The alpha-subunit of the maize F(1)-ATPase is synthesised in the mitochondrion

The F₁-ATPase complex has been purified from maize (Zea mays L.) mitochondria and shown to consist of five subunits with mol. wts. of 58 000 (α), 56 000 (β), 35 000 (γ), 22 000 (δ) and 8000 (ε). The α-subunit co-migrates on one- and two- dimensional isoelectric focussing-SDS polyacrylamide gels with the major polypeptide synthesised by isolated mitochondria. One-dimensional proteolytic peptide mapping and immunoprecipitation confirms that the α-subunit is a mitochondrial translation product and therefore presumably encoded in mitochondrial DNA. This contrasts with the situation in animal and fungal cells where all five subunits of the F₁-ATPase are encoded by the nuclear genome and synthesised on cytosolic ribosomes.     

Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria

The α subunit of the F₁-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpα, that appears to code for the F₁-ATPase α subunit of Zea mays. The atpα gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize α polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial α subunit and the tobacco chloroplast CF₁ and Escherichia coli α subunits are 54 and 51%, respectively. The origin of the atpα gene is discussed.     

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.     

Purification and Characterization of a Glycerol-Resistant CF(0)-CF(1) and CF(1)-ATPase from the Halotolerant Alga Dunaliella bardawil

The isolation of the chloroplast ATP synthase complex (CF₀-CF₁) and of CF₁ from Dunaliella bardawil is described. The subunit structure of the D. bardawil ATPase differs from that of the spinach in that the D. bardawil α subunit migrates ahead of the β subunit and ε-migrates ahead of subunit II of CF₀ when separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The CF₁ isolated from D. bardawil resembles the CF₁ isolated from Chladmydomonas reinhardi in that a reversible, Mg²⁺-dependent ATPase is induced by selected organic solvents. Glycerol stimulates cyclic photophosphorylation catalyzed by D. bardawil thylakoid membranes but inhibits photophosphorylation catalyzed by spinach thylakoid membranes. Glycerol (20%) also stimulates the rate of ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ proteoliposomes but inhibits the activity with the spinach enzyme. The ethanol-activated, Mg²⁺-ATPase of the D. bardawil CF₁ is more resistant to glycerol inhibition than the octylglucoside-activated, Mg²⁺-ATPase of spinach CF₁ or the ethanol-activated, Mg²⁺-dependent ATPase of the C. reinhardi CF₁. Both cyclic photophosphorylation and ATP-P_i exchange catalyzed by D. bardawil CF₀-CF₁ are more sensitive to high concentrations of NaCl than is the spinach complex.     

Protein Kinase C-α Interaction with F0F1-ATPase Promotes F0F1-ATPase Activity and Reduces Energy Deficits in Injured Renal Cells

We showed previously that active PKC-α maintains F₀F₁-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F₀F₁-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F₀F₁-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and β-subunits, were decreased by injury, an event associated with 54% inhibition of F₀F₁-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F₀F₁-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, β-, and γ-subunits, decreased F₀F₁-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, β-, and γ-subunits of F₀F₁-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F₀F₁-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or β- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, β-, and γ-subunits of F₀F₁-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser¹⁴⁶ on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F₀F₁-ATPase, 2) interacts with α-, β-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F₀F₁-ATPase activity and ATP content after injury in RPTC.     

The epsilon Subunit of the Chloroplast Coupling Factor 1 from Euglena gracilis: A Possible Role in Controlling ATPase Activity

The coupling factor from chloroplasts (CF₁) of Euglena gracilis Z strain is an active ATPase in situ, and its activity cannot be increased by treatment with trypsin or heating as is the case with the CF₁ from other sources. The smallest subunit of CF₁, the ε subunit, is supposed to be involved in controlling the ATPase activity. We have devised a simple technique for rapid and large-scale isolation of this subunit. The ε subunit from Euglena CF₁, although having only a limited inhibitory effect on Euglena CF₁, drastically inhibited the ATPase activity of heat-activated spinach CF₁. The inhibition of spinach CF₁ could be reversed by passage through Sephadex G-50 or by a second heat activation. An antibody to the ε subunit of Euglena CF₁ cross-reacted only weakly with CF₁ from spinach, Sorghum, Kalanchoë, or Anacystis nidulans, but reacted well with whole Euglena CF₁ in addition to its ε subunit. The antibody increased the ATPase activity of Euglena and Anacystis CF₁ and of unactivated or partially activated spinach CF₁. The results suggest that the function of the ε subunit in Euglena CF₁ is similar to its function in CF₁ from other sources. The data also suggest that changes induced in spinach CF₁ by activation involves modifications in subunits other than the ε one.     

