Hypothyroidism Leads to a Decreased Expression of Mitochondrial F0F1-ATP Synthase in Rat Liver

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F₀F₁-ATP synthase are accompanied by a decrease in the amount of immunodetected -F₁, F₀1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F₁ subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F₀F₁-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F₀F₁-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.     

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.     

Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes: DEVELOPMENT OF CELL-PERMEABLE,MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES*

The F₁F₀-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F₁F₀ activity via an interaction with the “d” subunit of F₁F₀-ATP synthase (dF₁F₀) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF₁F₀-derived peptide (NH₂-²AGRKLALKTIDWVSF¹⁶-COOH) that inhibits PKCδ binding to dF₁F₀ in overlay assays. We have also identified a second dF₁F₀-derived peptide (NH₂-¹¹¹RVREYEKQLEKIKNMI¹²⁶-COOH) that facilitates PKCδ binding to dF₁F₀. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF₁F₀ interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF₁F₀ by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F₁F₀-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF₁F₀ facilitator peptide by itself induced the PKCδ-dF₁F₀ co-immunoprecipitation and inhibited F₁F₀-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F₁F₀ inhibitor blocked PKCδ-mediated inhibition of F₁F₀-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF₁F₀ interaction in NCMs. These novel peptides will improve our understanding of cardiac F₁F₀ regulation and may have potential as therapeutics to attenuate cardiac injury.     

Single-Molecule Analysis of the Rotation of F1-ATPase under High Hydrostatic Pressure

F₁-ATPase is the water-soluble part of ATP synthase and is an ATP-driven rotary molecular motor that rotates the rotary shaft against the surrounding stator ring, hydrolyzing ATP. Although the mechanochemical coupling mechanism of F₁-ATPase has been well studied, the molecular details of individual reaction steps remain unclear. In this study, we conducted a single-molecule rotation assay of F₁ from thermophilic bacteria under various pressures from 0.1 to 140 MPa. Even at 140 MPa, F₁ actively rotated with regular 120° steps in a counterclockwise direction, showing high conformational stability and retention of native properties. Rotational torque was also not affected. However, high hydrostatic pressure induced a distinct intervening pause at the ATP-binding angles during continuous rotation. The pause was observed under both ATP-limiting and ATP-saturating conditions, suggesting that F₁ has two pressure-sensitive reactions, one of which is evidently ATP binding. The rotation assay using a mutant F₁(βE190D) suggested that the other pressure-sensitive reaction occurs at the same angle at which ATP binding occurs. The activation volumes were determined from the pressure dependence of the rate constants to be +100 Å³ and +88 Å³ for ATP binding and the other pressure-sensitive reaction, respectively. These results are discussed in relation to recent single-molecule studies of F₁ and pressure-induced protein unfolding.     

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.     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F₁F₀-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.     

The F0F1-ATP Synthase Complex Contains Novel Subunits and Is Essential for Procyclic Trypanosoma brucei

The mitochondrial F₀F₁ ATP synthase is an essential multi-subunit protein complex in the vast majority of eukaryotes but little is known about its composition and role in Trypanosoma brucei, an early diverged eukaryotic pathogen. We purified the F₀F₁ ATP synthase by a combination of affinity purification, immunoprecipitation and blue-native gel electrophoresis and characterized its composition and function. We identified 22 proteins of which five are related to F₁ subunits, three to F₀ subunits, and 14 which have no obvious homology to proteins outside the kinetoplastids. RNAi silencing of expression of the F₁ α subunit or either of the two novel proteins showed that they are each essential for the viability of procyclic (insect stage) cells and are important for the structural integrity of the F₀F₁-ATP synthase complex. We also observed a dramatic decrease in ATP production by oxidative phosphorylation after silencing expression of each of these proteins while substrate phosphorylation was not severely affected. Our procyclic T. brucei cells were sensitive to the ATP synthase inhibitor oligomycin even in the presence of glucose contrary to earlier reports. Hence, the two novel proteins appear essential for the structural organization of the functional complex and regulation of mitochondrial energy generation in these organisms is more complicated than previously thought.     

The depletion of F1 subunit ε in yeast leads to an uncoupled respiratory phenotype that is rescued by mutations in the proton-translocating subunits of F0

The central stalk of the ATP synthase is an elongated hetero-oligomeric structure providing a physical connection between the catalytic sites in F₁ and the proton translocation channel in F₀ for energy transduction between the two subdomains. The shape of the central stalk and relevance to energy coupling are essentially the same in ATP synthases from all forms of life, yet the protein composition of this domain changed during evolution of the mitochondrial enzyme from a two- to a three-subunit structure (γ, δ, ε). Whereas the mitochondrial γ- and δ-subunits are homologues of the bacterial central stalk proteins, the deliberate addition of subunit ε is poorly understood. Here we report that down-regulation of the gene (ATP15) encoding the ε-subunit rapidly leads to lethal F₀-mediated proton leaks through the membrane because of the loss of stability of the ATP synthase. The ε-subunit is thus essential for oxidative phosphorylation. Moreover, mutations in F₀ subunits a and c, which slow the proton translocation rate, are identified that prevent ε-deficient ATP synthases from dissipating the electrochemical potential. Cumulatively our data lead us to propose that the ε-subunit evolved to permit operation of the central stalk under the torque imposed at the normal speed of proton movement through mitochondrial F₀.     

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.     

The native F0F1-inhibitor protein complex from beef heart mitochondria and its reconstitution in liposomes

A functional F₀F₁ ATP synthase that contains the endogenous inhibitor protein (F₀F₁I) was isolated by the use of two combined techniques [Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975).Biochemistry 14, 1727–1735; Dreyfus, G., Celis, H., and Ramirez, J. (1984).Anal. Biochem. 142, 215–220]. The preparation is composed of 18 subunits as judged by SDS-PAGE. A steady-state kinetic analysis of the latent ATP synthase complex at various concentrations of ATP showed aV _max of 1.28mol min^–1 mg^–1, whereas theV _max of the complex without the inhibitor was 8.3mol min^–1 mg^–1. In contrast, theK _m for Mg-ATP of F₀F₁ I was 148M, comparable to theK _m value of 142M of the F₀F₁ complex devoid of IF₁. The hydrolytic activity of the F₀F₁I increased severalfold by incubation at 60C at pH 6.8, reaching a maximal ATPase activity of 9.5mol min^–1 mg^–1; at pH 9.0 a rapid increase in the specific activity of hydrolysis was followed by a sharp drop in activity. The latent ATP synthase was reconstituted into liposomes by means of a column filtration method. The proteoliposomes showed ATP-Pi exchange activity which responded to phosphate concentration and was sensitive to energy transfer inhibitors like oligomycin and the uncouplerp-trifluoromethoxyphenylhydrazone.     

The phototroph-specific β-hairpin structure of the γ subunit of FoF1-ATP synthase is important for efficient ATP synthesis of cyanobacteria

The F_oF₁ synthase produces ATP from ADP and inorganic phosphate. The γ subunit of F_oF₁ ATP synthase in photosynthetic organisms, which is the rotor subunit of this enzyme, contains a characteristic β-hairpin structure. This structure is formed from an insertion sequence that has been conserved only in phototrophs. Using recombinant subcomplexes, we previously demonstrated that this region plays an essential role in the regulation of ATP hydrolysis activity, thereby functioning in controlling intracellular ATP levels in response to changes in the light environment. However, the role of this region in ATP synthesis has long remained an open question because its analysis requires the preparation of the whole F_oF₁ complex and a transmembrane proton-motive force. In this study, we successfully prepared proteoliposomes containing the entire F_oF₁ ATP synthase from a cyanobacterium, Synechocystis sp. PCC 6803, and measured ATP synthesis/hydrolysis and proton-translocating activities. The relatively simple genetic manipulation of Synechocystis enabled the biochemical investigation of the role of the β-hairpin structure of F_oF₁ ATP synthase and its activities. We further performed physiological analyses of Synechocystis mutant strains lacking the β-hairpin structure, which provided novel insights into the regulatory mechanisms of F_oF₁ ATP synthase in cyanobacteria via the phototroph-specific region of the γ subunit. Our results indicated that this structure critically contributes to ATP synthesis and suppresses ATP hydrolysis.     

Functional and stoichiometric analysis of subunit e in bovine heart mitochondrial F<Subscript>0</Subscript>F<Subscript>1</Subscript>ATP synthase

The role of the integral inner membrane subunit e in self-association of F₀F₁ATP synthase from bovine heart mitochondria was analyzed by in situ limited proteolysis, blue native PAGE/iterative SDS-PAGE, and LC-MS/MS. Selective degradation of subunit e, without disrupting membrane integrity or ATPase capacity, altered the oligomeric distribution of F₀F₁ATP synthase, by eliminating oligomers and reducing dimers in favor of monomers. The stoichiometry of subunit e was determined by a quantitative MS-based proteomics approach, using synthetic isotope-labelled reference peptides IAQL*EEVK, VYGVGSL*ALYEK, and ELAEAQEDTIL*K to quantify the b, γ and e subunits, respectively. Accuracy of the method was demonstrated by confirming the 1:1 stoichiometry of subunits γ and b. Altogether, the results indicate that the integrity of a unique copy of subunit e is essential for self-association of mammalian F₀F₁ATP synthase. Elena Bisetto and Paola Picotti contributed equally to this work.     

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

The ζ subunit is a novel inhibitor of the F₁F_O-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF₁) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F₁-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F₁-ATPase and F₁-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F₁-ATPase at the open catalytic α_E/β_E interface, and two partial γ rotations lock the N terminus of ζ in an “inhibition-general core region,” blocking further γ rotation, while the ζ globular domain anchors it to the closed α_DP/β_DP interface. Heterologous inhibition of the F₁-ATPase of P. denitrificans by the mitochondrial IF₁ supported both the modeled ζ binding site at the α_DP/β_DP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF₁ or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.     

Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography

The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at ∼ 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F₁ headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles.     

Mitochondrial F1F0-ATP synthase and organellar internal architecture

The mitochondrial F₁F₀-ATP synthase adopts supramolecular structures. The interaction domains between monomers involve components belonging to the F₀ domains. In Saccharomyces cerevisiae, alteration of these components destabilizes the oligomeric structures, leading concomitantly to the appearance of monomeric species of ATP synthase and anomalous mitochondrial morphologies in the form of onion-like structures. The mitochondrial ultrastructure at the cristae level is thus modified. Electron microscopy on cross-sections of wild type mitochondria display many short cristae with narrowed intra-cristae space, whereas yeast mutants defected in supramolecular ATP synthases assembly present a low number of large lamellar cristae of constant thickness and traversing the whole organelle. The growth of these internal structures leads finally to mitochondria with sphere-like structures with a mean diameter of 1 μm that are easily identified by epifluorescence microscopy. As a result, ATP synthase is an actor of the mitochondrial ultrastructure in yeast. This paper reviews the ATP synthase components whose modifications lead to anomalous mitochondrial morphology and also provides a schema showing the formation of the so-called onion-like structures.     

On the mechanism of the reconstitution of F1-depleted ATPase complex with purified F1: Possible conformational effects

