Mutations in subunit C of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site.
Bafilomycin A1, a potent inhibitor of vacuolar H(+)-ATPases (V-ATPase), inhibited growth of Neurospora crassa in medium adjusted to alkaline pH. Ninety-eight mutant strains were selected for growth on medium (pH 7.2) containing 0.3 or 1.0 microm bafilomycin. Three criteria suggested that 11 mutant strains were altered in the V-ATPase: 1) these strains accumulated high amounts of arginine when grown at pH 5.8 in the presence of bafilomycin, 2) the mutation mapped to the locus of vma-3, which encodes the proteolipid subunit c of the V-ATPase, and 3) V-ATPase activity in purified vacuolar membranes was resistant to bafilomycin. Sequencing of the genomic DNA encoding vma-3 identified the following mutations: T32I (two strains), F136L (two strains), Y143H (two strains), and Y143N (five strains). Characterization of V-ATPase activity in the four kinds of mutant strains showed that the enzyme was resistant to bafilomycin in vitro, with half-maximal inhibition obtained at 80-400 nm compared with 6.3 nm for the wild-type enzyme. Surprisingly, the mutant enzymes showed only weak resistance to concanamycin. Interestingly, the positions of two mutations corresponded to positions of oligomycin-resistant mutations in the c subunit of F(1)F(0)-ATP synthases (F-ATPases), suggesting that bafilomycin and oligomycin utilize a similar binding site and mechanism of inhibition in the related F- and V-ATPases.
Bowman
Barry J
BJ
Department of Molecular, University of California, Santa Cruz, California 95064, USA. bowman@biology.ucsc.edu
Bowman
Emma Jean
EJ
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D018942:Macrolides; D008969:Molecular Sequence Data; D009154:Mutation; D009492:Neurospora crassa / Q000187:drug effects / Q000254:growth & development; D006180:Proton-Translocating ATPases; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D018942:Macrolides; C087287:VMA-3 protein, Neurospora crassa; D004024:Dicyclohexylcarbodiimide; C036978:concanamycin A; C040929:bafilomycin A1; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases