Mutations in Subunit c of the Vacuolar ATPase Confer Resistance to Bafilomycin and Identify a Conserved Antibiotic Binding Site

• Published in: The Journal of biological chemistry 2002-02, Vol.277 (6), p.3965-3972
• Main Authors: Bowman, Barry J., Bowman, Emma Jean
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anti-Bacterial Agents - pharmacology; Bafilomycin A1; Base Sequence; concanamycin; Dicyclohexylcarbodiimide - pharmacology; DNA Primers; Enzyme Inhibitors - pharmacology; Fungal Proteins - genetics; Macrolides; Molecular Sequence Data; Mutation; Neurospora crassa; Neurospora crassa - drug effects; Neurospora crassa - growth & development; oligomycin; Proton-Translocating ATPases; Sequence Homology, Amino Acid; Vacuolar Proton-Translocating ATPases - antagonists & inhibitors; Vacuolar Proton-Translocating ATPases - chemistry; Vacuolar Proton-Translocating ATPases - genetics; vma-3 gene
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109756200

Bafilomycin A1, a potent inhibitor of vacuolar H+-ATPases (V-ATPase), inhibited growth ofNeurospora 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 μm 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 ofvma-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 encodingvma-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 bafilomycinin 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 F1F0-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.