The membrane sector (F₀) of H⁺-ATPase was prepared by trypsin and urea treatment of F₁-F₀ and reconstituted with purified F₁. The oligomycin sensitivity of the reconstituted F₁-F₀ complex obtained by treating F₁ or F₀ with Mg²⁺ before binding is much higher than that obtained without Mg²⁺ treatment. The greater change in the intrinsic fluorescence of the reconstituted F₁-F₀ complex obtained by Mg²⁺ treatment suggests that conformational changes may occur during the reconstitution. We deduce that Mg²⁺ binds to membrane lipids, thus decreasing membrane fluidity and changing the physical state of the lipids to provide a suitable microenvironment for conformational changes in F₀. The data also suggest that the conformational change in the F₀ portion of the F₁-F₀ complex can be transmitted to the F₁ portion, the conformation of which is in turn altered, resulting in the formation of an F₁-F₀ complex with high oligomycin sensitivity. On the other hand, Mg²⁺ may act on F₁ directly to induce a suitable conformational change which is then trnsmitted to F₀, resulting in the formation of an H⁺-ATPase with greater sensitivity to oligomycin.Abbreviations STED 0.25 M sucrose, 10 mM Tris-SO₄, 0.2 mM EDTA, and 1 mM dithiothreitol, pH 8.0 - NADH nicotinamide adenine dinucleotide, reduced form - olig. oligomycin - OSCP oligomycin sensitivity conferring protein - F₆ coupling factor 6 - F₁ coupling factor one (or F₁-ATPase) - F₁ ^{+Mg 2+} and F₁ ^{–Mg 2+} the F₁ treated and untreated with 1 mM Mg²⁺ respectively - F₀ the membrane sector proteins of the H⁺-ATPase - TUF₀ trypsin-urea – F₀ - EUF₀ EDTA-urea – F₀ - F₀ ^{+Mg 2+} and F₀ ^{–Mg 2+} the F₀ treated and untreated with 1 mM Mg²⁺ respectively - (F₁ · F₀)^{+Mg 2+} and (F₁ · F₀)^{–Mg 2+} the reconstituted F₁ · F₀ complex containing Mg²⁺-treated F₁ and F₀ and untreated F₁ and F₀ respectively - F₁ · F₀ ^{+Mg 2+} and F₁ · F₀ ^{–Mg 2+} the reconstituted H⁺-ATPase complex derived from the binding of purified F₁ to the F₀ treated and untreated with Mg²⁺ respectively - F₁ ^{+Mg 2+} · F₀ and F₁ ^{–Mg 2+} · F₀ the reconstituted H⁺-ATPase derived from the binding of F₀ to the purified F₁ treated and untreated with Mg²⁺ respectively     

Bacterial Na(+)-ATP synthase has an undecameric rotor

Synthesis of adenosine triphosphate (ATP) by the F₁F₀ ATP synthase involves a membrane-embedded rotary engine, the F₀ domain, which drives the extra-membranous catalytic F₁ domain. The F₀ domain consists of subunits a₁b₂ and a cylindrical rotor assembled from 9–14 α-helical hairpin-shaped c-subunits. According to structural analyses, rotors contain 10 c-subunits in yeast and 14 in chloroplast ATP synthases. We determined the rotor stoichiometry of Ilyobacter tartaricus ATP synthase by atomic force microscopy and cryo-electron microscopy, and show the cylindrical sodium-driven rotor to comprise 11 c-subunits.     

Mitochondrial F1Fo-ATP Synthase: The Small Subunits e and g Associate with Monomeric Complexes to Trigger Dimerization

Mitochondrial F₁F_o-ATP synthase catalyzes the formation of ATP from ADP and inorganic phosphate. The enzyme is found in monomeric, dimeric and higher oligomeric forms in the inner mitochondrial membrane. Dimerization of ATP synthase complexes is a prerequisite for the generation of larger oligomers that promote membrane bending and formation of tubular cristae membranes. Two small proteins of the membrane-embedded F_o-domain, subunit e (Su e; Atp21) and Su g (Atp20), were identified as dimer-specific subunits of yeast ATP synthase and shown to be required for stabilization of the dimers. We have identified two distinct monomeric forms of yeast ATP synthase. Su e and Su g are present not only in the dimer but also in one of the monomeric forms. We demonstrate that Su e and Su g sequentially assemble with monomeric ATP synthase to form a dimerization-competent primed monomer. We conclude that association of Su e and Su g with monomeric F₁F_o-ATP synthase represents an initial step of oligomer formation.     

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.