Amino acid substitutions in mitochondrial ATPase subunit 6 of <i>Saccharomyces cerevisiae</i> leading to oligomycin resistance

John, Ulrik P.; Nagley, Phillip

doi:10.1016/0014-5793(86)80016-3

Cited by 62 publications

(34 citation statements)

References 20 publications

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“…Mutations in the a subunit of the F-ATPase were also found to confer resistance to oligomycin (38,39). These data were interpreted to show that the inhibitors bind to the interface between subunits c and a (37).…”

Section: Table IVmentioning

confidence: 92%

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

Bowman

2002

Journal of Biological Chemistry

131

100

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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...

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Section: Table IVmentioning

confidence: 92%

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

Bowman

2002

Journal of Biological Chemistry

131

100

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“…Indeed as previously shown through the analysis of numerous yeast ATP synthase mutants, the activity of this enzyme needs to be decreased by at least 80% to see an obvious respiratory growth defect, which indicates that ATP synthase is far from limiting for the proliferation of yeast cells producing ATP by oxidative phosphorylation [30,31]. However, when the rate of mitochondrial ATP production is diminished cells become more sensitive to chemical inhibition of ATP synthase with oligomycin [31], a compound that is presumed to target the F O because mutations in Atp9p and Atp6p can confer an increased resistance to it [32,33].…”

Section: Respiratory Growth and Genetic Stability Of Yeast Mutants Atmentioning

confidence: 99%

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C

et al. 2014

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Mutations in the human mitochondrial ATP6 gene encoding ATP synthase subunit a/6 (referred to as Atp6p in yeast) are at the base of neurodegenerative disorders like Neuropathy Ataxia Retinitis Pigmentosa (NARP), Leigh syndrome (LS), Charcot-Marie-Tooth (CMT), and ataxia telangiectasia. In previous studies, using the yeast Saccharomyces cerevisiae as a model we were able to better define how several of these mutations impact the ATP synthase.Here we report the construction of yeast models of two other ATP6 pathogenic mutations, T9185C and T9191C. The first one was reported as conferring a mild, sometimes reversible, CMT clinical phenotype; the second one has been described in a patient presenting with severe LS. We found that an equivalent of the T9185C mutation partially impaired the functioning of yeast ATP synthase, with only a 30% deficit in mitochondrial ATP production.An equivalent of the mutation T9191C had much more severe effects, with a nearly complete block in yeast Atp6p assembly and an >95% drop in the rate of ATP synthesis. These findings provide a molecular basis for the relative severities of the diseases induced by T9185C and T9191C.

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“…This suggested that these two classes of drugs were binding to related sites on the two classes of ATPase. Because mutations on F-ATPase subunit a have been identified that confer resistance to oligomycin (37)(38)(39), this suggested the possibility that V-ATPase subunit a, although not homologous to the F-ATPase subunit a, might similarly be participating in drug binding. The results of the present study support this conclusion and provide further insight into how bafilomycin may inhibit the V-ATPase.…”

mentioning

confidence: 99%

Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin

Wang

Inoué

Forgac

2005

Journal of Biological Chemistry

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The vacuolar (H ϩ )-ATPases (or V-ATPases) 2 are ATP-dependent proton pumps widely distributed among intracellular and plasma membranes of eukaryotic cells (1-11). They carry out ATP-driven transport of protons from the cytoplasm to either the lumen of internal compartments or to the extracellular space. V-ATPases present in intracellular membranes are important for membrane traffic processes such as receptor-mediated endocytosis and intracellular targeting of newly synthesized lysosomal enzymes (1). They also provide the acidic environment required for processing and degradation of macromolecules and the entry of certain envelope viruses and bacterial toxins, including anthrax toxin (12), as well as the driving force for coupled transport of small molecules, such as neurotransmitters. Plasma membrane V-ATPases function in a wide variety of normal and disease processes, including renal acidification, bone resorption, K ϩ secretion, sperm maturation, and tumor cell invasion (1, 7-11). V-ATPase inhibitors are thus potentially useful therapeutic agents for the treatment of a number of human diseases, including viral infection, osteoporosis, and cancer. V-ATPases are multisubunit complexes composed of two domains (1-9). The peripheral V 1 domain is composed of eight subunits (A-H) and functions to hydrolyze ATP. The integral V 0 domain is composed of six subunits (in yeast these are subunits a, c, cЈ, cЉ, d, and e) and is responsible for proton transport. The V-ATPases thus structurally resemble the ATP synthases (or F-ATPases) of mitochondria, chloroplasts, and bacteria that function in the synthesis of ATP (13-16).Like the F-ATPases (13-16), the V-ATPases have been shown to operate by a rotary mechanism (17, 18). ATP hydrolysis in the V 1 domain drives rotation of a central stalk composed of subunits D and F (17, 19). These subunits are linked to a ring of proteolipid subunits (c, cЈ, and cЉ) via subunit d (20). Each proteolipid subunit contains a single buried acidic residue that undergoes reversible protonation and deprotonation during the rotational catalysis (21). As the ring of proteolipid subunits rotates relative to subunit a, each buried carboxyl group on the ring is thought to pick up a proton from the cytoplasmic compartment via a hemi-channel located in subunit a (1, 15, 22, 23). Rotation of the ring brings this protonated carboxyl into contact with a second a subunit hemi-channel leading to the luminal or extracellular side of the membrane. Interaction between the carboxyl group and the positively charged guanidinium group of Arg 735 of subunit a (23) then causes release of the proton into the luminal hemi-channel.Subunit a is a 100-kDa integral membrane protein containing two domains (24). The N-terminal hydrophilic domain has a molecular mass of 50 kDa and is oriented toward the cytoplasmic side of the membrane (25). Together with subunits C, E, G, H, and the non-homologous domain of subunit A (19, 26 -29), the N-terminal domain forms a peripheral stalk or stator that keeps the catalytic domain of t...

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Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance

Cited by 62 publications

References 20 publications

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

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

Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C

Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin

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