Characterization of the Activation of Membrane-Bound and Soluble CF(1) by Thioredoxin

The activation of spinach (Spinacia oleracea) chloroplast coupling factor 1 (CF₁) by thioredoxin (ThR) was characterized using membrane-bound and soluble CF₁. Light generates an electrochemical proton gradient across the thylakoid membrane, which increases the accessibility of the disulfide bond on the γ-subunit of CF₁ to reduced ThR. The proton gradient substantially accelerates the activation of CF₁ compared with thylakoids incubated in the dark with similar concentrations of dithiothreitol and ThR. The interaction of soluble CF₁ with ThR was studied using fluorescent probes. CF₁ in solution, with and without its associated ε-subunit, was labeled at Cys-322 of the γ-subunit with fluoresceinyl maleimide. ThR from Escherichia coli was labeled with eosin isothiocyanate. Labeled ThR and CF₁ showed normal activities. Fluorescence energy transfer between donor fluoresceinyl maleimide and acceptor eosin isothiocyanate, manifested by a quenching of the donor fluorescence, was detected, suggesting that ThR and CF₁ form an intermolecular complex. When the ε-subunit was absent, quenching of donor fluorescence was approximately doubled, indicating that labeled ThR could approach more closely to the γ-subunit of CF₁. The distance between the fluorescent probes on CF₁ and ThR was calculated to be approximately 65 Å when ε-subunit was present and 52 Å when ε was absent. These values are consistent with other distance measurements and energy transfer values reported previously for fluorescent probes on CF₁. Whereas the extent of quenching increased by removal of the ε-subunit, the apparent dissociation constant was unchanged. The quenching effect was reversed when the ε-subunit was added back to the titration mixture. Similarly, the addition of unlabeled ThR decreased donor quenching.     

Purification and Characterization of the Soluble F(1)-ATPase of Oat Root Mitochondria

The properties of the soluble moiety (F₁) of the mitochondrial H⁺-ATPase from oat roots were examined and compared to those of the native mitochondrial membrane-bound enzyme. The chloroform soluble preparation was purified by Sephadex G-200 and DEAE-cellulose chromatography. The purified F₁ preparation contained major polypeptides corresponding to α, β, γ, δ, and ε of apparent molecular mass 58, 55, 35, 22, and 14 kilodaltons, respectively. The purified F₁-ATPase, like the native enzyme, was inhibited by azide (I₅₀ = 10 micromolar), nitrate (I₅₀ = 7-10 millimolar), 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid (I₅₀ = 1-3 micromolar), and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (I₅₀ = 3 micromolar). F₁-ATPase activity was stimulated by bicarbonate but not by chloride. In both the native and the F₁-form of the ATPase, ATP was hydrolyzed in preference to GTP. The results indicate that these properties of the native membrane-bound mitochondrial ATPase have been conserved in the purified F₁. In contrast to the membrane-bound enzyme, the F₁-ATPase was not inhibited by oligomycin or by N,N′-dicyclohexylcarbodiimide. The mitochondrial F₁-ATPase from oat roots is analogous to other known F₁F₀-ATPases.     

