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

Abstract

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 M 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 wildtype 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.The vacuolar (H ϩ )-ATPase (V-ATPase) 1 is a large, complex enzyme that couples the hydrolysis of ATP to the transport of protons across membranes. In eucaryotic cells, this enzyme plays a role in many physiological processes. It is present in several types of cellular organelles such as vacuoles, lysosomes, coated vesicles, Golgi, and secretory vesicles (reviewed in Refs. 1-3), and it is also the major proton pump in the plasma membrane of specialized acid-secreting cells such as osteoclasts and kidney intercalated cells and the intestinal epithelia cells of some insects (4). The role of the V-ATPase in physiological processes has often been examined by measuring the effects of bafilomycin and concanamycin, macrolide antibiotics that are potent inhibitors of the enzyme (5). Effective at nanomolar concentrations in vitro, these two drugs also inhibit the VATPase in living cells, although higher concentrations, typically 0.1-10.0 M, are required (6). At low concentrations, the two antibiotics appear to be highly specific for V-ATPases; at 10,000-fold higher concentrations, they inhibit some P-type ATPases in vitro (7,8).Because of their effectiveness and specificity in vivo, bafilomycin and concanamycin are attractive candidates for development as therapeutic agents (9, 10). For example, considerable effort has been made to develop bafilomycin derivatives for the treatment of osteoporosis (11-14). Bafilomycin and concanamycin are also potent anti-tumor agents that exhibit significant cell line specificity (15). Thus, derivatives of these inhibitors might be effective in treating cancer. The pharmacological potential of these drugs has pr...

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

“…Autoradiographies of gels after exposition to a phosphoscreen (lanes 2, 4, and 6). explain the discrepancy between this result and the results obtained with N. crassa mutants, which exhibited a higher tolerance to bafilomycin but not to concanamycin (10). Because labeling was not impaired by salicylihalamide, we conclude that the site(s) and mechanism of inhibition for benzolactone enamides may be different from that for plecomacrolides.…”

“…In the presence of 1 mM DCCD the V-ATPase did not bind to the column, indicating that the binding site for bafilomycin is near the binding site(s) for DCCD and may thus be located in the proteolipid, subunit c. However, because DCCD, especially at the rather high concentration of 1 mM, may bind to sites different from that in the proteolipid and may bind not only to carboxyl groups but also to sulfhydryl groups or to tyrosine residues (9), and since the 100-kDa subunit a was not clearly resolved in this study, the bafilomycin binding subunit was still not identified. Strong hints in favor of subunit c representing the bafilomycin binding subunit were provided by recent results from analysis of Neurospora crassa strains, indicating that those with point mutations in subunit c show a higher tolerance to bafilomycin (I 50 80 -400 nM) compared with the wild type (I 50 6.3 nM); interestingly, the tolerance to concanamycin was not changed simultaneously (10).…”

Activity of Plasma Membrane V-ATPases Is Critical for the Invasion of MDA-MB231 Breast Cancer Cells

“…Although sufficient quantities of streptavidin-biotin-bafilomycin were not available to perform the cytoplasmic pH and proton flux experiments described above, we assessed ATP-dependent proton transport in vacuolar membranes isolated from yeast in the presence and absence of this compound. Bafilomycin binds to the V-ATPase at a site within the ring of c subunits (36). We expected that when bound to streptavidin, biotin-bafilomycin would be able to inhibit activity from the luminal (or extracellular) surface but because of steric interference between streptavidin and the V 1 domain would be unable to inhibit the pump from the cytoplasmic surface.…”

cAMP regulates plasma membrane vacuolar-type H + -ATPase assembly and activity in blowfly salivary glands

“…V 0 forms a membranespanning proton-translocating complex; in yeast, it is composed of at least five different subunits termed a, c, cЈ, cЉ, and d, of which subunit c binds bafilomycin A 1 , a specific inhibitor of V-ATPases (5)(6)(7)(8). The V 1 sector is attached to the cytoplasmic side of the V 0 sector, consists of at least eight different subunits termed A-H, and contains catalytic and noncatalytic ATPbinding sites.…”