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.     

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.     

Cyclophilin D Modulates Mitochondrial F0F1-ATP Synthase by Interacting with the Lateral Stalk of the Complex

Blue native gel electrophoresis purification and immunoprecipitation of F₀F₁-ATP synthase from bovine heart mitochondria revealed that cyclophilin (CyP) D associates to the complex. Treatment of intact mitochondria with the membrane-permeable bifunctional reagent dimethyl 3,3-dithiobis-propionimidate (DTBP) cross-linked CyPD with the lateral stalk of ATP synthase, whereas no interactions with F₁ sector subunits, the ATP synthase natural inhibitor protein IF1, and the ATP/ADP carrier were observed. The ATP synthase-CyPD interactions have functional consequences on enzyme catalysis and are modulated by phosphate (increased CyPD binding and decreased enzyme activity) and cyclosporin (Cs) A (decreased CyPD binding and increased enzyme activity). Treatment of MgATP submitochondrial particles or intact mitochondria with CsA displaced CyPD from membranes and activated both hydrolysis and synthesis of ATP sustained by the enzyme. No effect of CsA was detected in CyPD-null mitochondria, which displayed a higher specific activity of the ATP synthase than wild-type mitochondria. Modulation by CyPD binding appears to be independent of IF1, whose association to ATP synthase was not affected by CsA treatment. These findings demonstrate that CyPD association to the lateral stalk of ATP synthase modulates the activity of the complex.     

Control of rotation of the F<Subscript>1</Subscript>F<Subscript>O</Subscript>-ATP synthase nanomotor by an inhibitory α-helix from unfolded ε or intrinsically disordered ζ and IF<Subscript>1</Subscript> proteins

The ATP synthase is a ubiquitous nanomotor that fuels life by the synthesis of the chemical energy of ATP. In order to synthesize ATP, this enzyme is capable of rotating its central rotor in a reversible manner. In the clockwise (CW) direction, it functions as ATP synthase, while in counter clockwise (CCW) sense it functions as an proton pumping ATPase. In bacteria and mitochondria, there are two known canonical natural inhibitor proteins, namely the ε and IF₁ subunits. These proteins regulate the CCW F₁F_O-ATPase activity by blocking γ subunit rotation at the α_DP/β_DP/γ subunit interface in the F₁ domain. Recently, we discovered a unique natural F₁-ATPase inhibitor in Paracoccus denitrificans and related α-proteobacteria denoted the ζ subunit. Here, we compare the functional and structural mechanisms of ε, IF₁, and ζ, and using the current data in the field, it is evident that all three regulatory proteins interact with the α_DP/β_DP/γ interface of the F₁-ATPase. In order to exert inhibition, IF₁ and ζ contain an intrinsically disordered N-terminal protein region (IDPr) that folds into an α-helix when inserted in the α_DP/β_DP/γ interface. In this context, we revised here the mechanism and role of the ζ subunit as a unidirectional F-ATPase inhibitor blocking exclusively the CCW F₁F_O-ATPase rotation, without affecting the CW-F₁F_O-ATP synthase turnover. In summary, the ζ subunit has a mode of action similar to mitochondrial IF₁, but in α-proteobacteria. The structural and functional implications of these intrinsically disordered ζ and IF₁ inhibitors are discussed to shed light on the control mechanisms of the ATP synthase nanomotor from an evolutionary perspective.     

Supramolecular organization of the yeast F1Fo-ATP synthase

Background information. The yeast mitochondrial F₁F_o‐ATP synthase is a large complex of 600 kDa that uses the proton electrochemical gradient generated by the respiratory chain to catalyse ATP synthesis from ADP and P_i. For a large range of organisms, it has been shown that mitochondrial ATP synthase adopts oligomeric structures. Moreover, several studies have suggested that a link exists between ATP synthase and mitochondrial morphology. Results and discussion. In order to understand the link between ATP synthase oligomerization and mitochondrial morphology, more information is needed on the supramolecular organization of this enzyme within the inner mitochondrial membrane. We have conducted an electron microscopy study on wild‐type yeast mitochondria at different levels of organization from spheroplast to isolated ATP synthase complex. Using electron tomography, freeze‐fracture, negative staining and image processing, we show that cristae form a network of lamellae, on which ATP synthase dimers assemble in linear and regular arrays of oligomers. Conclusions. Our results shed new light on the supramolecular organization of the F₁F_o‐ATP synthase and its potential role in mitochondrial morphology.     

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.     

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.     

The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo‐ATP synthase

Mitochondrial F₁F_o‐ATP synthase generates the bulk of cellular ATP. This molecular machine assembles from nuclear‐ and mitochondria‐encoded subunits. Whereas chaperones for formation of the matrix‐exposed hexameric F₁‐ATPase core domain have been identified, insight into how the nuclear‐encoded F₁‐domain assembles with the membrane‐embedded F_o‐region is lacking. Here we identified the INA complex (INAC) in the inner membrane of mitochondria as an assembly factor involved in this process. Ina22 and Ina17 are INAC constituents that physically associate with the F₁‐module and peripheral stalk, but not with the assembled F₁F_o‐ATP synthase. Our analyses show that loss of Ina22 and Ina17 specifically impairs formation of the peripheral stalk that connects the catalytic F₁‐module to the membrane embedded F_o‐domain. We conclude that INAC represents a matrix‐exposed inner membrane protein complex that facilitates peripheral stalk assembly and thus promotes a key step in the biogenesis of mitochondrial F₁F_o‐ATP synthase.     

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.     

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.     

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.     

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.     

The role of the ATPase inhibitor factor 1 (IF1) in cancer cells adaptation to hypoxia and anoxia

The physiological role of the mitochondrial ATP synthase complex is to generate ATP through oxidative phosphorylation. Indeed, the enzyme can reverse its activity and hydrolyze ATP under ischemic conditions, as shown in isolated mitochondria and in mammalian heart and liver. However, what occurs when cancer cells experience hypoxia or anoxia has not been well explored. In the present study, we investigated the bioenergetics of cancer cells under hypoxic/anoxic conditions with particular emphasis on ATP synthase, and the conditions driving it to work in reverse. In this context, we further examined the role exerted by its endogenous inhibitor factor, IF₁, that it is overexpressed in cancer cells. Metabolic and bioenergetic analysis of cancer cells exposed to severe hypoxia (down to 0.1% O₂) unexpectedly showed that Δψ_m is preserved independently of the presence of IF₁ and that ATP synthase still phosphorylates ADP though at a much lower rate than in normoxia. However, when we induced an anoxia-mimicking condition by collapsing Δμ_Η⁺ with the FCCP uncoupler, the IF₁-silenced clones only reversed the ATP synthase activity hydrolyzing ATP in order to reconstitute the electrochemical proton gradient. Notably, in cancer cells IF₁ overexpression fully prevents ATP synthase hydrolytic activity activation under uncoupling conditions. Therefore, our results suggest that IF₁ overexpression promotes cancer cells survival under temporary anoxic conditions by preserving cellular ATP despite mitochondria dysfunction.     

ATP synthase: Subunit–subunit interactions in the stator stalk

In ATP synthase, proton translocation through the F_o subcomplex and ATP synthesis/hydrolysis in the F₁ subcomplex are coupled by subunit rotation. The static, non-rotating portions of F₁ and F_o are attached to each other via the peripheral “stator stalk”, which has to withstand elastic strain during subunit rotation. In Escherichia coli, the stator stalk consists of subunits b₂δ; in other organisms, it has three or four different subunits. Recent advances in this area include affinity measurements between individual components of the stator stalk as well as a detailed analysis of the interaction between subunit δ (or its mitochondrial counterpart, the oligomycin-sensitivity conferring protein, OSCP) and F₁. The current status of our knowledge of the structure of the stator stalk and of the interactions between its subunits will be discussed in this review.     

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     

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.     

Reconstitution of mitochondrial ATP synthase into lipid bilayers for structural analysis

Mitochondrial F₁F_o-ATP synthase is a molecular motor that couples the energy generated by oxidative metabolism to the synthesis of ATP. Direct visualization of the rotary action of the bacterial ATP synthase has been well characterized. However, direct observation of rotation of the mitochondrial enzyme has not been reported yet. Here, we describe two methods to reconstitute mitochondrial F₁F_o-ATP synthase into lipid bilayers suitable for structure analysis by electron and atomic force microscopy (AFM). Proteoliposomes densely packed with bovine heart mitochondria F₁F_o-ATP synthase were obtained upon detergent removal from ternary mixtures (lipid, detergent and protein). Two-dimensional crystals of recombinant hexahistidine-tagged yeast F₁F_o-ATP synthase were grown using the supported monolayer technique. Because the hexahistidine-tag is located at the F₁ catalytic subcomplex, ATP synthases were oriented unidirectionally in such two-dimensional crystals, exposing F₁ to the lipid monolayer and the F_o membrane region to the bulk solution. This configuration opens a new avenue for the determination of the c-ring stoichiometry of unknown hexahistidine-tagged ATP synthases and the organization of the membrane intrinsic subunits within F_o by electron microscopy and AFM.     

The Structural and Functional Connection between the Catalytic and Proton Translocating Sectors of the Mitochondrial F 1 F 0 -ATP Synthase

The structural and functional connection between the peripheral catalytic F₁ sector and theproton-translocating membrane sector F₀ of the mitochondrial ATP synthase is reviewed. Theobservations examined show that the N-terminus of subunit , the carboxy-terminal and centralregion of F₀I-PVP(b), OSCP, and part of subunit d constitute a continuous structure, the lateralstalk, which connects the peripheries of F₁ to F₀ and surrounds the central element of thestalk, constituted by subunits and . The ATPase inhibitor protein (IF₁) binds at one sideof the F₁F₀ connection. The carboxy-terminal segment of IF₁ apparently binds to OSCP. The42L-58K segment of IF₁, which is per se the most active domain of the protein, binds at thesurface of one of the three / pairs of F₁, thus preventing the cyclic interconversion of thecatalytic sites required for ATP hydrolysis.     

Structure of Dimeric F1F0-ATP Synthase

The structure of the dimeric ATP synthase from yeast mitochondria was analyzed by transmission electron microscopy and single particle image analysis. In addition to the previously reported side views of the dimer, top view and intermediate projections served to resolve the arrangement of the rotary c₁₀ ring and the other stator subunits at the F₀-F₀ dimeric interface. A three-dimensional reconstruction of the complex was calculated from a data set of 9960 molecular images at a resolution of 27 Å. The structural model of the dimeric ATP synthase shows the two monomers arranged at an angle of ∼45°, consistent with our earlier analysis of the ATP synthase from bovine heart mitochondria (Minauro-Sanmiguel, F., Wilkens, S., and Garcia, J. J. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 12356–12358). In the ATP synthase dimer, the two peripheral stalks are located near the F₁-F₁ interface but are turned away from each other so that they are not in contact. Based on the three-dimensional reconstruction, a model of how dimeric ATP synthase assembles to form the higher order oligomeric structures that are required for mitochondrial cristae biogenesis is discussed.     

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.