Identification of a Novel Isoform of the Chloroplast-Coupling Factor α-Subunit

Studies were conducted to identify a 64-kD thylakoid membrane protein of unknown function. The protein was extracted from chloroplast thylakoids under low ionic strength conditions and purified to homogeneity by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four peptides generated from the proteolytic cleavage of the wheat 64-kD protein were sequenced and found to be identical to internal sequences of the chloroplast-coupling factor (CF₁) α-subunit. Antibodies for the 64-kD protein also recognized the α-subunit of CF₁. Both the 64-kD protein and the 61-kD CF₁ α-subunit were present in the monocots barley (Hordeum vulgare), maize (Zea mays), oat (Avena sativa), and wheat (Triticum aestivum); but the dicots pea (Pisum sativum), soybean (Glycine max Merr.), and spinach (Spinacia oleracea) contained only a single polypeptide corresponding to the CF₁ α-subunit. The 64-kD protein accumulated in response to high irradiance (1000 μmol photons m⁻² s⁻¹) and declined in response to low irradiance (80 μmol photons m⁻² s⁻¹) treatments. Thus, the 64-kD protein was identified as an irradiance-dependent isoform of the CF₁ α-subunit found only in monocots. Analysis of purified CF₁ complexes showed that the 64-kD protein represented up to 15% of the total CF₁ α-subunit.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

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.     

Crystallographic structure of the turbine C-ring from spinach chloroplast F-ATP synthase

In eukaryotic and prokaryotic cells, F-ATP synthases provide energy through the synthesis of ATP. The chloroplast F-ATP synthase (CF₁F_O-ATP synthase) of plants is integrated into the thylakoid membrane via its F_O-domain subunits a, b, b’ and c. Subunit c with a stoichiometry of 14 and subunit a form the gate for H⁺-pumping, enabling the coupling of electrochemical energy with ATP synthesis in the F₁ sector.Here we report the crystallization and structure determination of the c14-ring of subunit c of the CF₁F_O-ATP synthase from spinach chloroplasts. The crystals belonged to space group C2, with unit-cell parameters a=144.420, b=99.295, c=123.51 Å, and β=104.34° and diffracted to 4.5 Å resolution. Each c-ring contains 14 monomers in the asymmetric unit. The length of the c-ring is 60.32 Å, with an outer ring diameter 52.30 Å and an inner ring width of 40 Å.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

The Arabidopsis Protein CONSERVED ONLY IN THE GREEN LINEAGE160 Promotes the Assembly of the Membranous Part of the Chloroplast ATP Synthase

The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) couples ATP synthesis to the light-driven electrochemical proton gradient. The cpATPase is a multiprotein complex and consists of a membrane-spanning protein channel (comprising subunit types a, b, b′, and c) and a peripheral domain (subunits α, β, γ, δ, and ε). We report the characterization of the Arabidopsis (Arabidopsis thaliana) CONSERVED ONLY IN THE GREEN LINEAGE160 (AtCGL160) protein (AtCGL160), conserved in green algae and plants. AtCGL160 is an integral thylakoid protein, and its carboxyl-terminal portion is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins that are thought to function in ATP synthase assembly. Plants without AtCGL160 display an increase in xanthophyll cycle activity and energy-dependent nonphotochemical quenching. These photosynthetic perturbations can be attributed to a severe reduction in cpATPase levels that result in increased acidification of the thylakoid lumen. AtCGL160 is not an integral cpATPase component but is specifically required for the efficient incorporation of the c-subunit into the cpATPase. AtCGL160, as well as a chimeric protein containing the amino-terminal part of AtCGL160 and Synechocystis sp. PCC6803 Atp1, physically interact with the c-subunit. We conclude that AtCGL160 and Atp1 facilitate the assembly of the membranous part of the cpATPase in their hosts, but loss of their functions provokes a unique compensatory response in each organism.The majority of cellular energy is stored in the form of ATP synthesized by the ubiquitous F₁F_o-ATP synthase (F₁ stands for coupling factor 1, F_o for coupling factor o), which is found in the energy-transducing membranes of bacteria, mitochondria, and chloroplasts. The chloroplast F₁F_o-ATP synthase/ATPase (cpATPase) is a rotary motor that is responsible for coupling ATP synthesis (and hydrolysis) to the light-driven electrochemical proton gradient. The cpATPase comprises two physically separable parts, chloroplast coupling factor o (CF_o), which is an integral membrane-spanning proton channel, and chloroplast coupling factor 1 (CF₁), which is located peripheral to the membrane and contains the catalytic site(s) for reversible ATP synthesis (for review, see von Ballmoos et al., 2009). CF_o comprises four different subunit types, designated b (synonymously, I or AtpF), b′ (II or AtpG), c (III or AtpH), and a (IV or AtpI), and contains one each of subunits a, b, and b′ and a ring made up of 14 copies of subunit c. CF₁ comprises five different subunits, α (AtpA), β (AtpB), γ (AtpC), δ (AtpD), and ε (AtpE), and its subunit composition is α₃β₃γδε (for review, see von Ballmoos et al., 2009).The passage of protons through the CF_o motor drives rotation of the ring of c-subunits, which together form a rotor. The c-ring is connected to subunit γ, and rotation of γ causes conformational changes in the catalytic nucleotide-binding sites of the CF₁ motor, resulting in the synthesis and release of ATP (for review, see Okuno et al., 2011). This process is made possible by the fact that CF₁ and CF_o are physically connected by two stalks, a central one containing the ε- and γ-subunits and a peripheral one made up of δ, b, and b′ (for review, see Böttcher and Gräber, 2000; Weber, 2007). There are six nucleotide-binding sites in CF₁, one at each of the αβ-subunit interfaces about halfway along the vertical axis of the hexamer. Three of the sites are located primarily on the β-subunits and are catalytic; the other three are noncatalytic and probably regulatory. While the three-dimensional structure of the α₃β₃ hexamer in chloroplasts has been solved to a resolution of 3.2 Å (Groth and Pohl, 2001), the structure of the entire CF_o has not yet been determined. However, the conformation of the ring-forming part of CF_o from spinach (Spinacia oleracea) chloroplasts has been defined and found to consist of 14 c-units (Vollmar et al., 2009), whereas the c-ring of the ATP synthase from the cyanobacterium Spirulina platensis contains 15 units (Pogoryelov et al., 2009).Similar to other thylakoid multiprotein complexes like PSII and PSI as well as the cytochrome b₆f complex (Cyt b₆f), the assembly of the ATP synthase must be tightly regulated. Moreover, the variable stoichiometry of the constituents of F₁ (three α/β-subunits versus one each of γ, δ, and ε) and F_o (10–15 c-subunits versus one each of a, b, and b′) requires coordination of the expression of the corresponding genes. This is particularly important in eukaryotes, where the genes are located in different compartments, for instance, in the case of the cpATPase, in the plastid (for α, β, ε, a, b, and c) and the nucleus (for b′, γ, and δ).The assembly of ATP synthase has been most extensively studied in Saccharomyces cerevisiae mitochondria, leading to the identification of several factors involved in this process (for review, see Rak et al., 2009). Thus, three proteins in yeast are known to be involved in the assembly of the α₃β₃ hexamer of F₁. Atp11p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1996) and Atp12p (Ackerman and Tzagoloff, 1990a; Wang and Ackerman, 1998) code for mitochondrial proteins that interact with the β- and α-subunits, respectively, to promote their assembly into the oligomeric F₁-ATPase, and the absence of