Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic ∼9 nmF ₁ (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the “etched” surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with the freeze-dried TU particles (trypsin-urea treated SMP) that are devoid ofF ₁ (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-“etching.”     

Mg2+-induced ADP-dependent inhibition of the ATPase activity of beef heart mitochondrial coupling factor F1.

Incubation of F₁ in the presence of Mg²⁺ results in a pronounced lag in its ATPase activity measured with the ATP-regenerating system. A decrease of the initial rate of ATPase induced by Mg²⁺ is also observed when free nucleotides were separated from the enzyme by Sephadex gel filtration. No inhibition is observed when F₁ treated to remove tightly bound nucleotides was preincubated in the presence of Mg²⁺. Mg²⁺-induced inhibition of ATPase activity of nucleotide-depleted F₁ can be restored by an addition of low concentrations of ADP. In all cases the inhibited ATPase can be activated by the ADP-removing system /phosphoenol pyruvate + pyruvate kinase/. It is concluded that i/ Mg²⁺-induced inhibition of the ATPase activity of F₁ is due to the formation of an inactive F₁. ADP complex; and ii/ unusual inhibition of oligomycin-sensitive ATPase by ADP /Fitin et al., Biochem. Biophys. Res. Communs. 1979, $\text{86}$, 434/ is directed to F₁ component of the complete mitochondrial ATPase system.     

Binding of ADP to beef-heart mitochondrial ATPase (F1)

1. ADP binding to beef-heart mitochondrial ATPase (F1), in the absence of Mg2+, has been determined by separating the free ligand by ultrafiltration and determining it in the filtrate by a specially modified isotachophoretic procedure. 2. Since during the binding experiments the 'tightly' bound ADP (but not the ATP) dissociates, it is necessary to take this into account in calculating the binding parameters. 3. The binding data show that only one tight binding site (Kd about 0.5 microM) for ADP is present. 4. It is not possible to calculate from the binding data alone the number of or the dissociation constants for the weak binding sites. It can be concluded, however, that the latter is not less than about 50 microM.     

The stimulation of coupling factor 1 ATPase by tentoxin.

Tentoxin at 10--1000 micrometer causes a marked species-selective stimulation of coupling factor 1 Ca2+-dependent ATPase activity (Ka 6.3 . 10(3) M-1). This effect decreases the Km for ATP to about 0.3 mM and increases V 2.75-fold. Above 1.6 micrometer tentoxin the rate of coupled electron transport was reduced to basal without uncoupling.     

Structure of ATPase (coupling factor TF1) from a thermophilic bacterium.

Sheets of soluble ATPase from a thermophilic bacterium, identified with coupling factor TF₁, were shown to be two-dimensional crystals. Computer filtering of an electron micrograph showed that the shape of ATPase (TF₁) is hexagonal and has pseudo 6-fold and 3-fold symmetry. The length of each side of a hexagon is about 38 Å.These observations suggest that there are six rather than four large subunits in ATPase.     

Inhibition of mitochondrial F1 ATPase and sarcoplasmic reticulum ATPase by hydrophobic molecules

The hydrophobic nature of the active site of two energy-transducing ATPases was explored by comparing interactions between Pi and each of three hydrophobic drugs in the absence and presence of organic solvents. The drugs tested were the Fe . bathophenanthroline complex and the anticalmodulin drugs, calmidazolium and trifluoperazine. All inhibit the Pi in equilibrium with ATP exchange reaction catalyzed by submitochondrial particles and the ATPase activity of both submitochondrial particles and soluble F1 ATPase. The inhibition by the three drugs is reversed by either raising the Pi concentration or by adding organic solvent (dimethylsulfoxide, ethyleneglycol or methanol) to the medium. The inhibition of the Pi in equilibrium with ATP exchange by trifluoperazine becomes more pronounced when the electrochemical proton gradient formed across the membrane of the submitochondrial particles is decreased by the addition to the medium of the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone. The ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum vesicles are inhibited by the Fe . bathophenanthroline complex, calmidazolium and trifluoperazine. Phosphorylation of the ATPases by Pi, synthesis of ATP from ADP and Pi and the fast efflux of Ca2+ observed during reversal of the Ca2+ pump are inhibited by the three drugs. The inhibition is reversed by raising the concentration of Pi or dimethylsulfoxide. The three drugs tested appear to compete with Pi for a common binding site on the Ca2+-ATPase. The data presented are interpreted according to the proposal that the catalytic site of an enzyme involved in energy transduction undergoes a hydrophobic-hydrophilic transition during the catalytic cycle.     

The non-catalytic nucleotide-binding site of mitochondrial ATPase is localised on the alpha-subunit(s) of factor F1

The incubation of isolated factor F1 with the di-aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9--1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 micro M. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the alpha-subunit(s) of factor F1. When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30 000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane-bound factor F1 covalently binds 0.2--0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the alpha subunit(s) of factor F1. The modification of membrane-bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non-catalytic site of mitochondrial ATP ase located on the alpha-subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane-bound ATPase.     

