The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1

When mitochondrial respiration is compromised, the F₁F_o-ATP synthase reverses and consumes ATP, serving to maintain the mitochondrial membrane potential (Δψ_m). This process is mitigated by IF₁. As little is known of the cell biology of IF₁, we have investigated the functional consequences of varying IF₁ expression. We report that, (1) during inhibition of respiration, IF₁ conserves ATP at the expense of Δψ_m; (2) overexpression of IF₁ is protective against ischemic injury; (3) relative IF₁ expression level varies between tissues and cell types and dictates the response to inhibition of mitochondrial respiration; (4) the density of mitochondrial cristae is increased by IF₁ overexpression and decreased by IF₁ suppression; and (5) IF₁ overexpression increases the formation of dimeric ATP synthase complexes and increases F₁F_o-ATP synthase activity. Thus, IF₁ regulates mitochondrial function and structure under both physiological and pathological conditions.     

In vitro and in vivo studies of F0F1ATP synthase regulation by inhibitor protein IF1 in goat heart

A method has been developed to allow the level of F₀F₁ATP synthase capacity and the quantity of IF₁ bound to this enzyme be measured in single biopsy samples of goat heart. ATP synthase capacity was determined from the maximal mitochondrial ATP hydrolysis rate and IF₁ content was determined by detergent extraction followed by blue native gel electrophoresis, two-dimensional SDS-PAGE and immunoblotting with anti-IF₁ antibodies.Anaesthetized open-chest goats were subjected to ischemic preconditioning and/or sudden increases of coronary blood flow (CBF) (reactive hyperemia). When hyperemia was induced before ischemic preconditioning, a steep increase in synthase capacity, followed by a deep decrease, was observed. In contrast, hyperemia did not affect synthase capacity when applied after ischemic preconditioning. Similar effects could be produced in vitro by treatment of heart biopsy samples with anoxia (down-regulation of the ATP synthase) or high-salt or high-pH buffers (up-regulation). We show that both in vitro and in vivo the same close inverse correlation exists between enzyme activity and IF₁ content, demonstrating that under all conditions tested the only significant modulator of the enzyme activity was IF₁. In addition, both in vivo and in vitro, 1.3-1.4 mol of IF₁ was predicted to fully inactivate 1 mol of synthase, thus excluding the existence of significant numbers of non-inhibitory binding sites for IF₁ in the F₀ sector.     

Mitochondrial F1FO ATP synthase determines the local proton motive force at cristae rims

The classical view of oxidative phosphorylation is that a proton motive force (PMF) generated by the respiratory chain complexes fuels ATP synthesis via ATP synthase. Yet, under glycolytic conditions, ATP synthase in its reverse mode also can contribute to the PMF. Here, we dissected these two functions of ATP synthase and the role of its inhibitory factor 1 (IF1) under different metabolic conditions. pH profiles of mitochondrial sub‐compartments were recorded with high spatial resolution in live mammalian cells by positioning a pH sensor directly at ATP synthase’s F₁ and F_O subunits, complex IV and in the matrix. Our results clearly show that ATP synthase activity substantially controls the PMF and that IF1 is essential under OXPHOS conditions to prevent reverse ATP synthase activity due to an almost negligible ΔpH. In addition, we show how this changes lateral, transmembrane, and radial pH gradients in glycolytic and respiratory cells.     

Regulation of the mitochondrial ATPasein situ in cardiac muscle: Role of the inhibitor subunit

The mitochondrial F₁-ATPase inhibitor protein, IF₁, binds to subunits of the F₁-ATPase bothin vitro andin situ under nonenergizing conditions, i.e., under conditions that allow a net hydrolysis of ATP by the mitochondrial ATPase to take place. This reversible IF₁ binding occurs in a wide variety of cell types including (anaerobic) baker's yeast cells and (ischemic) mammalian cardiomyocytes under conditions that limit oxidative phosphorylation. The binding of inhibitor results in a marked slowing of ATP hydrolysis by the undriven mitochondrial ATP synthase. An apparent main function of this reversible IF₁ binding, at least in cells that undergo aerobic-anaerobic switching, is the mitigation of a wasteful hydrolysis of ATP produced by glycolysis during anoxic or ischemic intervals, by the mitochondrial ATPase. While this apparent main function is probably of considerable importance in cells that normally either can or must undergo aerobic-anaerobic switching such as baker's yeast cells and skeletal myocytes, one wonders why a full complement of IF₁ has been retained in certain cells that normally do not undergo such aerobic-anaerobic switching, cells such as adult mammalian cardiomyocytes of many species. While some mammalian species have, indeed, not retained a functional complement of IF₁ in their cardiomyocytes, those that have can benefit significantly from its presence during intervals of myocardial ischemia.This mini-review is dedicated to the memory of Professor Efraim Racker.     

