Michel Satre

Recent developments on structural and functional aspects of the F1 sector of H+-linked ATPases

SummaryThis review concerns the catalytic sector of F1 factor of the H+-dependent ATPases in mitochondria (MF1), bacteria (BF1) and chloroplasts (CF1). The three types of Ft have many similarities with respect to the structural parameters, subunit composition and catalytic mechanism. An α3β3γ2δ2ε2 stoichiometry is now accepted for MF1 and BF1; the α2β2γ2δ2ɛ2 stoichiometry for CFI remains as matter of debate. The major subunits α, β and γ are equivalent in MF1, BF1 and CF1; this is not the case for the minor subunits δ and ε. The δ subunit of MFI corresponds to the ε subunit of BF1 and CF1, whereas the mitochondria) subunit equivalent to the δ subunit of BF1 and CF1 is probably the oligomycin sensitivity conferring protein (OSCP). The a β γ assembly is endowed with ATPase activity, β being considered as the catalytic subunit and y as a proton gate. On the other hand, the 6 and E subunits of BFI and CFI most probably act as links between the F1 and F0 sectors of the ATPase complex. The natural mitochondria) ATPase inhibitor, which is a separate protein loosely attached to MF1, could have its counterpart in the E subunit of BF1 and CF1. The generally accepted view that the catalytic subunit in the different F1 species is β comes from a number of approaches, including chemical modification, specific photolabeling and, in the case of BF1, use of mutants. The a subunit also plays a central role in catalysis, since structural alteration of a by chemical modification or mutation results in loss of activity of the whole molecule of F1. The notion that the proton motive force generated by respiration is required for conformational changes of the F1 sector of the H+-ATPase complex has gained acceptance. During the course of ATP synthesis, conversion of bound ADP and Pi into bound ATP probably requires little energy input; only the release of the F1-bound ATP would consume energy. ADP and Pi most likely bind at one catalytic site of F1, while ATP is released at another site. This mechanism, which underlines the alternating cooperativity of subunits in F1, is supported by kinetic data and also by the demonstration of partial site reactivity in inactivation experiments performed with selective chemical modifiers. One obvious advantage of the alternating site mechanism is that the released ATP cannot bind to its original site. The chemistry of the condensation reaction of ADP and Pi to form ATP has not yet been elucidated. Although implicitly admitted, definite evidence that the condensation reaction does not involve a phosphorylated intermediate has been acquired recently by analysis of the stereochemical course of the phosphoric residue transfer in ATP synthesis or hydrolysis. Whereas the catalytic events of ATP synthesis are well understood, the regulatory mechanism, and particularly the role of the so-called inhibitory peptides, remain enigmatic.

Cytotoxicity of dichloromethane diphosphonate and of 1-hydroxyethane-1,1-diphosphonate in the amoebae of the slime mould Dictyostelium discoideum: A 31P NMR study

Two pyrophosphate analogues, dichloromethane diphosphonate (Cl2MDP), and 1-hydroxyethane-1,1-diphosphonate (EHDP), at concentrations of 0.5-1 mM, efficiently inhibited the growth of amoebae of the slime mould Dictyostelium discoideum. Cell viability decreased markedly upon incubation with the diphosphonates. The mechanism of toxicity was investigated by in vivo 31P NMR spectroscopy and the formation of analogues of ATP [adenosine 5-(beta, gamma-dichloromethane triphosphate) and adenosine 5-(beta, gamma-1-hydroxyethane triphosphate)] was demonstrated. These two compounds were identified from their 31P NMR spectra in perchloric acid extracts prepared from amoebae poisoned with Cl2MDP or EHDP and may have been synthesized by reversible pyrophosphate exchange catalysed by cytosolic aminoacyl-tRNA synthetases.

Spontaneous aggregation of the mitochondrial natural ATPase inhibitor in salt solutions as demonstrated by gel filtration and neutron scattering. Application to the concomitant purification of the ATPase inhibitor and F1-ATPase

(1) The natural ATPase inhibitor (IF1) from beef heart mitochondria has a tendency to form aggregates in aqueous solutions. The extent of aggregation and the structure of the aggregates were assessed by gel filtration and small-angle neutron scattering. IF1 polymerization was found to depend on the salt concentrations, pH of the medium and concentration of IF1. The higher the salt concentration, the lower the aggregation state. Aggregation of IF1 was decreased at slightly acidic pH. It increased with the concentration of IF1 as expected from the law of mass action. (2) Neutron scattering showed the aggregation of IF1 in 2 M ammonium sulfate solutions. The predominant species is the dimer which has a somewhat elongated shape. (3) The Sephadex G-50 chromatography that is supposed to deprive beef heart submitochondrial particles of loosely bound IF1 (Racker, E. and Horstman, L.L. (1967) J. Biol. Chem. 242, 2547-2551) was shown to have a limited effectiveness as a trap for IF1. The reason was that IF1 released from the particles formed high molecular weight aggregates that were not separated from the membrane vesicles by Sephadex G-50 chromatography. (4) The above observations provide the basis for a simple method of purification of beef heart IF1 which combines the recovery of the supernatant from submitochondrial particles with the last three steps of the IF1 preparation described by Horstman and Racker (J. Biol. Chem. (1970) 265, 1336-1344). The particles recovered in the sediment were deprived of IF1 and could therefore be used for preparation of F1-ATPase. The advantage of this method is that both IF1 and F1-ATPase can be prepared from the same batch of mitochondria.

Structure of beef heart mitochondrial F1-ATPase Arrangement of subunits as disclosed by cross-linking reagents and selective labeling by radioactive ligands

1. The following bifunctional reagents, dimethylsuberimidiate, dimethyladipimidate, methylmercaptobutyrimidate have been used to produce dimers between the neighboring subunits of beef heart F1-ATPase. 2. Treatment of beef heart F1-ATPase with dimethylsuberimidate or dimethyladipimidate resulted in the formation of four cross-linked products. Their molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 11 500, 105 000, 95 000 and 80 000, respectively. The products of molecular weight 115 000 and 105 000 were predominant and could be detected at the early stage of the cross-linking reaction. Treatment of beef heart F1-ATPase with methylmercaptobutyrimidate resulted in the accumulation of the product of molecular weight 115 000 and in traces of products of lower molecular weight. When the cross-linked products obtained with methylmercaptobutyrimidate were cleaved by beta-mercaptoethanol, the original gel electrophoresis pattern was restored. 3. Cross-linking of beef heart F1-ATPase by dimethylsuberimidate, dimethyladipimidate and methylmercaptobutyrimidate was accompanied by a loss of the ATPase activity. Cleavage of the cross-linked products obtained with methylmercaptobutyrimidate did not restore the original ATPase activity. 4. Identification of subunits A and B in the products of molecular weight 115 000 and 105 000 was achieved by specific labeling of subunit A with N-[14C]ethylmaleimide and of subunit B by chloronitro [14C]benzooxodiazole. Both products were able to bind N-[14C]ethylmaleimide; only the 105 000 dalton product was able to bind chloronitro [14C]benzooxodiazole. 5. The product of molecular weight 115 000 obtained by treatment of beef heart ATPase with methylmercaptobutyrimidate could bind N-[14C]ethylmaleimide. Its cleavage, following N-[14C]ethylmaleimide binding, yielded one labeled peptide identified with subunit A by polyacrylamide gel electrophoresis. 6. The above results indicate that the product of molecular weight 115 000 is a dimer containing two subunits A and that the product of molecular weight 105 000 is a dimer containing one subunit A and one subunit B. It can therefore be concluded that, in beef heart F1-ATPase, the A subunits are close to each other and that subunit A is close to subunit B. In contrast the B sublnits are probably too far from each other to be cross-linked by dimethylsuberimidate, dimethyladipimidate or methylmercaptobutyrimidate.

Characterization of dicyclohexylcarbodiimide binding site on coupling factor 1 of mitochondrial and bacterial membrane-bound ATPases

The H’-linked ATPases (mitochondrial, chloroplastic, and bacterial ATPases) are made of two sectors, a membrane sector Fe which is considered a proton pump, and an extrinsic sector (factor Fr) which possesses the catalytic site [ 1,2]. Until recently, it was thought that dicyclohexylcarbodiimide (DCCD), a carboxylic reagent, bound specifically to a hydrophobic small rel. mol. mass protein (8000 M,) of the membrane sector (DCCD-binding protein) and concomitantly inactivated the entire ATPase complex (reviewed [3]). However, in [4,5] evidence was given that DCCD was also able to bind to and inactivate isolated factor 1 (Fr in mitochondria and BFr in bacteria). Inactivation was much more rapid at acid pH than at alkaline pH, the half-maximum effect being obtained at pH -7. Inactivation was accompanied by the covalent binding of DCCD to the /.l subunit of mitochondrial and Escherichia coli Fr. Evidence for binding of DCCD to a carboxyl group was provided by the interfering effect of a nucleophilic reagent, glycine ethyl ester, and by demonstration, in electrofocusing experiments, of the loss of a negative charge on the 0 subunit of E. coli F1. Similar effects of DCCD on the soluble chloroplastic factor F, were reported [6]. That DCCD may bind to two sites on H’hnked ATPases, i.e., the proteolipid of factor F0 and the 0 subunit of factor Fr, is not yet fully accepted; interaction of DCCD with F, may be peculiar to the soluble Fr and not occur with the membrane-bound

Titration of the binding sites for the oligomycin-sensitivity conferring protein in beef heart submitochondrial particles

The binding parameters of the oligomycin‐sensitivity conferring protein (OSCP) in inside‐out particles from beef heart mitochondria have been tested by means of two assays, the oligomycin‐sensitive ATP—Pi exchange, and the oligomycin‐sensitive ATP hydrolysis. The total number of OSCP binding sites in A particles was equal to 220 pmol/mg particle protein. Each mole of ATPase active site was able to bind 1.1±0.5 mol OSCP with K d 1.7 nM.

The effect of asteltoxin and citreomontanine, two polyenic α-pyrone mycotoxins, on Escherichia coli adenosine triphosphatase

Abstract Asteltoxin and citreomontanine, two polyenic α-pyrone mycotoxins closely related to aurovertins and citreoviridin, have been tested for their effect on the ATPase activity of E. coli BF1. Citreomontanine was inactive towards E. coli ATPase. Asteltoxin inhibited E. coli BF1-ATPase activity with a potency intermediate between that of citreoviridin and aurovertins. Asteltoxin had no inhibitory effect on BF1 isolated from an aurovertin-resistant mutant. Like for aurovertin, large enhancement of the fluorescence of asteltoxin was observed upon interaction of asteltoxin with BF1. There was no enhancement of fluorescence when citreomontanine was added to BF1. The fluorescence response of asteltoxin was further stimulated by ADP and quenched by Mg2+. The binding data showed one binding site for asteltoxin per BF1 in the presence of ADP. No fluorescent complex was formed when asteltoxin was added to BF1 isolated from an aurovertin-resistant mutant. In contrast to aurovertin, asteltoxin did not enhance the binding affinity of BF1 for inorganic phosphate. Data presented in the paper indicate that, in the aurovertin family derivatives, the terminal ring system opposite to the α-pyrone end of the molecule plays a decisive role in inhibitory and binding properties with respect to ATPase.

Inactivation of beef heart mitochondrial F1-ATPase by the 2′,3′-dialdehyde derivatives of adenine nucleotides

Beef heart mitochondrial F1‐ATPase was inactivated by the 2′,3′‐dialdehyde derivatives of ATP, ADP and AMP (oATP, oADP, oAMP). In the absence of Mg2+, inactivation resulted from the binding of 1 mol nucleotide analog per active unit of F1. The most efficient analog was oADP, followed by oAMP and oATP. Complete inactivation was correlated with the binding of about 11 mol [14C]oADP/mol F1. After correction for non‐specific labeling, the number of specifically bound [14C]oADP was 2–3 mol per mol F1. By SDS‐polyacrylamide gel electrophoresis, [14C]oADP was found to bind covalently mainly to the α and β subunits. In the presence of Mg2+, oATP behaved as a substrate and was slowly hydrolyzed.

The ϵ subunit as an ATPase inhibitor of the F1-ATPase in Escherichia coli

Abstract The isolation of protein ATPase inhibitor was attempted directly from Escherichia coli membrane extracts to examine the possible presence of a Pullman-Monroy-type inhibitor [ M. E. Pullman and G. C. Monroy (1963) J. Biol. Chem. 238 , 3762–3769] distinct from the ϵ subunit of E. coli ATPase. Purification to homogeneity was achieved in a sequence of steps involving trichloracetic acid precipitation, DEAE-cellulose, Sephadex G75 chromatography, and a terminal isoelectric focusing step. An inhibitory protein was obtained and was identified by its physicochemical and inhibitory properties as the ϵ subunit of E. coli ATPase. The other inhibitory fraction observed in the purification procedure consisted of aggregated ϵ subunits.

A kinetic and binding study of the reactivity of Escherichia coli ATPase to N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.

Escherichia coli H+-ATPase (ECF1) was inactivated in a time- and concentration-dependent manner by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a selective carboxyl group reagent. Among the subunits of ECF1, only the beta subunit was modified by EEDQ. The reaction of 1 mol of EEDQ per mol of ECF1 resulted in total inactivation, in spite of the fact that the enzyme possesses three beta subunits.