Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

The interaction of nucleotides with F1-ATPase inactivated with 4-chloro-7-nitrobenzofurazan

In common with the F₁-ATPase from other sources, yeast mitochondrial F₁-ATPase was inhibited by 4-chloro-7-nitrobenzofurazan. Total inhibition of the F₁-ATPase activity was compatible with the modification of a single tyrosine residue per F₁-ATPase molecule. Radioactive labelling experiments localized this modification on a β-subunit. The inactive modified enzyme retained the capacity to bind the photoaffinity label 8-azido-1,N⁶-etheno-ATP, which has previously been shown to bind nucleotide sites of low affinity. As well, the inactive modified enzyme bound MgATP with high affinity, yielding a K_d of 14 μM. The results are consistent with the hypothesis of alternating, or cooperative, site catalysis by F₁-ATPase.     

Reconstitution of Oxidative Phosphorylation and of Oligomycin-Sensitive ATPase by Five- and Six-Subunit Forms of Pea Mitochondrial F(1)-ATPase

Five- and six-subunit forms of F₁-ATPase were purified from pea (Pisum sativum L. cv Homesteader) cotyledon submitochondrial particles. Apart from the usual complement of five subunits, the six-subunit enzyme contained an additional 26,500-dalton protein. Both forms of the F₁-ATPase were used to reconstitute oxidative phosphorylation in F₁-depleted (ASU) as well as in F₁ and oligomycin-sensitivity conferring protein (OSCP)-depleted (ASUA) bovine mitochondrial membranes. The six-subunit enzyme was considerably more efficient in reconstituting the ATP synthesis than the five-subunit enzyme. Both forms of the enzyme were also able to reconstitute the ATPase activity in ASU- as well as in ASUA-particles. There were substantial differences, however, in the oligomycin sensitivity of the ATPase bound to the ASUA-particles: 20 and 60% inhibition by oligomycin was obtained in the case of the five-subunit and six-subunit enzyme, respectively. We conclude, that the 26,500-dalton protein present in the six-subunit F₁-ATPase is responsible for the increase in oligomycin sensitivity of the bound enzyme and functions, therefore, as the plant OSCP.     

Arylazido aminopropionyl ATP, and active site directed photoaffinity reagent for mitochondrial adenosine triphosphatase.

The photoaffinity label, arylazido aminopropionyl ATP¹, brings about a photodependent inhibition of mitochondrial F₁-ATPase and an associated specific covalent labeling of the soluble enzyme. In addition the ATP analog can act as a substrate when incubated with F₁-ATPase in the dark.     

Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase

Corn mitochondrial F₁-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4°C was substantially activated by ATP, while enzyme stored at −70°C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P_i released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60°C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F₁-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (α), 55,000 (β), 35,000 (γ), 22,000 (δ), and 12,000 (ε). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F₁-ATPase was antigenically distinct from the chloroplastic CF₁-ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF₁-ATPase β and γ subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F₁-ATPase.     

Presence of an extramitochondrial anion-stimulated ATPase in the rabbit kidney: Localization along the nephron and effect of corticosteroids

Summary To determine whether kidney membrane fractions contain an extramitochondrial anion-stimulated ATPase, we compared the pharmacological and kinetic properties of HCO₃-ATPase activities in mitochondrial and microsomal fractions prepared from rabbit kidney cortex and outer medulla. The results indicated that this activity differed markedly in each type of fraction. Microsomal HCO₃-ATPase was less sensitive than mitochondrial ATPase to azide, oligomycin, DCCD and thiocyanate, but was more sensitive to filipin and displayed different dependency towards ATP, magnesium and pH. Microsomal ATPase activity was stimulated by sulfite much more strongly than by bicarbonate, whereas mitochondrial activity was stimulated by both these anions to a similar extent. These results demonstrate the presence of an extramitochondrial HCO₃-ATPase in kidney membrane fractions. HCO₃-ATPase was also measured in single microdissected segments of the rabbit nephron using a radiochemical microassay previously developed for tubular Na, K-ATPase activity. An enzyme with the pharmacological and kinetic properties of the microsomal enzyme was detected in both proximal tubule, distal convoluted tubule and collecting duct, but the thick ascending limb was devoid of any detectable activity. Long-term DOCA administration markedly increased HCO₃-ATPase activity in the distal convoluted and collecting tubule. The insensitivity of microsomal HCO₃-ATPase to vanadate indicates that it belongs to the F₀–F₁ class of ATPases, and might therefore be involved in proton transport. This hypothesis is also supported by the localization of tubular HCO₃-ATPase activity at the sites of urinary acidification.     