The affinity purification and characterization of ATP synthase complexes from mitochondria

The mitochondrial F₁-ATPase inhibitor protein, IF₁, inhibits the hydrolytic, but not the synthetic activity of the F-ATP synthase, and requires the hydrolysis of ATP to form the inhibited complex. In this complex, the α-helical inhibitory region of the bound IF₁ occupies a deep cleft in one of the three catalytic interfaces of the enzyme. Its N-terminal region penetrates into the central aqueous cavity of the enzyme and interacts with the γ-subunit in the enzyme''s rotor. The intricacy of forming this complex and the binding mode of the inhibitor endow IF₁ with high specificity. This property has been exploited in the development of a highly selective affinity procedure for purifying the intact F-ATP synthase complex from mitochondria in a single chromatographic step by using inhibitor proteins with a C-terminal affinity tag. The inhibited complex was recovered with residues 1–60 of bovine IF₁ with a C-terminal green fluorescent protein followed by a His-tag, and the active enzyme with the same inhibitor with a C-terminal glutathione-S-transferase domain. The wide applicability of the procedure has been demonstrated by purifying the enzyme complex from bovine, ovine, porcine and yeast mitochondria. The subunit compositions of these complexes have been characterized. The catalytic properties of the bovine enzyme have been studied in detail. Its hydrolytic activity is sensitive to inhibition by oligomycin, and the enzyme is capable of synthesizing ATP in vesicles in which the proton-motive force is generated from light by bacteriorhodopsin. The coupled enzyme has been compared by limited trypsinolysis with uncoupled enzyme prepared by affinity chromatography. In the uncoupled enzyme, subunits of the enzyme''s stator are degraded more rapidly than in the coupled enzyme, indicating that uncoupling involves significant structural changes in the stator region.     

Content and binding characteristics of the mitochondrial ATPase inhibitor,IF1 in the tissues of several slow and fast heart-rate homeothermic species and in two poikilotherms

We determined the IF₁ contents of pig, rabbit, rat, mouse, guinea pig, pigeon, turtle, and frog heart mitochondria and the effects of varying ionic strength upon the IF₁-mediated inhibition of the ATPase activity of IF₁-depleted rabbit heart mitochondrial particles (RHMP) by IF₁-containing extracts from these same eight species. The IF₁ binding experiments were run at both species-endogenous IF₁ levels and at an IF₁ level normalized to that present in rabbit heart mitochondria. When species-endogenous levels of rabbit heart IF₁ or either speciesendogenous or normalized levels of pig heart IF₁ were incubated with RHMP over a range of KCl concentrations, increasing the [KCl] to 150 mM had relatively little effect on IF₁-mediated ATPase inhibition. When either species-endogenous or normalized levels of guinea pig, pigeon, turtle, or frog heart IF₁ were incubated with RHMP under the same conditions, increasing [KCl] to 150 mM nearly completely blocked IF₁-mediated ATPase inhibition. While species-endogenous levels of rat and mouse heart IF₁ inhibited the ATPase activity of RHMP virtually not at all at any [KCl] examined, normalized levels of rat and mouse IF₁ inhibited the ATPase activity of RHMP to the same extents as species-endogenous levels of pig and rabbit heart IF₁, respectively, in the presence of increasing [KCl]. These experiments suggest that, while pig and rabbit heart mitochondria contain a full complement of higher-affinity IF₁, pigeon, guinea pig, turtle, and frog heart mitochondria cell contain essentially a full complement of a lower-affinity form of IF₁. In contrast, rat and mouse heart mitochondria contain only low levels of IF₁ which exhibit binding characteristics similar to those of the pig and rabbit heart inhibitor. The guinea pig is the only mammal thus far examined that contains a loweraffinity form of IF₁. In the present study we also determined the IF₁ contents and IF₁-to-F₁ ATPase activity ratios of cardiac muscle, skeletal muscle, liver, and brain mitochondria of rabbit, pigeon, and rat, species representative of the three homeothermic regulatory classes.     

