Investigation of the Role and Mechanism of IF1 and STF1 Proteins, Twin Inhibitory Peptides Which Interact with the Yeast Mitochondrial ATP Synthase

Venard, Renée; Brèthes, Daniel; Giraud, Marie‐France; Vaillier, Jacques; Velours, Jean; Haraux, Francis

doi:10.1021/bi034394t

Cited by 53 publications

(52 citation statements)

References 66 publications

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“…5b), yet much slower than at high MgATP concentration (see below). Assuming that k app is proportional to the IF1 concentration (37,41) Fig. 6 shows the relationship between MgATP concentrations and the rate of IF1 inhibition.…”

Section: Resultsmentioning

confidence: 99%

“…Because of the presence of the N-terminal enterokinase cleavage site, the first three amino acids of the recombinant and processed enzyme were Ala-Met-Ala, as compared with the natural IF1, which starts with Ser. However, the inhibitory activity of the recombinant IF1, as measured by the k on rate constant of formation of the IF1-MF 1 complex in the presence of 1 mM MgATP, was the same as the natural IF1 from yeast or a chemically synthesized product made by Neosystem (Strasbourg, France) (37).…”

Section: Methodsmentioning

confidence: 99%

“…ATP Hydrolysis Measurement and Kinetic Analysis-Continuous monitoring of ATP hydrolysis coupled to NADH oxidation was carried out spectrophotometrically as described (37). The reaction was in a stirred and thermostatted cuvette (25°C) containing 50 mM MES, pH 6.5, 20 mM KCl, 1 mM MgCl 2 , 1 mM phosphoenolpyruvate, 20 units/ml pyruvate kinase, 50 units/ml lactate dehydrogenase, 0.4 mM NADH and MgATP at from 0.5 M to 20 mM.…”

Section: Methodsmentioning

confidence: 99%

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The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide

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Venard

et al. 2005

Journal of Biological Chemistry

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The mechanism of inhibition of yeast mitochondrial F 1 -ATPase by its natural regulatory peptide, IF1, was investigated by correlating the rate of inhibition by IF1 with the nucleotide occupancy of the catalytic sites. Nucleotide occupancy of the catalytic sites was probed by fluorescence quenching of a tryptophan, which was engineered in the catalytic site (␤-Y345W). Fluorescence quenching of a ␤-Trp 345 indicates that the binding of MgADP to F 1 can be described as 3 binding sites with dissociation constants of K d1 ‫؍‬ 10 ؎ 2 nM, K d2 ‫؍‬ 0.22 ؎ 0.03 M, and K d3 ‫؍‬ 16.3 ؎ 0.2 M. In addition, the ATPase activity of the ␤-Trp 345 enzyme followed simple Michaelis-Menten kinetics with a corresponding K m of 55 M. Values for the K d for MgATP were estimated and indicate that the K m (55 M) for ATP hydrolysis corresponds to filling the third catalytic site on F 1 . IF1 binds very slowly to F 1 -ATPase depleted of nucleotides and under unisite conditions. The rate of inhibition by IF1 increased with increasing concentration of MgATP to about 50 M, but decreased thereafter. The rate of inhibition was half-maximal at 5 M MgATP, which is 10-fold lower than the K m for ATPase. The variations of the rate of IF1 binding are related to changes in the conformation of the IF1 binding site during the catalytic reaction cycle of ATP hydrolysis. A model is proposed that suggests that IF1 binds rapidly, but loosely to F 1 with two or three catalytic sites filled, and is then locked in the enzyme during catalytic hydrolysis of ATP.F 0 F 1 1 proton ATPases (or ATP synthases), present in energytransducing membranes, are molecular motors powered by a protonmotive force generated by an electron transport chain (1). ATP synthases catalyze the phosphorylation of ADP forming ATP. The ATP synthase consists of two subcomplexes: F 0 and F 1 . F 0 is an integral membrane protein complex minimally composed of a 1 b 1 c 10 and it acts as a proton-driven turbine (2). The extrinsic F 1 moiety is composed of five different subunits with the stoichiometry ␣ 3 ␤ 3 ␥␦⑀. F 1 contains three catalytic and three non-catalytic nucleotide-binding sites, located at ␣/␤ interfaces (3-4). F 1 and F 0 are connected by a central stalk composed of the ␥, ␦, and ⑀ subunits.In the mitochondrion, movement of protons from the cytosol to the matrix drives the F 0 turbine, which rotates the central stalk. There is a second or peripheral stalk, which holds together the static parts of the machine including the core F 1 unit. Rotation of the central stalk in F 1 sequentially modifies the interactions of the ␥-subunit with each of the catalytic sites thereby altering the nucleotide affinity of each of the three catalytic sites. Thus, F 1 has 3 catalytic sites each with different catalytic properties including their affinity for nucleotides.F 1 couples ATP hydrolysis to the rotation of the ␥ subunit in the opposite direction of that observed during ATP synthesis (5-6). Single-molecule studies indicated that hydrolysis of ATP is coupled with 120°rotation of the ␥-subuni...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide

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Sigalat

Venard

et al. 2005

Journal of Biological Chemistry

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“…This dye accumulates inside the mitochondrial matrix, where its fluorescence is quenched,in response to an established  [27]. Before testing for ATP-driven proton translocation, the mitochondria were energized with ethanol in order to elicit release of the natural inhibitory peptide (IF1) that binds F 1 F O in the resting state and prevents ATP hydrolysis [37]. The imposed  was then collapsed with KCN and less than 2 minutes later ATP was added, i.e.…”

Section: C) Atp-driven Translocation Of Protons Across the Mitochondrmentioning

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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“…The respiratory activity was then blocked with KCN, and less than 1 min later (thus well before rebinding of IF1, see Ref. 47) ATP was added. In wild type mitochondria, the ATP addition promoted a large and stable fluorescence quenching of the dye that was oligomycin-sensitive (Fig.…”

Section: Influence Of Atp6-l183r Mutation Onmentioning

confidence: 99%

A Yeast Model of the Neurogenic Ataxia Retinitis Pigmentosa (NARP) T8993G Mutation in the Mitochondrial ATP Synthase-6 Gene

Rak

Tétaud

Duvezin-Caubet

et al. 2007

Journal of Biological Chemistry

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NARP (neuropathy, ataxia, and retinitis pigmentosa) and MILS (maternally inherited Leigh syndrome) are mitochondrial disorders associated with point mutations of the mitochondrial DNA (mtDNA) in the gene encoding the Atp6p subunit of the ATP synthase. The most common and studied of these mutations is T8993G converting the highly conserved leucine 156 into arginine. We have introduced this mutation at the corresponding position (183) of yeast Saccharomyces cerevisiae mitochondrially encoded Atp6p. The "yeast NARP mutant" grew very slowly on respiratory substrates, possibly because mitochondrial ATP synthesis was only 10% of the wild type level. The mutated ATP synthase was found to be correctly assembled and present at nearly normal levels (80% of the wild type). Contrary to what has been reported for human NARP cells, the reverse functioning of the ATP synthase, i.e. ATP hydrolysis in the F 1 coupled to F 0 -mediated proton translocation out of the mitochondrial matrix, was significantly compromised in the yeast NARP mutant. Interestingly, the oxygen consumption rate in the yeast NARP mutant was decreased by about 80% compared with the wild type, due to a selective lowering in cytochrome c oxidase (complex IV) content. This finding suggests a possible regulatory mechanism between ATP synthase activity and complex IV expression in yeast mitochondria. The availability of a yeast NARP model could ease the search for rescuing mechanisms against this mitochondrial disease.

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Investigation of the Role and Mechanism of IF1 and STF1 Proteins, Twin Inhibitory Peptides Which Interact with the Yeast Mitochondrial ATP Synthase

Cited by 53 publications

References 66 publications

The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide

The Binding Mechanism of the Yeast F1-ATPase Inhibitory Peptide

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

A Yeast Model of the Neurogenic Ataxia Retinitis Pigmentosa (NARP) T8993G Mutation in the Mitochondrial ATP Synthase-6 Gene

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