The complex of mitochondrial F1-ATPase with the natural inhibitor protein is unable to catalyze single-site ATP hydrolysis

Interaction of mitochondrial F₁-ATPase with the isolated natural inhibitor protein resulting in the inhibition of multi-site ATP hydrolysis is accompanied by the loss of activity at low ATP concentrations when single-site hydrolysis should occur. Catalytic site occupancy by [¹⁴C]nucleotides in F₁-ATPase during steady-state [¹⁴C]ATP hydrolysis, which is saturated in parallel with single-site catalysis, is prevented after blocking the enzyme with the inhibitor protein.     

Isolation and comparative studies of mitochondrial F1-ATPase from Morris hepatoma and rat liver.

A simple method of isolating mitochondrial ATPase from rat liver and Morris hepatoma cell lines by chloroform extraction and chromatography on DEAE-Sephadex is described. This method is suitable even when small amounts of starting material with relatively low specific ATPase activity (in the case of hepatoma mitochondria and submitochondrial particles) are available. The isolated enzyme from both rat liver and hepatomas had a high specific activity, was similarly activated by bicarbonate and 2,4-dinitrophenol, and had a typical five-band pattern in sodium dodecyl sulfate electrophoresis. Prior to DEAE-Sephadex chromatography, an additional protein band which migrates between the δ and ? subunits in the tumor F₁-ATPase preparation was observed. The purified enzymes were cold labile and restored oxidative phosphorylation function of F₁-ATPase depleted submitochondrial particles prepared from rat liver. The ATPase activity of the isolated enzymes was inhibited by mitochondrial ATPase inhibitor protein. The apparent stoichiometry of the inhibitor protein to the purified ATPase was extrapolated to be 2:1.     

Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

Mitochondria as ATP consumers in cellular pathology

ATP provided by oxidative phosphorylation supports highly complex and energetically expensive cellular processes. Yet, in several pathological settings, mitochondria could revert to ATP consumption, aggravating an existing cellular pathology. Here we review (i) the pathological conditions leading to ATP hydrolysis by the reverse operation of the mitochondrial F_oF₁-ATPase, (ii) molecular and thermodynamic factors influencing the directionality of the F_oF₁-ATPase, (iii) the role of the adenine nucleotide translocase as the intermediary adenine nucleotide flux pathway between the cytosol and the mitochondrial matrix when mitochondria become ATP consumers, (iv) the role of the permeability transition pore in bypassing the ANT, thereby allowing the flux of ATP directly to the hydrolyzing F_oF₁-ATPase, (v) the impact of the permeability transition pore on glycolytic ATP production, and (vi) endogenous and exogenous interventions for limiting ATP hydrolysis by the mitochondrial F_oF₁-ATPase.     

The inhibitor peptide of the mitochondrial F1 · F0-ATPase interacts with calmodulin and stimulates the calmodulin-dependent Ca2+-ATPase of erythrocytes

The binding of calmodulin to the mitochondrial F₁ · F₀-ATPase has been studied. [^125I]Iodoazidocalmodulin binds to the ε-subunit and to the endogeneous ATPase inhibitor peptide in a Ca²⁺-dependent reaction. The effect of the mitochondrial ATPase inhibitor peptide on the purified Ca²⁺-ATPase of erythrocytes has also been analyzed. The inhibitor peptide stimulates the ATPase when pre-incubated with the enzyme. The activation of the Ca²⁺-ATPase by calmodulin is not influenced by the inhibitor peptide, indicating that the two mechanisms of activation are different. These in vitro effects of the two regulatory proteins may reflect a common origin of the two ATPases considered and/or of the regulatory proteins.     

