Mitochondrial ATP synthase. Crystal structure of the catalytic F1 unit in a vanadate-induced transition-like state and implications for mechanism

ATP synthesis from ADP, P(i), and Mg2+ takes place in mitochondria on the catalytic F1 unit (alpha3beta3gammedeltaepsilon) of the ATP synthase complex (F0F1), a remarkable nanomachine that interconverts electrochemical and mechanical energy, producing the high energy terminal bond of ATP. In currently available structural models of F1, the P-loop (amino acid residues 156GGAGVGKT163) contributes to substrate binding at the subunit catalytic sites. Here, we report the first transition state-like structure of F1 (ADP.V(i).Mg.F1) from rat liver that was crystallized with the phosphate (P(i)) analog vanadate (VO(3-)4 or V(i)). Compared with earlier "ground state" structures, this new F1 structure reveals that the active site region has undergone significant remodeling. P-loop residue alanine 158 is located much closer to V(i) than it is to P(i) in a previous structural model. No significant movements of P-loop residues of the subunit were observed at its analogous but noncatalytic sites. Under physiological conditions, such active site remodeling involving the small hydrophobic alanine residue may promote ATP synthesis by lowering the local dielectric constant, thus facilitating the dehydration of ADP and P(i). This new crystallographic study provides strong support for the catalytic mechanism of ATP synthesis deduced from earlier biochemical studies of liver F1 conducted in the presence of V(i) (Ko, Y. H., Bianchet, M., Amzel, L. M., and Pedersen, P. L. (1997) J. Biol. Chem. 272, 18875-18881; Ko, Y. H., Hong, S., and Pedersen, P. L. (1999) J. Biol. Chem. 274, 28853-28856).     

The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits

It is now clearly established that dimerization of the F(1)F(o) ATP synthase takes place in the mitochondrial inner membrane. Interestingly, oligomerization of this enzyme seems to be involved in cristae morphogenesis. As they were able to form homodimers, subunits 4, e, and g have been proposed as potential ATP synthase dimerization subunits. In this paper, we provide evidence that subunit h, a peripheral stalk component, is located either at or near the ATP synthase dimerization interface. Subunit h homodimers were formed in mitochondria and were found to be associated to ATP synthase dimers. Moreover, homodimerization of subunit h and of subunit i turned out to be independent of subunits e and g, confirming the existence of an ATP synthase dimer in the mitochondrial inner membrane in the absence of subunits e and g. For the first time, this dimer has been observed by BN-PAGE. Finally, from these results we are now able to update our model for the supramolecular organization of the ATP synthase in the membrane and propose a role for subunits e and g, which stabilize the ATP synthase dimers and are involved in the oligomerization of the complex.     

Mitochondrial ATP synthasome: three-dimensional structure by electron microscopy of the ATP synthase in complex formation with carriers for Pi and ADP/ATP

The terminal steps involved in making ATP in mitochondria require an ATP synthase (F(0)F(1)) comprised of two motors, a phosphate carrier (PIC), and an adenine nucleotide carrier (ANC). Under mild conditions, these entities sub-fractionate as an ATP synthase/PIC/ANC complex or "ATP synthasome" (Ko, Y.H., Delannoy, M, Hullihen, J., Chiu, W., and Pedersen, P.L. (2003) J. Biol. Chem. 278, 12305-12309). As a first step toward obtaining three-dimensional information about this large complex or "metabolon" and the locations of PIC and ANC therein, we dispersed ATP synthasomes into single complexes and visualized negatively stained images by electron microscopy (EM) that showed clearly the classical headpiece, central stalk, and basepiece. Parallel immuno-EM studies revealed the presence of PIC and ANC located non-centrally in the basepiece, and other studies implicated an ATP synthase/PIC/ANC stoichiometry near 1:1:1. Single ATP synthasome images (7506) were boxed, and, using EMAN software, a three-dimensional model was obtained at a resolution of 23 A. Significantly, the basepiece is oblong and contains two domains, the larger of which connects to the central stalk, whereas the smaller appears as an extension. Docking studies with known structures together with the immuno-EM studies suggest that PIC or ANC may be located in the smaller domain, whereas the other transporter resides nearby in the larger domain. Collectively, these finding support a mechanism in which the entry of the substrates ADP and P(i) into mitochondria, the synthesis of ATP on F(1), and the release and exit of ATP are very localized and highly coordinated events.     

Cytosolic subunits of ATP synthase are localized to the cortical endoplasmic reticulum‐rich domain of the ascidian egg myoplasm

Previously, we revealed that p58, one of the ascidian maternal factors, is identical to the alpha‐subunit of F1‐ATP synthase (ATPα), a protein complex of the inner mitochondrial membrane. In the current study, we used immunological probes for ascidian mitochondria components to show that the ascidian ATPα is ectopically localized to the cytosol. Virtually all mitochondrial components were localized to the mitochondria‐rich myoplasm. However, in detail, ATP synthase subunits and the matrix proteins showed different localization patterns. At least at the crescent stage, transmission electron microscopy (TEM) distinguished the mitochondria‐less, endoplasmic reticulum (ER)‐rich cortical region and the mitochondria‐rich internal region. ATPα was enriched in the cortical region and MnSOD was limited to the internal region. Using subcellular fractionation, although all of the mitochondria components were highly enriched in the mitochondria‐enriched fraction, a considerable amount of ATPα and F1‐ATP synthase beta‐subunit (ATPβ) were recovered in the insoluble cytoplasmic fraction. Even under these conditions, F1‐ATP synthase gamma‐subunit (ATPγ) and F0‐ATP synthase subunit b (ATPb) were not recovered in the insoluble cytoplasmic fraction. This result strongly supports the exomitochondrial localization of both ATPα and ATPβ. In addition, the detergent extraction of eggs supports the idea that these cytosolic ATP synthase subunits are associated with the egg cytoskeleton. These results suggest that the subunits of ATP synthase might play dual roles at different subcellular compartments during early development.     

Topological and functional study of subunit h of the F1Fo ATP synthase complex in yeast Saccharomyces cerevisiae

Subunit h, a 92-residue-long, hydrophilic, acidic protein, is a component of the yeast mitochondrial F1Fo ATP synthase. This subunit, homologous to the mammalian factor F6, is essential for the correct assembly and/or functioning of this enzyme since yeast cells lacking it are not able to grow on nonfermentable carbon sources. Chemical cross-links between subunit h and subunit 4 have previously been shown, suggesting that subunit h is a component of the peripheral stalk of the F1Fo ATP synthase. The construction of cysteine-containing subunit h mutants and the use of bismaleimide reagents provided insights into its environment. Cross-links were obtained between subunit h and subunits alpha, f, d, and 4. These results and secondary structure predictions allowed us to build a structural model and to propose that this subunit occupies a central place in the peripheral stalk between the F1 sector and the membrane. In addition, subunit h was found to have a stoichiometry of one in the F1Fo ATP synthase complex and to be in close proximity to another subunit h belonging to another F1Fo ATP synthase in the inner mitochondrial membrane. Finally, functional characterization of mitochondria from mutants expressing different C-terminal shortened subunit h suggested that its C-terminal part is not essential for the assembly of a functional F1Fo ATP synthase.     

Accumulation of newly synthesized F1 in vivo in arabidopsis mitochondria provides evidence for modular assembly of the plant F1Fo ATP synthase

F(1) subcomplex in mitochondrial samples is often considered to be a breakage product of the F(1)F(O) ATP synthase during sample handling and electrophoresis. We have used a progressive (15)N incorporation strategy to investigate the plant F(1)F(O) ATP synthase assembly model and the apparently free F(1) in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F(1) in the inner membrane and matrix had a relatively higher (15)N incorporation rate than corresponding subunits in intact membrane F(1)F(O). This demonstrates that free F(1) was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [(35)S]Met-labeled F(1) subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F(1) assembly intermediates. The different (15)N incorporation rate in matrix F(1), inner membrane F(1) and intact F(1)F(O) demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F(1) in mitochondria are discussed.     

Turnover of ATP synthase subunits in F1-depleted HeLa and yeast cells

Mitochondrial translation of the Saccharomyces cerevisiae Atp6p subunit of F(1)-F(0) ATP synthase is regulated by the F(1) ATPase. Here we show normal expression of Atp6p in HeLa cells depleted of the F(1) β subunit. Instead of being translationally down-regulated, HeLa cells lacking F(1) degrade Atp6p, thereby preventing proton leakage across the inner membrane. Mammalian mitochondria also differ in the way they minimize the harmful effect of unassembled F(1) α subunit. While yeast mutants lacking β subunit have stable aggregated F(1) α subunit in the mitochondrial matrix, the human α subunit is completely degraded in cells deficient in F(1) β subunit. These results are discussed in light of the different properties of the proteins and environments in which yeast and human mitochondria exist.     

Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology

Within the yeast mitochondrial ATP synthase, subunit h is a small nuclear encoded protein belonging to the so-called "peripheral stalk" that connects the enzyme catalytic F(1) component to the mitochondrial inner membrane. This study examines the role of subunit h in ATP synthase function and assembly using a regulatable, doxycycline-repressible subunit h gene to overcome the strong instability of the mtDNA previously observed in strains lacking the native subunit h gene. Yeast cells expressing less than 3% of subunit h, but still containing intact mitochondrial genomes, grew poorly on respiratory substrates because of a major impairment of ATP synthesis originating from the ATP synthase, whereas the respiratory chain complexes were not affected. The lack of ATP synthesis in the subunit h-depleted (deltah) mitochondria was attributed to defects in the assembly/stability of the ATP synthase. A main feature of deltah-mitochondria was a very low content (<6%) in the mitochondrially encoded Atp6p subunit, an essential component of the enzyme proton channel, which was in large part because of a slowing down in translation. Interestingly, depletion of subunit h resulted in dramatic changes in mitochondrial cristae morphology, which further supports the existence of a link between the ATP synthase and the folding/biogenesis of the inner mitochondrial membrane.     

Location of subunit d in the peripheral stalk of the ATP synthase from Saccharomyces cerevisiae

ATP synthase from Saccharomyces cerevisiae is an approximately 600 kDa membrane protein complex. The enzyme couples the proton motive force across the mitochondrial inner membrane to the synthesis of ATP from ADP and inorganic phosphate. The peripheral stalk subcomplex acts as a stator, preventing the rotation of the soluble F 1 region relative to the membrane-bound F O region during ATP synthesis. Component subunits of the peripheral stalk are Atp5p (OSCP), Atp4p (subunit b), Atp7p (subunit d), and Atp14p (subunit h). X-ray crystallography has defined the structure of a large fragment of the bovine peripheral stalk, including 75% of subunit d (residues 3-123). Docking the peripheral stalk structure into a cryo-EM map of intact yeast ATP synthase showed that residue 123 of subunit d lies close to the bottom edge of F 1. The 37 missing C-terminal residues are predicted to either fold back toward the apex of F 1 or extend toward the membrane. To locate the C terminus of subunit d within the peripheral stalk of ATP synthase from S. cerevisiae, a biotinylation signal was fused to the protein. The biotin acceptor domain became biotinylated in vivo and was subsequently labeled with avidin in vitro. Electron microscopy of the avidin-labeled complex showed the label tethered close to the membrane surface. We propose that the C-terminal region of subunit d spans the gap from F 1 to F O, reinforcing this section of the peripheral stalk.     

Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles

Mitochondria are one of the hallmarks of eukaryotic cells, exporting ATP in exchange for cytosolic ADP using ADP/ATP carriers (AAC) located in the inner mitochondrial membrane. In contrast, several evolutionarily important anaerobic eukaryotes lack mitochondria but contain hydrogenosomes, peculiar organelles of controversial ancestry that also supply ATP but, like some fermentative bacteria, make molecular hydrogen in the process. We have now identified genes from two species of the hydrogenosome-containing fungus Neocallimastix that have three-fold sequence repeats and signature motifs that, along with phylogenetic analysis, identify them as AACs. When expressed in a mitochondrial AAC- deficient yeast strain, the hydrogenosomal protein was correctly targeted to the yeast mitochondria inner membrane and yielded mitochondria able to perform ADP/ATP exchange. Characteristic inhibitors of mitochondrial AACs blocked adenine nucleotide exchange by the Neocallimastix protein. Thus, our data demonstrate that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange. These experiments provide some of the strongest evidence yet that yeast mitochondria and Neocallimastix hydrogenosomes are but two manifestations of the same fundamental organelle.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Membrane deformation under local pH gradient: mimicking mitochondrial cristae dynamics

Mitochondria are cell substructures (organelles) critical for cell life, because biological fuel production, the ATP synthesis by oxidative phosphorylation, occurs in them driven by acidity (pH) gradients. Mitochondria play a key role as well in the cell death and in various fatigue and exercise intolerance syndromes. It is clear now that mitochondria present an astonishing variety of inner membrane morphologies, dynamically correlated with their functional state, coupled with the rate of the ATP synthesis, and characteristic for normal as well as for pathological cases. Our work offers some original insights into the factors that determine the dynamical tubular structures of the inner membrane cristae. We show the possibility to induce, by localized proton flow, a macroscopic cristae-like shape remodeling of an only-lipid membrane. We designed a minimal membrane system (GUV) and experimentally showed that the directional modulation of local pH gradient at membrane level of cardiolipin-containing vesicles induces dynamic cristae-like membrane invaginations. We propose a mechanism and theoretical model to explain the observed tubular membrane morphology and suggest the underlying role of cardiolipin. Our results support the hypothesis of localized bioenergetic transduction and contribute to showing the inherent capacity of cristae morphology to become self-maintaining and to optimize the ATP synthesis.     

Two ATP synthases can be linked through subunits i in the inner mitochondrial membrane of Saccharomyces cerevisiae

Cross-linking experiments showed that the supernumerary subunit i is close to the interface between two ATP synthases. These data were used to demonstrate the presence of ATP synthase dimers in the inner mitochondrial membrane of Saccharomyces cerevisiae. A cysteine residue was introduced into the inter-membrane space located C-terminal part of subunit i. Cross-linking experiments revealed a dimerization of subunit i. This cross-linking occurred only with the dimeric form of the enzyme after incubating intact mitochondria with a bis-maleimide reagent, thus indicating an inter-ATP synthase cross-linking, whereas the monomeric form of the enzyme exhibited only an intra-ATP synthase cross-linking with subunit 6, another component of the membranous domain of the ATP synthase.     

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.     

Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria

There is increasing evidence now that F(1)F(0) ATP synthase is arranged in dimers in the inner mitochondrial membrane of several organisms. The dimers are also considered to be the building blocks of oligomers. It was recently found that the monomers in beef and the alga Polytomella ATP synthase dimer make an angle of approximately 40 degrees and approximately 70 degrees, respectively. This arrangement is considered to induce a strong local bending of the membrane. To further understand the packing of dimers into oligomers we performed an electron microscopy analysis of ATP synthase dimers purified from Saccharomyces cerevisiae. Two types of dimers were found in which the angle between the monomers is either approximately 90 degrees or approximately 35 degrees. According to our interpretation, the wide-angle dimers (70-90 degrees) are "true-dimers" whereas the small-angle dimers (35-40 degrees) rather are "pseudo-dimers", which represent breakdown products of two adjacent true dimers in the oligomer. Ultrathin sectioning of intact Polytomella mitochondria indicates that the inner mitochondrial or cristae membrane is folded into lamellae and tubuli. Oligomers of ATP synthase can arrange in a helical fashion in tubular-shaped cristae membranes. These results strongly support the hypothesized role of ATP synthase oligomers in structural determination of the mitochondrial inner membrane.     

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.     

The alpha-subunit of Leishmania F1 ATP synthase hydrolyzes ATP in presence of tRNA

Import of tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania requires the tRNA-dependent hydrolysis of ATP leading to the generation of membrane potential through the pumping of protons. Subunit RIC1 of the inner membrane RNA import complex is a bi-functional protein that is identical to the alpha-subunit of F1F0 ATP synthase and specifically binds to a subset (Type I) of importable tRNAs. We show that recombinant, purified RIC1 is a Type I tRNA-dependent ATP hydrolase. The activity was insensitive to oligomycin, sensitive to mutations within the import signal of the tRNA, and required the cooperative interaction between the ATP-binding and C-terminal domains of RIC1. The ATPase activity of the intact complex was inhibited by anti-RIC1 antibody, while knockdown of RIC1 in Leishmania tropica resulted in deficiency of the tRNA-dependent ATPase activity of the mitochondrial inner membrane. Moreover, RIC1 knockdown extracts failed to generate a membrane potential across reconstituted proteoliposomes, as shown by a rhodamine 123 uptake assay, but activity was restored by adding back purified RIC1. These observations identify RIC1 as a novel form of the F1 ATP synthase alpha-subunit that acts as the major energy transducer for tRNA import.     

Environmental study of subunit i, a F(o) component of the yeast ATP synthase

The topology of subunit i, a component of the yeast F(o)F(1)-ATP synthase, was determined by the use of cysteine-substituted mutants. The N(in)-C(out) orientation of this intrinsic subunit was confirmed by chemical modification of unique cysteine residues with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid. Near-neighbor relationships between subunit i and subunits 6, f, g, and d were demonstrated by cross-link formation following sulfhydryl oxidation or reaction with homobifunctional and heterobifunctional reagents. Our data suggest interactions between the unique membrane-spanning segment of subunit i and the first transmembranous alpha-helix of subunit 6 and a stoichiometry of 1 subunit i per complex. Cross-linked products between mutant subunits i and proteins loosely bound to the F(o)F(1)-ATP synthase suggest that subunit i is located at the periphery of the enzyme and interacts with proteins of the inner mitochondrial membrane that are not involved in the structure of the yeast ATP synthase.     

ATP synthase in slow- and fast-growing mycobacteria is active in ATP synthesis and blocked in ATP hydrolysis direction

ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ? and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.     

A minimum structured adenine nucleotide for ADP/ATP transport in mitochondria

An ADP analogue, i.e. 3H- or 14C-labeled 5'-diphosphate of 6-(beta-D-ribofuranosyl-methyl)-4-pyrimidinamine, was synthesized, the structure of which was deduced from structure-activity studies on the substrate specificity of the nucleotide-binding center of the inner mitochondrial membrane integrated ADP/ATP carrier protein. Bearing only the minimal but substrate-essential recognition structures, minimum structured ADP (msADP) is bound to the cytosol-facing active center and transported by the highly specific carrier system across the inner mitochondrial membrane.