Point Mutation; D006180:Proton-Translocating ATPases / Q000235:genetics*; D013012:Sorbitol / Q000494:pharmacology; D013172:Spores, Fungal; D013489:Suppression, Genetic; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology* / Q000648:ultrastructure
D013012:Sorbitol; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases