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.     

Dimer ribbons of ATP synthase shape the inner mitochondrial membrane

ATP synthase converts the electrochemical potential at the inner mitochondrial membrane into chemical energy, producing the ATP that powers the cell. Using electron cryo-tomography we show that the ATP synthase of mammalian mitochondria is arranged in long approximately 1-microm rows of dimeric supercomplexes, located at the apex of cristae membranes. The dimer ribbons enforce a strong local curvature on the membrane with a 17-nm outer radius. Calculations of the electrostatic field strength indicate a significant increase in charge density, and thus in the local pH gradient of approximately 0.5 units in regions of high membrane curvature. We conclude that the mitochondrial cristae act as proton traps, and that the proton sink of the ATP synthase at the apex of the compartment favours effective ATP synthesis under proton-limited conditions. We propose that the mitochondrial ATP synthase organises itself into dimer ribbons to optimise its own performance.     

Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to approximately 35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.     

Single copies of subunits d, oligomycin-sensitivity conferring protein, and b are present in the Saccharomyces cerevisiae mitochondrial ATP synthase

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14-18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case.     

Overexpression of a mitochondrial ATP synthase small subunit gene (AtMtATP6) confers tolerance to several abiotic stresses in Saccharomyces cerevisiae and Arabidopsis thaliana

Mitochondrial F(1)F(0)-ATPase is a key enzyme in plant metabolism, providing cells with ATP that uses the transmembrane electrochemical proton gradient to drive synthesis of ATP. A 6 kDa protein (At3g46430) has been previously purified from Arabidopsis thaliana mitochondrial F(1)F(0)-ATPase. In this study, the gene (AtMtATP6; GenBank accession no. AK117680) encoding this protein was isolated from Arabidopsis and characterized. Northern blot analyses showed that the expression of AtMtATP6 gene in Arabidopsis suspension-cultured cells was induced by several abiotic stresses from salts, drought, and cold. Over-expression of AtMtATP6 gene in transgenic yeast and Arabidopsis plants increased the resistance to salts, drought, oxidative and cold stresses. Taken together, our data raise the possibility that induction of the F(1)F(0)-ATPase plays a role in stress tolerance.     

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.     

Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits

Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae , required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.     

Identity problems concerning subunits of the membrane factor of the mitochondrial ATPase of Saccharomyces cerevisiae

As confirmed by fingerprint analysis, high-resolution acrylamide gel electrophoresis identifies subunits of the mitochondrial ATPase complex directly at the level of Coomassie-blue-stained yeast mitochondria. Using this technique, the following results were obtained. 1. Three classes of subunits have been found in the ATPase complex. (a) The classical F1 peptides of cytoribosomal origin (57, 53 and 30.5 kDa). (b) Two peptides of mitoribosomal origin with high cycloheximide-resistant label: one of 7.8 kDa (the OLII gene product), the second of 20 kDa (the OLI2 gene product). Neither of these peptides is readily stained by Coomassie blue in the purified ATPase complex, (c) Four Coomassie-blue-stained peptides (27.5, 25, 23.5 and 22.5 kDa), the last one being subunit 6, which (like class b) functionally qualify as subunits of the membrane factor of the ATPase complex. As shown by labeling with 35SO24- and 3H-labeled amino acids, their biosynthesis is only very slightly cycloheximide-resistant, although it is submitted to coordinate regulation distinct from that of the class F1 peptides. 2. Consequently it was shown that the OLI2 gene product, a 20-kDa peptide with high cycloheximide-resistant label, which was generally taken to be 'subunit 6' of the ATPase, is not in fact identical to this peptide.     

Isolation and complete amino acid sequence of the mitochondrial ATP synthase epsilon-subunit of the yeast Saccharomyces cerevisiae

All five subunits of yeast mitochondrial F1-ATPase have been isolated by reverse-phase high performance liquid chromatography. This procedure allows micro-preparative purification of all the subunits with 60% recoveries. The complete amino acid sequence of the epsilon-subunit has been established. This has been achieved by the sequence analysis of subnanomole amounts of the intact molecule and that of peptides derived by enzymatic digestion with endoproteinase Arg-C and by chemical cleavage with hydroxylamine. Yeast ATP synthase epsilon-subunit is composed of 61 residues with a calculated molecular mass of 6612 Da. This polypeptide is rather basic since it contains 7 basic residues and 3 acidic residues. This study shows a slight similarity with the bovine epsilon-subunit ATP synthase since there are 16 identical residues.     

Membrane topography of the subunits of ubiquinol-cytochrome-c oxidoreductase of Saccharomyces cerevisiae. The 14-kDa and the 11-kDa subunits face opposite sides of the mitochondrial inner membrane

The topology of the subunits of ubiquinol-cytochrome-c oxidoreductase of the yeast Saccharomyces cerevisiae has been determined using a digitonin/proteinase K assay. With this assay we were able selectively to disrupt the mitochondrial membranes and to identify the subunits which became proteinase-K sensitive after disruption of either the outer or both outer and inner membranes. This approach confirmed previous indications for the localization of the core I protein, cytochrome c1, cytochrome b, the FeS protein and the 17-kDa subunit, while it also provided direct evidence for the site of accessibility to proteinase K of the 14-kDa and 11-kDa subunits. The 14-kDa subunit faces the mitochondrial matrix and the 11-kDa subunit faces the intermembrane space.     

Identification of camelid specific residues in mitochondrial ATP synthase subunits

ATP synthase is an enzyme involved in oxidative phosphorylation from prokaryotic to eukaryotic cells. In mammals it comprises at least 16 subunits from which the mitochondrial encoded ATP6 and ATP8 are essential. Mitochondrial genes variations have been suggested to allow rapid human and animal adaptation to new climates and dietary conditions (Mishmar et al. 2003). Camelidae taxa are uniquely adapted to extremely hot and dry climates of African-Asian territories and to cold and hypoxic environments of the South American Andean region. We sequenced and analyzed ATP6 and ATP8 genes in all camelid species. Based on the available structural data and evolutionary conservation of the deduced proteins we identified features proper of the group. In Old World camels the ATP8, important in the assembly of the F0 complex, showed a number of positively charged residues higher than in the other aligned species. In ATP6 we found the camelid specific substitutions Q47H and I106V that occur in sites highly conserved in other species. We speculate that these changes may have functional importance.     

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.     

Localized energization of the mitochondrial inner membrane by ATP

Studies were made to determine whether the energy-dependent binding of ethidium to the mitochondrial inner membrane reflects the membrane potential or the energization of localized regions of the membrane.The number of binding sites of ethidium in mitochondria energized with ATP was 72 nmol/mg protein and decreased with increase in the amount of the ATPase system (F₁ · F_o) inactivated by oligomycin. These findings clearly show that the energy-dependent binding of ethidium to the mitochondrial inner membrane energized with ATP does not reflect the membrane potential, in good accord with the previous conclusion (Higuti, T., Yokota, M., Arakaki, N., Hattori, A. and Tani, I. (1978) Biochim. Biophys. Acta 503, 211–222), but that ethidium binds to localized regions of the energized membrane that are directly affected by ATPase (F₁), reflecting the localized energization of the membrane by ATP.     

Rows of ATP synthase dimers in native mitochondrial inner membranes

The ATP synthase is a nanometric rotary machine that uses a transmembrane electrochemical gradient to form ATP. The structures of most components of the ATP synthase are known, and their organization has been elucidated. However, the supramolecular assembly of ATP synthases in biological membranes remains unknown. Here we show with submolecular resolution the organization of ATP synthases in the yeast mitochondrial inner membranes. The atomic force microscopy images we have obtained show how these molecules form dimers with characteristic 15 nm distance between the axes of their rotors through stereospecific interactions of the membrane embedded portions of their stators. A different interaction surface is responsible for the formation of rows of dimers. Such an organization elucidates the role of the ATP synthase in mitochondrial morphology. Some dimers have a different morphology with 10 nm stalk-to-stalk distance, in line with ATP synthases that are accessible to IF1 inhibition. Rotation torque compensation within ATP synthase dimers stabilizes the ATP synthase structure, in particular the stator-rotor interaction.     

In vivo and in vitro evidence for a proton leakage through the inner mitochondrial membrane in a mutant of Saccharomyces cerevisiae

Mutants of Saccharomyces cerevisiae were isolated which supported three mutations: two unlinked chromosomic mutations conferring thermosensitivity and cold sensitivity respectively, and a mitochondrial mutation conferring paromomycin sensitivity. When studied on isolated mitochondria, these mutants exhibited low phosphorylation efficiency and great proton permeability of their inner mitochondrial membrane. Experiments were carried out on whole cells: determination of growth rates, cellular yields and cellular respiration, either in the presence of triethyltin, an ATP synthase inhibitor, or in the presence of uncoupler, demonstrating that the proton leakage is actually a physiological phenomenon linked to the cold-sensitive phenotype. Experiments performed on isolated mitochondria confirmed the existence of such a proton leakage.