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.     

The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology

Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.     

The ATP synthase is involved in generating mitochondrial cristae morphology

The inner membrane of the mitochondrion folds inwards, forming the cristae. This folding allows a greater amount of membrane to be packed into the mitochondrion. The data in this study demonstrate that subunits e and g of the mitochondrial ATP synthase are involved in generating mitochondrial cristae morphology. These two subunits are non-essential components of ATP synthase and are required for the dimerization and oligomerization of ATP synthase. Mitochondria of yeast cells deficient in either subunits e or g were found to have numerous digitations and onion-like structures that correspond to an uncontrolled biogenesis and/or folding of the inner mitochondrial membrane. The present data show that there is a link between dimerization of the mitochondrial ATP synthase and cristae morphology. A model is proposed of the assembly of ATP synthase dimers, taking into account the oligomerization of the yeast enzyme and earlier data on the ultrastructure of mitochondrial cristae, which suggests that the association of ATP synthase dimers is involved in the control of the biogenesis of the inner mitochondrial membrane.     

The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane.

A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.     

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.     

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.     

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.     

Partial Assembly of the Yeast Mitochondrial ATP Synthase 1

The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F₁ catalytic domain, the F₀ proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F₁ to F₀, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F₀ into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F₀. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.     

Cardiolipin affects the supramolecular organization of ATP synthase in mitochondria

F₁F₀ ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane.     

Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae?

Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of detergent mitochondrial extracts have provided evidence that the yeast ATP synthase could form dimers. Cross-linking experiments performed on a modified version of the i-subunit of this enzyme indicate the existence of such ATP synthase dimers in the yeast inner mitochondrial membrane. We also show that the first transmembrane segment of the eukaryotic b-subunit (bTM1), like the two supernumerary subunits e and g, is required for dimerization/oligomerization of ATP synthases. Unlike mitochondria of wild-type cells that display a well-developed cristae network, mitochondria of yeast cells devoid of subunits e, g, or bTM1 present morphological alterations with an abnormal proliferation of the inner mitochondrial membrane. From these observations, we postulate that an anomalous organization of the inner mitochondrial membrane occurs due to the absence of ATP synthase dimers/oligomers. We provide a model in which the mitochondrial ATP synthase is a key element in cristae morphogenesis.     

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.     

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.     

Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase

Several human neurological disorders have been associated with various mutations affecting mitochondrial enzymes involved in cellular ATP production. One of these mutations, T9176C in the mitochondrial DNA (mtDNA), changes a highly conserved leucine residue into proline at position 217 of the mitochondrially encoded Atp6p (or a) subunit of the F₁F_O-ATP synthase. The consequences of this mutation on the mitochondrial ATP synthase are still poorly defined. To gain insight into the primary pathogenic mechanisms induced by T9176C, we have investigated the consequences of this mutation on the ATP synthase of yeast where Atp6p is also encoded by the mtDNA. In vitro, yeast atp6-T9176C mitochondria showed a 30% decrease in the rate of ATP synthesis. When forcing the F₁F_O complex to work in the reverse mode, i.e. F₁-catalyzed hydrolysis of ATP coupled to proton transport out of the mitochondrial matrix, the mutant showed a normal proton-pumping activity and this activity was fully sensitive to oligomycin, an inhibitor of the ATP synthase proton channel. However, under conditions of maximal ATP hydrolytic activity, using non-osmotically protected mitochondria, the mutant ATPase activity was less efficiently inhibited by oligomycin (60% inhibition versus 85% for the wild type control). Blue Native Polyacrylamide Gel Electrophoresis analyses revealed that atp6-T9176C yeast accumulated rather good levels of fully assembled ATP synthase complexes. However, a number of sub-complexes (F₁, Atp9p-ring, unassembled α-F₁ subunits) could be detected as well, presumably because of a decreased stability of Atp6p within the ATP synthase. Although the oxidative phosphorylation capacity was reduced in atp6-T9176C yeast, the number of ATP molecules synthesized per electron transferred to oxygen was similar compared with wild type yeast. It can therefore be inferred that the coupling efficiency within the ATP synthase was mostly unaffected and that the T9176C mutation did not increase the proton permeability of the mitochondrial inner membrane.     

Attenuated ADP-inhibition of FOF1 ATPase mitigates manifestations of mitochondrial dysfunction in yeast