Unidirectional regulation of the F1FO-ATP synthase nanomotor by the ζ pawl-ratchet inhibitor protein of Paracoccus denitrificans and related α-proteobacteria

The ATP synthase is a reversible nanomotor that gyrates its central rotor clockwise (CW) to synthesize ATP and in counter clockwise (CCW) direction to hydrolyse it. In bacteria and mitochondria, two natural inhibitor proteins, namely the ε and IF₁ subunits, prevent the wasteful CCW F₁F_O-ATPase activity by blocking γ rotation at the α_DP/β_DP/γ interface of the F₁ portion. In Paracoccus denitrificans and related α-proteobacteria, we discovered a different natural F₁-ATPase inhibitor named ζ. Here we revise the functional and structural data showing that this novel ζ subunit, although being different to ε and IF₁, it also binds to the α_DP/β_DP/γ interface of the F₁ of P. denitrificans. ζ shifts its N-terminal inhibitory domain from an intrinsically disordered protein region (IDPr) to an α-helix when inserted in the α_DP/β_DP/γ interface. We showed for the first time the key role of a natural ATP synthase inhibitor by the distinctive phenotype of a Δζ knockout mutant in P. denitrificans. ζ blocks exclusively the CCW F₁F_O-ATPase rotation without affecting the CW-F₁F_O-ATP synthase turnover, confirming that ζ is important for respiratory bacterial growth by working as a unidirectional pawl-ratchet PdF₁F_O-ATPase inhibitor, thus preventing the wasteful consumption of cellular ATP. In summary, ζ is a useful model that mimics mitochondrial IF₁ but in α-proteobacteria. The structural, functional, and endosymbiotic evolutionary implications of this ζ inhibitor are discussed to shed light on the natural control mechanisms of the three natural inhibitor proteins (ε, ζ, and IF₁) of this unique ATP synthase nanomotor, essential for life.     

Regulation of the F1F0-ATP Synthase Rotary Nanomotor in its Monomeric-Bacterial and Dimeric-Mitochondrial Forms

The F₁F₀-adenosine triphosphate (ATP) synthase rotational motor synthesizes most of the ATP required for living from adenosine diphosphate, Pi, and a proton electrochemical gradient across energy-transducing membranes of bacteria, chloroplasts, and mitochondria. However, as a reversible nanomotor, it also hydrolyzes ATP during de-energized conditions in all energy-transducing systems. Thus, different subunits and mechanisms have emerged in nature to control the intrinsic rotation of the enzyme to favor the ATP synthase activity over its opposite and commonly wasteful ATPase turnover. Recent advances in the structural analysis of the bacterial and mitochondrial ATP synthases are summarized to review the distribution and mechanism of the subunits that are part of the central rotor and regulate its gyration. In eubacteria, the ε subunit works as a ratchet to favor the rotation of the central stalk in the ATP synthase direction by extending and contracting two α-helixes of its C-terminal side and also by binding ATP with low affinity in thermophilic bacteria. On the other hand, in bovine heart mitochondria, the so-called inhibitor protein (IF₁) interferes with the intrinsic rotational mechanism of the central γ subunit and with the opening and closing of the catalytic β-subunits to inhibit its ATPase activity. Besides its inhibitory role, the IF₁ protein also promotes the dimerization of the bovine and rat mitochondrial enzymes, albeit it is not essential for dimerization of the yeast F₁F₀ mitochondrial complex. High-resolution electron microscopy of the dimeric enzyme in its bovine and yeast forms shows a conical shape that is compatible with the role of the ATP synthase dimer in the formation of tubular the cristae membrane of mitochondria after further oligomerization. Dimerization of the mitochondrial ATP synthase diminishes the rotational drag of the central rotor that would decrease the coupling efficiency between rotation of the central stalk and ATP synthesis taking place at the F₁ portion. In addition, F₁F₀ dimerization and its further oligomerization also increase the stability of the enzyme to natural or experimentally induced destabilizing conditions.     

Characterization of two cDNA clones encoding isozymes of the F1F0-ATPase inhibitor protein of rice mitochondria

Two cDNA clones encoding F₁F₀-ATPase inhibitor proteins, which are loosely associated with the F₁ part of the mitochondrial F₁F₀-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF ₁ -1 and IF ₁ -2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF ₁ genes and the amino acid sequence of the mature IF₁ protein from potato revealed that IF₁-1 and IF₁-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF₁-1 or IF₁-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-β-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF₁ proteins are targeted to mitochondria. Received: 9 July 1999 / Accepted: 17 August 1999     

Binding of the inhibitor protein IF(1) to bovine F(1)-ATPase

In the structure of bovine F₁-ATPase inhibited with residues 1-60 of the bovine inhibitor protein IF₁, the α-helical inhibitor interacts with five of the nine subunits of F₁-ATPase. In order to understand the contributions of individual amino acid residues to this complex binding mode, N-terminal deletions and point mutations have been introduced, and the binding properties of each mutant inhibitor protein have been examined. The N-terminal region of IF₁ destabilizes the interaction of the inhibitor with F₁-ATPase and may assist in removing the inhibitor from its binding site when F₁F_o-ATPase is making ATP. Binding energy is provided by hydrophobic interactions between residues in the long α-helix of IF₁ and the C-terminal domains of the β_DP-subunit and β_TP-subunit and a salt bridge between residue E30 in the inhibitor and residue R408 in the C-terminal domain of the β_DP-subunit. Several conserved charged amino acids in the long α-helix of IF₁ are also required for establishing inhibitory activity, but in the final inhibited state, they are not in contact with F₁-ATPase and occupy aqueous cavities in F₁-ATPase. They probably participate in the pathway from the initial interaction of the inhibitor and the enzyme to the final inhibited complex observed in the structure, in which two molecules of ATP are hydrolysed and the rotor of the enzyme turns through two 120° steps. These findings contribute to the fundamental understanding of how the inhibitor functions and to the design of new inhibitors for the systematic analysis of the catalytic cycle of the enzyme.     

Cross-linking of the endogenous inhibitor protein (IF1) with rotor (gamma,epsilon) and stator (alpha) subunits of the mitochondrial ATP synthase

The location of the endogenous inhibitor protein ( IF₁) in the rotor/stator architecture of the bovine mitochondrial ATP synthase was studied by reversible cross-linking with dithiobis(succinimidylpropionate) in soluble F₁I and intact F₁F₀I complexes of submitochondrial particles. Reducing two-dimensional electrophoresis, Western blotting, and fluorescent cysteine labeling showed formation of –IF₁, IF₁–IF₁, –IF₁, and –IF₁ cross-linkages in soluble F₁I and in native F₁F₀I complexes. Cross-linking blocked the release of IF₁ from its inhibitory site and therefore the activation of F₁I and F₁F₀I complexes in a dithiothreitol-sensitive process. These results show that the endogenous IF₁ is at a distance 12 Å,to and subunits of the central rotor of the native mitochondrial ATP synthase. This finding strongly suggests that, without excluding the classical assumption that IF₁ inhibits conformational changes of the catalytic subunits, the inhibitory mechanism of IF₁ may involve the interference with rotation of the central stalk.     

Mitochondrial and cell-surface F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase in innate and acquired cardioprotection

Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F₀F₁ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F₀F₁ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F₀F₁ATPsynthase regulation by the inhibitory protein IF₁ in heart preconditioning strategies; ii) the structure and function of mitochondrial F₀F₁ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F₀F₁ ATP synthase in search for possible actors of its regulation, such as IF₁ and calmodulin, at cell surface.     

F1FO ATP Synthase Is Expressed at the Surface of Embryonic Rat Heart‐Derived H9c2 Cells and Is Affected by Cardiac‐Like Differentiation