either protein causes the α- and β-subunits to aggregate into insoluble inclusion bodies in the mitochondrial matrix. Lack of the third protein, FORMATION OF MITOCHONDRIAL COMPLEXES1 (Fmc1p), is associated with aggregation of the α- and β-subunits under heat stress, suggesting that Fmc1p is required for correct folding of Atp12p at elevated temperatures (Lefebvre-Legendre et al., 2001). Originally, the c-ring was assumed to form spontaneously (Arechaga et al., 2002), but subsequent studies have indicated that the assembly of this structural component is also a protein-assisted process. Thus, Atp25p is required for both the synthesis of the c-subunit and its oligomerization into a ring structure of the proper size (Zeng et al., 2008). Moreover, Atp10p (Ackerman and Tzagoloff, 1990b), Atp23p (Osman et al., 2007), and OXIDASE ASSEMBLY1 (Oxa1p) (Jia et al., 2007) are involved in F_o assembly in yeast mitochondria.In prokaryotes, two ATP synthase assembly factors have been described in detail. The membrane protein insertase YidC belongs to the Oxa1 family, is required in vitro for the membrane insertion of subunit c, and assists in the formation of the c-ring from monomers (van der Laan et al., 2004; Kol et al., 2008). In bacterial genomes, the atp1/uncI genes typically precede the genes encoding the structural subunits of the F₁F_o-ATP synthase (for review, see Kol et al., 2008). Moreover, in Synechocystis sp. PCC6803, sll1321/atp1 is coordinately expressed with the seven other genes in the ATP synthase operon (Grossman et al., 2010), implying that Sll1321/Atp1 might have a function associated with the ATP synthase. The genes atp1 and uncI code for small proteins; for instance, Synechocystis sp. PCC6803 Sll1321 has 117 amino acids, and Escherichia coli UncI has 130 amino acids. The function of Atp1/UncI has long remained elusive because deletion of uncI in E. coli results merely in a slightly reduced growth yield (Gay, 1984), indicating that the protein is not essential for the formation of the F₁F_o-ATP synthase complex. Similarly, in the alkaliphilic Bacillus pseudofirmus OF4, Atp1/UncI is not absolutely required for ATP synthase function, and a B. pseudofirmus strain deleted for the atp1 gene could still grow nonfermentatively and its purified ATP synthase had a c-ring of normal size (Liu et al., 2013). Recently, a hybrid F₁F_o (F₁ from Bacillus PS3 and F_o from Propionigenium modestum) was expressed in E. coli. In this system, P. modestum Atp1/UncI was found to be indispensable for c-ring formation and coupled ATPase activity (Suzuki et al., 2007). Similarly, functional production of the Na⁺ F₁F_o-ATP synthase from Acetobacterium woodii in E. coli required the A. woodii atp1/uncI gene for proper assembly (Brandt et al., 2013). Moreover, because subunit c monomers, as well as assembled c-rings, can be copurified together with P. modestum UncI/Atp1 (Suzuki et al., 2007) and the oligomerization of P. modestum c-subunits into c₁₁-rings is mediated by Atp1/UncI in vitro (Ozaki et al., 2008), Atp1/UncI seems to play a role in c-ring assembly for some bacterial ATP synthases.In plants and green algae, regulation of the biogenesis of the cpATPase is well understood at the level of translation of CF₁ subunits (Drapier et al., 2007). Thus, synthesis of the nucleus-encoded subunit γ is required for sustained translation of the chloroplast-encoded subunit β, which in turn transactivates the translation of chloroplast-encoded subunit α. Translational down-regulation of subunit β or α, when not assembled, involves the 5′ untranslated regions (UTRs) of their own mRNAs, pointing to control at the level of translation initiation. In addition, a negative feedback exerted by α/β assembly intermediates on the translation of subunit β can be released when subunit γ assembles with α₃β₃ hexamers.Our knowledge of the nature of true assembly factors for the cpATPase is scarce. So far, only the ALBINO3 homolog Alb4 protein, which can functionally substitute for YidC in E. coli, has been shown to play a role in the biogenesis of the cpATPase, possibly by stabilizing or promoting the assembly of CF₁ during its attachment to the CF_o portion (Benz et al., 2009). Thus, Alb4-Oxa1p-YidC represents an ATP synthase assembly factor family that is conserved between prokaryotes, yeast, and plants. For the bacterial Atp1/UncI protein, one homolog exists in yeast, Vma21p, which is an integral membrane protein localized to the endoplasmic reticulum and is required for vacuolar H⁺-ATPase biogenesis (Graham et al., 1998).In this study, we have identified and characterized a knockout mutant for Arabidopsis (Arabidopsis thaliana) CGL160, a protein that displays moderate similarity to prokaryotic Atp1/UncI proteins in its C-terminal domain. AtCGL160 is required for the efficient assembly of the cpATPase, but lack of AtCGL160 in Arabidopsis has more severe effects on cpATPase assembly than those reported in the literature for inactivation of its prokaryotic relatives and can be located to the assembly of c-subunits into the membranous subcomplex. AtCGL160 physically interacts with the c-subunit of CF_o, and, interestingly, Atp1 can replace the C-terminal part of AtCGL160 in such interactions, indicating that the function of Atp1 and CGL160 proteins is conserved.     