Proton-translocating F_OF₁ ATP synthase (F-ATPase) couples ATP synthesis or hydrolysis to transmembrane proton transport in bacteria, chloroplasts, and mitochondria. The primary function of the mitochondrial F_OF₁ is ATP synthesis driven by protonmotive force (pmf) generated by the respiratory chain. However, when pmf is low or absent (e.g. during anoxia), F_OF₁ consumes ATP and functions as a proton-pumping ATPase.Several regulatory mechanisms suppress the ATPase activity of F_OF₁ at low pmf. In yeast mitochondria they include special inhibitory proteins Inh1p and Stf1p, and non-competitive inhibition of ATP hydrolysis by MgADP (ADP-inhibition). Presumably, these mechanisms help the cell to preserve the ATP pool upon membrane de-energization. However, no direct evidence was presented to support this hypothesis so far.Here we report that a point mutation Q263L in subunit beta of Saccharomyces cerevisiae ATP synthase significantly attenuated ADP-inhibition of the enzyme without major effect on the rate of ATP production by mitochondria. The mutation also decreased the sensitivity of the enzyme ATPase activity to azide. Similar effects of the corresponding mutations were observed in earlier studies in bacterial enzymes. This observation indicates that the molecular mechanism of ADP-inhibition is probably the same in mitochondrial and in bacterial F_OF₁.The mutant yeast strain had lower growth rate and had a longer lag period preceding exponential growth phase when starved cells were transferred to fresh growth medium. However, upon the loss of mitochondrial DNA (ρ⁰) the βQ263L mutation effect was reversed: the βQ263L ρ⁰ mutant grew faster than the wild-type ρ⁰ yeast. The results suggest that ADP-inhibition might play a role in prevention of wasteful ATP hydrolysis in the mitochondrial matrix.     

HcRed, a genetically encoded fluorescent binary cross-linking agent for cross-linking of mitochondrial ATP synthase in Saccharomyces cerevisiae

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase.     

Formation of cristae and crista junctions in mitochondria depends on antagonism between Fcj1 and Su e/g

Crista junctions (CJs) are important for mitochondrial organization and function, but the molecular basis of their formation and architecture is obscure. We have identified and characterized a mitochondrial membrane protein in yeast, Fcj1 (formation of CJ protein 1), which is specifically enriched in CJs. Cells lacking Fcj1 lack CJs, exhibit concentric stacks of inner membrane in the mitochondrial matrix, and show increased levels of F₁F_O–ATP synthase (F₁F_O) supercomplexes. Overexpression of Fcj1 leads to increased CJ formation, branching of cristae, enlargement of CJ diameter, and reduced levels of F₁F_O supercomplexes. Impairment of F₁F_O oligomer formation by deletion of its subunits e/g (Su e/g) causes CJ diameter enlargement and reduction of cristae tip numbers and promotes cristae branching. Fcj1 and Su e/g genetically interact. We propose a model in which the antagonism between Fcj1 and Su e/g locally modulates the F₁F_O oligomeric state, thereby controlling membrane curvature of cristae to generate CJs and cristae tips.     

Novel insights into the mitochondrial permeability transition

Alavian and colleagues recently provided further evidence in support of the notion that the c subunit of the mitochondrial F₁F_O ATP synthase constitutes the long-sought pore-forming unit of the supramolecular complex responsible for the so-called ‘mitochondrial permeability transition’ (MPT). Besides shedding new light on the molecular mechanisms that underlie the MPT, these findings corroborate the notion that several components of the cell death machinery, including cytochrome c and the F₁F_O ATP synthase, mediate critical metabolic activities.     

The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase

The supernumerary subunit g is found in all mitochondrial ATP synthases. Most of the conserved amino acid residues are present in the membrane C-terminal part of the protein that contains a dimerization motif GXXXG. In yeast, alteration of this motif leads to the loss of subunit g and of supramolecular structures of the ATP synthase with concomitant appearance of anomalous mitochondrial morphologies. Disulfide bond formation involving an engineered cysteine in position 109 of subunit g and the endogenous cysteine 28 of subunit e promoted g + g, e + g, and e + e adducts, thus revealing the proximity in the mitochondrial membrane of several subunits e and g. Disulfide bond formation between two subunits g in mitochondria increased the stability of an oligomeric structure of the ATP synthase in digitonin extracts. These data suggest the participation of the dimerization motif of subunit g in the formation of supramolecular structures and is in favor of the existence of ATP synthase associations, in the inner mitochondrial membrane, whose masses are higher than those of ATP synthase dimers.     

Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas

Mitochondrial F₁F_O ATP synthase (Complex V) catalyses ATP synthesis from ADP and inorganic phosphate using the proton-motive force generated by the substrate-driven electron transfer chain. In this work, we investigated the impact of the loss of activity of the mitochondrial enzyme in a photosynthetic organism. In this purpose, we inactivated by RNA interference the expression of the ATP2 gene, coding for the catalytic subunit β, in the green alga Chlamydomonas reinhardtii. We demonstrate that in the absence of β subunit, complex V is not assembled, respiratory rate is decreased by half and ATP synthesis coupled to the respiratory activity is fully impaired. Lack of ATP synthase also affects the morphology of mitochondria which are deprived of cristae. We also show that mutants are obligate phototrophs and that rearrangements of the photosynthetic apparatus occur in the chloroplast as a response to ATP synthase deficiency in mitochondria. Altogether, our results contribute to the understanding of the yet poorly studied bioenergetic interactions between organelles in photosynthetic organisms.     

Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity

Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using ¹H NMR and ³¹P NMR techniques, we observed that increasing the temperature (8 °C to 37 °C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F₀ sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F₀ sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F₀ sector, and thereby increase ATP synthesis.