Suppression of mitochondrial ATPase inhibitor protein (IF1) in the liver of late septic rats

Sepsis and ensuing multiple organ failure continue to be the most leading cause of death in critically ill patients. Despite hepatocyte-related dysfunctions such as necrosis, apoptosis as well as mitochondrial damage are observed in the process of sepsis, the molecular mechanism of pathogenesis remains uncertain. We recently identified one of the differentially expressed genes, mitochondrial ATPase inhibitor protein (IF1) which is down-regulated in late septic liver. Hence, we further hypothesized that the variation of IF1 protein may be one of the causal events of the hepatic dysfunction during late sepsis. The results showed that the elevated mitochondrial F₀F₁-ATPase activity is concomitant with the decline of intramitochondrial ATP concentration in late septic liver. In addition, the key finding of this study showed that the mRNA and the mitochondrial content of IF1 were decreased in late sepsis while no detectable IF1 was found in cytoplasm. When analyzed by immunoprecipitation, it seems reasonable to imply that the association capability of IF1 with F₁-ATPase β-subunit is not affected. These results confirm the first evidence showing that the suppression of IF1 expression and subsequent elevated mitochondrial F₀F₁-ATPase activity might contribute to the bioenergetic failure in the liver during late sepsis.     

The serum- and glucocorticoid-induced protein kinase-1 (Sgk-1) mitochondria connection: Identification of the IF-1 inhibitor of the F1F0-ATPase as a mitochondria-specific binding target and the stress-induced mitochondrial localization of endogenous Sgk-1

The expression, localization and activity of the serum- and glucocorticoid-induced protein kinase, Sgk-1, are regulated by multiple hormonal and environmental cues including cellular stress. Biochemical fractionation and indirect immunofluorescence demonstrated that sorbitol induced hyperosmotic stress stimulated expression and triggered the localization of endogenous Sgk-1 into the mitochondria of NMuMG mammary epithelial cells. The immunofluorescence pattern of endogenous Sgk-1 was similar to that of a green fluorescent linked fusion protein linked to the N-terminal Sgk-1 fragment that encodes the mitochondrial targeting signal. In the presence or absence of cellular stress, exogenously expressed wild type Sgk-1 efficiently compartmentalized into the mitochondria demonstrating the mitochondrial import machinery per se is not stressed regulated. Co-immunoprecipitation and GST-pull down assays identified the IF-1 mitochondrial matrix inhibitor of the F₁F₀-ATPase as a new Sgk-1 binding partner, which represents the first observed mitochondrial target of Sgk-1. The Sgk-1/IF-1 interaction requires the 122-176 amino acid region within the catalytic domain of Sgk-1 and is pH dependent, occurring at neutral pH but not at slightly acidic pH, which suggests that this interaction is dependent on mitochondrial integrity. An in vitro protein kinase assay showed that the F₁F₀-ATPase can be directly phosphorylated by Sgk-1. Taken together, our results suggest that stress-induced Sgk-1 localizes to the mitochondria, which permits access to physiologically appropriate mitochondrial interacting proteins and substrates, such as IF-1 and the F₁F₀-ATPase, as part of the cellular stressed induced program.     

Identification of the F1-ATPase at the Cell Surface of Colonic Epithelial Cells: ROLE IN MEDIATING CELL PROLIFERATION*

F1 domain of F₁F_o-ATPase was initially believed to be strictly expressed in the mitochondrial membrane. Interestingly, recent reports have shown that the F1 complex can serve as a cell surface receptor for apparently unrelated ligands. Here we show for the first time the presence of the F₁-ATPase at the cell surface of normal or cancerous colonic epithelial cells. Using surface plasmon resonance technology and mass spectrometry, we identified a peptide hormone product of the gastrin gene (glycine-extended gastrin (G-gly)) as a new ligand for the F₁-ATPase. By molecular modeling, we identified the motif in the peptide sequence (E(E/D)XY), that directly interacts with the F₁-ATPase and the amino acids in the F₁-ATPase that bind this motif. Replacement of the Glu-9 residue by an alanine in the E(E/D)XY motif resulted in a strong decrease of G-gly binding to the F₁-ATPase and the loss of its biological activity. In addition we demonstrated that F₁-ATPase mediates the growth effects of the peptide. Indeed, blocking F₁-ATPase activity decreases G-gly-induced cell growth. The mechanism likely involves ADP production by the membrane F₁-ATPase, which is induced by G-gly. These results suggest an important contribution of cell surface F₁-ATPase in the pro-proliferative action of this gastrointestinal peptide.     