The alpha-subunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p

The Atp12p protein of Saccharomyces cerevisiae is required for the assembly of the F(1) component of the mitochondrial F(1)F(0) ATP synthase. In this report, we show that the F(1) alpha-subunit co-precipitates and co-purifies with a tagged form of Atp12p adsorbed to affinity resins. Moreover, sedimentation analysis indicates that in the presence of the F(1) alpha-subunit, Atp12p behaves as a particle of higher mass than is observed in the absence of the alpha-subunit. Yeast two-hybrid screens confirm the direct association of Atp12p with the alpha-subunit and indicate that the binding site for the assembly factor lies in the nucleotide-binding domain of the alpha-subunit, between Asp133 and Leu322. These studies provide the basis for a model of F(1) assembly in which Atp12p is released from the alpha-subunit in exchange for a beta-subunit to form the interface that contains the non-catalytic adenine nucleotide-binding site.     

Evidence supporting the identity of beef heart mitochondrial chloroform-released adenosine triphosphatase (ATPase) with coupling factor I.

Highly purified mitochondrial chloroform-released beef heart ATPase had molecular weight 330 000, five bands (alpha, beta, gamma, delta, epsilon) in sodium dodecyl sulfate gel electrophoresis and could restore the oxidative-phosphorylation function of A particles. Maximal inhibition (90%) of the enzyme by N,N'-dicyclohexylcarbodiimide was achieved at a molar ratio of inhibitor to protein of 30 : 1. Chloroform introduced into an aqueous solution of beef heart coupling factor I protected it from cold inactivation.     

Evidence for essential primary amino groups in a bacterial coupling factor F1ATPase

We have found that the binding of pyridoxal-5′-phosphate to 6 primary amino groups leads to the inactivation of the enzyme. A preferential reaction of pyridoxal-5′-phosphate with the α-subunits of this enzyme can be demonstrated. The reactivity of the amino groups is influenced by various effectors. In the presence of ATP the inhibition of the ATPase activity is noncompetitive.     

Correspondence of minor subunits of plant mitochondrial F1ATPase to F1F0ATPase subunits of other organisms

In addition to two major alpha- and beta-subunits, the soluble oligomycin-insensitive F1ATPase purified from sweet potato root mitochondria contains four different minor subunits of gamma (Mr = 35,500), delta (Mr = 27,000), delta' (Mr = 23,000), and epsilon (Mr = 12,000) (Iwasaki, Y., and Asashi, T. (1983) Arch. Biochem. Biophys. 227, 164-173). Among these minor subunits, the delta-subunit specifically cross-reacted with an antibody against the delta-subunit of maize mitochondrial F1 which contains only three minor gamma-, delta- and epsilon-subunits like F1ATPases from other organisms, indicating that the delta'-subunit is an extra subunit of sweet potato F1 which is absent in the maize F1. All of the four minor subunits of sweet potato F1 were purified and their N-terminal amino acid sequences of 30-36 residues were determined. The N-terminal sequence of gamma-subunit was homologous to those of the gamma-subunits of bacterial F1 and mammalian mitochondrial F1. The N-terminal sequence of the delta-subunit was homologous to those of the delta-subunits of bacterial F1, chloroplast CF1, and oligomycin sensitivity conferring protein of bovine mitochondrial F1F0. A sequence homology was also observed between the sweet potato epsilon-subunit and the epsilon-subunit of bovine mitochondrial F1. The N-terminal sequence of the delta'-subunit did not show any significant sequence homology to known protein sequences. These subunit correspondences place plant mitochondrial F1 at an unique position in the evolution of F1ATPase.     

The binding of aurovertin to isolated F1 (mitochondrial ATPase).

1. Isolated F1 contains 14.9% N, indicating the presence of at least 8% non-protein material. The Lowry method, standardized with bovine serum albumin, correctly measures the protein content. 2. An extinction coefficient of 28.5 mM-1.cm-1 at 367.5 nm was found for aurovertin D in ethanol. 3. The fluorescence enhancement of aurovertin bound to F1 at pH 7.5 was found to be more than 100-fold. 4. Binding parameters calculated from the fluorescence enhancement with fixed F1 and variable aurovertin concentrations, and vice versa, indicate two binding sites per F1 molecule. 5. The fluorescence data are not readily interpreted on the basis of successive binding of aurovertin by 3-component binding reactions of the form E + A in equilibrium EA, but fit closely a model of two non-interacting sites binding aurovertin in a 4-component reaction, EF + A in equilibrium EA + F, with an equilibrium constant of about 2.     

Evidence supporting the identity of beef heart mitochondrial chloroform-released adenosine triphosphatase (ATPase) with coupling factor I

Highly purified mitochondrial chloroform-released beef heart ATPase had molecular weight 330 000, five bands (α, β, γ, δ, ε) in sodium dodecyl sulfate gel electrophoresis and could restore the oxidative-phosphorylation function of A particles. Maximal inhibition (90%) of the enzyme by N,N′-dicyclohexylcarbodiimide was achieved at a molar ratio of inhibitor to protein of 30 : 1. Chloroform introduced into an aqueous solution of beef heart coupling factor I protected it from cold inactivation.     

The mitochondrial F1ATPase alpha-subunit is necessary for efficient import of mitochondrial precursors.

The mitochondrial import and assembly of the F1ATPase subunits requires, respectively, the participation of the molecular chaperones hsp70SSA1 and hsp70SSC1 and other components operating on opposite sides of the mitochondrial membrane. In previous studies, both the homology and the assembly properties of the F1ATPase alpha-subunit (ATP1p) compared to the groEL homologue, hsp60, have led to the proposal that this subunit could exhibit chaperone-like activity. In this report the extent to which this subunit participates in protein transport has been determined by comparing import into mitochondria that lack the F1ATPase alpha-subunit (delta ATP1) versus mitochondria that lack the other major catalytic subunit, the F1ATPase beta-subunit (delta ATP2). Yeast mutants lacking the alpha-subunit but not the beta-subunit grow much more slowly than expected on fermentable carbon sources and exhibit delayed kinetics of protein import for several mitochondrial precursors such as the F1 beta subunit, hsp60MIF4 and subunits 4 and 5 of the cytochrome oxidase. In vitro and in vivo the F1 beta-subunit precursor accumulates as a translocation intermediate in absence of the F1 alpha-subunit. In the absence of both the ATPase subunits yeast grows at the same rate as a strain lacking only the beta-subunit, and import of mitochondrial precursors is restored to that of wild type. These data indicate that the F1 alpha-subunit likely functions as an "assembly partner" to influence protein import rather than functioning directly as a chaperone. These data are discussed in light of the relationship between the import and assembly of proteins in mitochondria.     

Interaction of beef-heart mitochondrial ATPase,coupling factor F1, with aurovertin

The antibiotic aurovertin binds to beef-heart mitochondrial ATPase, coupling Factor F₁, with biphasic fluorescence enhancement. Specific binding effects, polarity and viscosity changes may all contribute to the enhancement. Evidence is presented that it stems from aurovertin binding followed by a slow conformational change in F₁. This occurs more rapidly in dissociated F₁. The effect of substrates of the enzyme on the fluorescence enhancement is examined. Evidence is presented that in the absence of added magnesium, F₁ can hydrolyse low concentrations of added ATP.     

Increased conformational stability of mitochondrial soluble ATPase (F1) by substitution of H2O for D2O.

Between 20 and 40 °C D₂O inhibits the hydrolytic activity of soluble mitochondrial ATPase F₁. The effect of D₂O is proportional to its concentration in the incubation mixture and at nearly 100% D₂O in the incubation mixture the ATPase activity is inhibited by 50–60%. The effect of D₂O is mainly on the V of the reaction. At temperatures above 45 °C, D₂O does not inhibit the activity. D₂O protects against the denaturation of the enzyme that is observed at relatively high temperatures and against the cold-induced inactivation of F₁. The intensity of fluorescence of 8-anilino-1-naphthalene sulfonate incubated with F₁ increases as the enzyme becomes inactivated by low temperatures; in D₂O the changes of fluorescence are almost nil. These observations indicate that H (or D) bonding between the solvent and the protein as well as the strength of the hydrophobic interactions within the enzyme as determined by the solvent are of central importance in determining the overall activity of F₁ and the stability of the enzyme to denaturing conditions. Moreover, the data indicate that the enzyme may exist in two different conformations, each with a characteristic activation energy. It is also proposed that D₂O may be employed with success in the isolation and purification of labile enzymes.     

Specificity of acidic phospholipids (CL & PA) in the activation of mitochondrial F0F1 ATPase by Mg2+

The interaction of Mg2+ with native F0F1 ATPase was studied. The hydrolytic activity of F0F1 ATPase could be competitively activated by Mg2+, but the preincubation of F0F1 ATPase with cholate eliminated the Mg2+ effect. The result from the comparison of the effect of Mg2+ on F0F1 ATPase with that on soluble F1 ATPase, and the fact that the activation of Mg2+ on cholate-treated F0F1 ATPase could be reconstituted only by divalent acidic phospholipid cardiolipin, indicate that there exists a specificity between the acidic phospholipids of the mitochondrial inner membrane and Mg2+ enhancement of ATP-hydrolyzing activity of F0F1 ATPase.     

Inhibition of chloroplast coupling factor ATPase by 5'-p-fluorosulfonylbenzoyl adenosine.

Incubation of chloroplast coupling factor with 5′-p-fluorosulfonylbenzoyl adenosine in the 1 to 2 mM range inhibits subsequently measured ATPase activity. The inhibition is probably due to covalent binding since it survives ammonium sulfate fractionation and dialysis. The kinetics of the inhibited enzyme with respect to substrate show a decrease in V_max with no change in K_m for ATP. The presence of ATP or ADP together with the inhibitor provides some protection against inhibition. The results suggest a possible covalent attack at a nucleotide binding site, leading to inhibition of activity.