Optimization of the purification of mitochondrial F1-adenosine triphosphatase

A simple technique of purification of the soluble pig heart mitochondrial F₁-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammonium sulfate fractionation. A high degree of purity, an excellent stability and a good yield are attained after gel filtration through an Ultrogel ACA 34 column equilibrated in the presence of 50% glycerol. The tested properties of the F₁-ATPase prepared by this method are similar to those of the same enzyme extracted by sonication. The enzyme is virtually devoid of tightly bound nucleotides. In addition, some characteristics of the behaviour of the β subunit are shown.     

ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F₁-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F₁-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F₁-ATPase from Bacillus subtilis (BF₁), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF₁. We conclude that the ε subunit relieves BF₁ from MgADP inhibition.     

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α_DP- and β_DP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β_E-subunits. The nucleotide binding site in β_E-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.     

Crystal Structure of the Mg·ADP-inhibited State of the Yeast F1c10-ATP Synthase

The F₁c₁₀ subcomplex of the yeast F₁F₀-ATP synthase includes the membrane rotor part c₁₀-ring linked to a catalytic head, (αβ)₃, by a central stalk, γδϵ. The Saccharomyces cerevisiae yF₁c₁₀·ADP subcomplex was crystallized in the presence of Mg·ADP, dicyclohexylcarbodiimide (DCCD), and azide. The structure was solved by molecular replacement using a high resolution model of the yeast F₁ and a bacterial c-ring model with 10 copies of the c-subunit. The structure refined to 3.43-Å resolution displays new features compared with the original yF₁c₁₀ and with the yF₁ inhibited by adenylyl imidodiphosphate (AMP-PNP) (yF₁(I–III)). An ADP molecule was bound in both β_DP and β_TP catalytic sites. The α_DP-β_DP pair is slightly open and resembles the novel conformation identified in yF₁, whereas the α_TP-β_TP pair is very closed and resembles more a DP pair. yF₁c₁₀·ADP provides a model of a new Mg·ADP-inhibited state of the yeast F₁. As for the original yF₁ and yF₁c₁₀ structures, the foot of the central stalk is rotated by ∼40 ° with respect to bovine structures. The assembly of the F₁ central stalk with the F₀ c-ring rotor is mainly provided by electrostatic interactions. On the rotor ring, the essential cGlu⁵⁹ carboxylate group is surrounded by hydrophobic residues and is not involved in hydrogen bonding.     

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.     

ATPaseTb2, a Unique Membrane-bound FoF1-ATPase Component,Is Essential in Bloodstream and Dyskinetoplastic Trypanosomes

In the infectious stage of Trypanosoma brucei, an important parasite of humans and livestock, the mitochondrial (mt) membrane potential (Δψ_m) is uniquely maintained by the ATP hydrolytic activity and subsequent proton pumping of the essential F_oF₁-ATPase. Intriguingly, this multiprotein complex contains several trypanosome-specific subunits of unknown function. Here, we demonstrate that one of the largest novel subunits, ATPaseTb2, is membrane-bound and localizes with monomeric and multimeric assemblies of the FoF1-ATPase. Moreover, RNAi silencing of ATPaseTb2 quickly leads to a significant decrease of the Δψ_m that manifests as a decreased growth phenotype, indicating that the F_oF₁-ATPase is impaired. To further explore the function of this protein, we employed a trypanosoma strain that lacks mtDNA (dyskinetoplastic, Dk) and thus subunit a, an essential component of the proton pore in the membrane F_o-moiety. These Dk cells generate the Δψ_m by combining the hydrolytic activity of the matrix-facing F₁-ATPase and the electrogenic exchange of ATP⁴- for ADP³- by the ATP/ADP carrier (AAC). Surprisingly, in addition to the expected presence of F1-ATPase, the monomeric and multimeric F_oF₁-ATPase complexes were identified. In fact, the immunoprecipitation of a F₁-ATPase subunit demonstrated that ATPaseTb2 was a component of these complexes. Furthermore, RNAi studies established that the membrane-bound ATPaseTb2 subunit is essential for maintaining normal growth and the Δψ_m of Dk cells. Thus, even in the absence of subunit a, a portion of the F_oF₁-ATPase is assembled in Dk cells.