Taking advantage from the peculiar features of the embryonic rat heart‐derived myoblast cell line H9c2, the present study is the first to provide evidence for the expression of F₁F_O ATP synthase and of ATPase Inhibitory Factor 1 (IF₁) on the surface of cells of cardiac origin, together documenting that they were affected through cardiac‐like differentiation. Subunits of both the catalytic F₁ sector of the complex (ATP synthase‐β) and of the peripheral stalk, responsible for the correct F₁‐F_O assembly/coupling, (OSCP, b, F6) were detected by immunofluorescence, together with IF₁. The expression of ATP synthase‐β, ATP synthase‐b and F6 were similar for parental and differentiated H9c2, while the levels of OSCP increased noticeably in differentiated cells, where the results of in situ Proximity Ligation Assay were consistent with OSCP interaction within ecto‐F₁F_O complexes. An opposite trend was shown by IF₁ whose ectopic expression appeared greater in the parental H9c2. Here, evidence for the IF₁ interaction with ecto‐F₁F_O complexes was provided. Functional analyses corroborate both sets of data. i) An F₁F_O ATP synthase contribution to the exATP production by differentiated cells suggests an augmented expression of holo‐F₁F_O ATP synthase on plasma membrane, in line with the increase of OSCP expression and interaction considered as a requirement for favoring the F₁‐F_O coupling. ii) The absence of exATP generation by the enzyme, and the finding that exATP hydrolysis was largely oligomycin‐insensitive, are in line in parental cells with the deficit of OSCP and suggest the occurrence of sub‐assemblies together evoking more regulation by IF₁. J. Cell. Biochem. 9999: 1–13, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Biochem. 117: 470–482, 2016. © 2015 Wiley Periodicals, Inc.     

Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

Overexpression of F0F1-ATP synthase α suppresses mutant huntingtin aggregation and toxicity in vitro

Huntington’s disease (HD) and other polyglutamine (polyQ) neurodegenerative diseases are characterized by neuronal accumulation of the disease protein, suggesting that the cellular ability to handle abnormal proteins is compromised. As a multi-subunit protein localized in the mitochondria of eukaryotic cells, the F₀F₁-ATP synthase α belongs to the family of stress proteins HSP60. Currently, mounting evidences indicate F₀F₁-ATP synthase α may play a role in neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Recently, ATP synthase α was reported to have protective and therapeutic roles in primary cardiacmyocytes of iron-overloaded rats by lowering ROS production. However, little is understood about the role of ATP synthase α in cell death and neurodegeneration. Here, we demonstrate that overexpression of ATP synthase α suppresses huntingtin (htt) polyQ aggregation and toxicity in transfected SH-SY5Y cell lines. Overexpression of ATP synthase α is able to protect cell death caused by polyglutamine-expanded htt. Transient overexpression of ATP synthase α suppresses the aggregate formation by estimation of polyQ aggregation, Western blot analysis, and filter trap assay (FTA) in transfected SH-SY5Y cells. These results indicated that ATP synthase α has a strong inhibitory effect on polyglutamine aggregate formation and toxicity in vitro, and suggest a novel neuroprotective role of ATP synthase α.     

IF<Subscript>1</Subscript> distribution in HepG2 cells in relation to ecto–F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATPsynthase and calmodulin

F₀F₁ATPsynthase is now known to be expressed as a plasma membrane receptor for several extracellular ligands. On hepatocytes, ecto–F₀F₁ATPsynthase binds apoA–I and triggers HDL endocytosis concomitant with ATP hydrolysis. Considering that inhibitor protein IF₁ was shown to regulate the hydrolytic activity of ecto–F₀F₁ATPsynthase and to interact with calmodulin (CaM) in vitro, we investigated the subcellular distributions of IF₁, calmodulin (CaM), OSCP and β subunits of F₀F₁ATPsynthase in HepG2 cells. Using immunofluorescence and Western blotting, we found that around 50% of total cellular IF₁ is localized outside mitochondria, a relevant amount of which is associated to the plasma membrane where we also found Ca²⁺–CaM, OSCP and β. Confocal microscopy showed that IF₁ colocalized with Ca²⁺–CaM on plasma membrane but not in mitochondria, suggesting that Ca²⁺–CaM may modulate the cell surface availability of IF₁ and thus its ability to inhibit ATP hydrolysis by ecto–F₀F₁ATPsynthase. These observations support a hypothesis that the IF₁–Ca²⁺–CaM complex, forming on plasma membrane, functions in the cellular regulation of HDL endocytosis by hepatocytes.     

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Here we study ATP synthase from human ρ⁰ (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF₁ inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF₁ still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in ρ⁰ cells and not to the dimeric form. This supports previous suggestions that IF₁ plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.     

Structural and functional characterization of FoF1-ATP synthase on the extracellular surface of rat hepatocytes

Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-F_oF₁-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F₁ (α/β and γ) and F_o (F_oI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF₁ is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H⁺ and are inhibited by F₁ and F_o-targeting inhibitors. We hypothesise that ecto-F_oF₁-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.