Temperature-dependence of spectroscopic and catalytic properties of the eel (Anguilla anguilla) liver mitochondrial F1-ATPase

• 1.1. F₁-ATPase from eel liver mitochondria at low concentrations preserves unaltered the enzymatic activity for more than 20 min over a temperature range of 6–36°C.
• 2.2. The Arrhenius plot of ATP hydrolysis at saturating substrate concentration appears biphasic with a break-point at 16°C and activation energies of 14.4 and 56.1 kJ/mol.
• 3.3. The ultraviolet, fluorescence and circular dichroism spectra of the enzyme, below and above 16°C, have been recorded; the fluorescence emission spectra of F₁-ATPase excited at 275 nm, and the circular dichroism spectra, are different at the two temperatures examined.
• 4.4. It is concluded that temperature induces two different conformational states of F₁-ATPase with different catalytic properties.
• 5.5. Ultraviolet spectroscopic features and temperature-dependence of eel liver mitochondrial F₁-ATPase are discussed in relation to mammalian F₁-ATPases.

     

Multi-site TBT binding skews the inhibition of oligomycin on the mitochondrial Mg-ATPase in Mytilus galloprovincialis

Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F₁F₀-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F₀, namely oligomycin and N,N′-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F₁, maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F₁ and F₀ domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F₀ proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F₁F₀-ATPase oligomycin sensitivity loss.     

Antitumor activity of efrapeptins,alone or in combination with 2-deoxyglucose,in breast cancer in vitro and in vivo

Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F₁F₀-ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F₁F₀-ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC₅₀ value ranging from 6 nM to 3.4 μM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F₁F₀-ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F₁F₀-ATPase by 2DG.     

On the mechanism of inhibition of the bovine heart F1-ATPase by local anesthetics

The rate of inactivation of the mitochondrial F₁-ATPase by dicyclohexylcarbodiimide is slowed by concentrations of chlorpromazine, dibucaine, or tetracaine which have been shown by others (B. Chazotte, G. Vanderkooi, and D. Chignell (1982)$\text{Biochim. Biophys. Acta}\text{680}$, 310–316) to inhibit the hydrolytic reaction catalyzed by the enzyme. The order of effectiveness of the drugs as protectors of the enzyme against inactivation by dicyclohexylcarbodiimide is: chlorpromazine > dibucaine > tetracaine. Examination of the steady state kinetics showed the chlorpromazine inhibits the ATPase competitively at concentrations up to 18.5 μM while complex kinetic behavior is exhibited at chlorpromazine concentrations from 25–50 μM. These results suggest that the drugs inhibit the F₁-ATPase by interacting with the catalytic site of the enzyme and not by promoting its dissociation.     

Phosphorylation/dephosphorylation of maize mitochondrial proteins with different localization

Phosphorylation and dephosphorylation of the proteins residing in the outer mitochondrial membrane, mitoplasts and whole mitochondria of maize (Zea mays L.) were investigated in order to reveal the possible participation of these processes in mitochondrial signaling. A mitochondrial protein of around 57 kD was identified by immunocytochemistry as α-subunit of the F₁-ATPase complex. In isolated mitochondria of maize, phosphorylation of this protein could be visualized only after treating mitochondria with endotholl, an inhibitor of the PP1a and PP2A protein phosphatases. A phosphorylated protein of 46.6 kD was identified as β-subunit of the F₁-ATPase complex. Ca²⁺ is the most common second messenger participating in mitochondrial signaling. We conclude that the transmission of the Ca²⁺ signal to the plant mitochondria occurs via proteins of the outer mitochondrial membrane. The systems perceiving this signal could include the protein phosphatases residing in the outer mitochondrial membrane, which preferentially dephosphorylate the proteins in the inner membrane.