Mutations of pma-1, the gene encoding the plasma membrane H+-ATPase of Neurospora crassa, suppress inhibition of growth by concanamycin A, a specific inhibitor of vacuolar ATPases.
Concanamycin A (CCA), a specific inhibitor of vacuolar ATPases, inhibited growth of Neurospora crassa in medium adjusted to pH 7 or above. Mutant strains were selected for growth on medium containing 1.0 microM CCA. Sixty-four (of 66) mutations mapped in the region of the pma1 locus, which encodes the plasma membrane H+-ATPase. Analysis of V-ATPase activity in isolated vacuolar membranes from the mutant strains showed wild-type activity and sensitivity to CCA. In contrast, plasma membrane H+-ATPase activity in isolated plasma membranes from the mutants was reduced as compared with wild-type, and in four strains the activity showed increased resistance to vanadate. The most interesting change in the plasma membrane H+-ATPase was in kinetic behavior. The wild-type enzyme showed sigmoid dependence on MgATP concentration with a Hill number of 2.0, while the seven mutants tested exhibited hyperbolic kinetics with a Hill number of 1.0. One interpretation of these data was that the enzyme had changed from a functional dimer to a functional monomer. Mutation of the plasma membrane H+-ATPase did not confer resistance by preventing uptake of CCA. In the presence of CCA both wild-type and mutant strains were unable to accumulate arginine, failed to concentrate chloroquine in acidic vesicles, and exhibited gross alterations in hyphal morphology, indicating that the CCA had entered the cells and inactivated the V-ATPase. Instead, we hypothesize that the mutations conferred resistance because the altered plasma membrane H+-ATPase could more efficiently rid the cell of toxic levels of Ca2+ or protons or other ions accumulated in the cytoplasm following inactivation of the V-ATPase by CCA.
Bowman
E J
EJ
Department of Biology, Sinsheimer Laboratories, University of California, Santa Cruz, California 95064, USA. rbowman@biology.ucsc.edu
O'Neill
F J
FJ
Bowman
B J
BJ
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D002462:Cell Membrane / Q000201:enzymology; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D004352:Drug Resistance, Microbial; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005800:Genes, Fungal / Q000187:drug effects*; D006921:Hygromycin B / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D007700:Kinetics; D018942:Macrolides; D008928:Mitochondria / Q000201:enzymology; D016296:Mutagenesis; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000096:biosynthesis* / Q000235:genetics*; D013045:Species Specificity; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D018942:Macrolides; D006921:Hygromycin B; C036978:concanamycin A; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases