Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.     

Organization and nucleotide sequence of the atp genes encoding the ATP synthase from alkaliphilic Bacillus firmus OF4

Summary The atp operon from the extreme alkaliphile Bacillus firmus OF4 was cloned and sequenced, and shown to contain genes for the eight structural subunits of the ATP synthase, preceded by a ninth gene predicted to encode a 14 kDa hydrophobic protein. The arrangement of genes is identical to that of the atp operons from Escherichia coli, Bacillus megaterium, and thermophilic Bacillus PS3. The deduced amino acid sequences of the subunits of the enzyme are also similar to their homologs in other ATP synthases, except for several unusual substitutions, particularly in the a and c subunits. These substitutions are in domains that have been implicated in the mechanism of proton translocation through F₀-ATPase, and therefore could contribute to the gating properties of the alkaliphile ATP synthase or its capacity for proton capture.     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

What Is the Role of ε in the Escherichia coli ATP Synthase?

The ATP synthase from Escherichia coli is a prototype of the ATP synthases that are found in many bacteria, in the mitochondria of eukaryotes, and in the chloroplasts of plants. It contains eight different types of subunits that have traditionally been divided into F₁, a water-soluble catalytic sector, and F_o, a membrane-bound ion transporting sector. In the current rotary model for ATP synthesis, the subunits can be divided into rotor and stator subunits. Several lines of evidence indicate that is one of the three rotor subunits, which rotate through 360 degrees. The three-dimensional structure of is known and its interactions with other subunits have been explored by several approaches. In light of recent work by our group and that of others, the role of in the ATP synthase from E. coli is discussed.     

The ATP Synthase atpHAGDC (F1) Operon from Rhodobacter capsulatus

The atpHAGDC operon of Rhodobacter capsulatus, containing the five genes coding for the F₁ sector of the ATP synthase, has been cloned and sequenced. The promoter region has been defined by primer extension analysis. It was not possible to obtain viable cells carrying atp deletions in the R. capsulatus chromosome, indicating that genes coding for ATP synthase are essential, at least under the growth conditions tested. We were able to circumvent this problem by combining gene transfer agent transduction with conjugation. This method represents an easy way to construct strains carrying mutations in indispensable genes.     

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.     

Regulation of Proton Flow and ATP Synthesis in Chloroplasts

The chloroplast ATP synthase is strictly regulated so that it is very active in the light (rates of ATP synthesis can be higher than 5 mol/min/mg protein), but virtually inactive in the dark. The subunits of the catalytic portion of the ATP synthase involved in activation, as well as the effects of nucleotides are discussed. The relation of activation to proton flux through the ATP synthase and to changes in the structure of enzyme induced by the proton electrochemical gradient are also presented. It is concluded that the and subunits of CF₁ play key roles in both regulation of activity and proton translocation.     

The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli

Summary The genes for the eight subunits of the membrane bound ATP synthase of Escherichia coli (Ca⁺⁺, Mg⁺⁺ dependent ATPase, EC 3.6.1.3) were mapped through genetic, physical and functional analysis of specialized transducing phages asn (von Meyenburg et al. 1978). The ATP synthase genes, designated atp ¹, are located at 83.2 min in a segment of the chromosome between 3.5 and 11.3 kb left (counterclockwise) of the origin of replication oriC. The counterclockwise order of the genes for the eight subunits, the expression of which starts from a control region at 3.5 kb-L, was found to be: a, (c, b, ), , , (, ) which in the notation of Downie et al. (1981) reads atpB (E F H) A G (C D). The analysis was in part based on the isolation of new types of atp (unc, Suc^-) mutations. We made use of the fact that specialized transducing phages asn carrying oriC can establish themselves as minichromosomes rendering asnA cells Asn⁺, and that the resulting Asn⁺ cells grow slowly if the asn carries part or all of the atp operon. Selecting for fast growing strains mutations were isolated on the asn which either eliminated atp genes or affected their expression (promoter mutations). The relationship between these atp mutations and the cop mutations of Ogura et al. (1980), which also appear to map in front of or within the atp genes, is discussed.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803

The two operons atp1 and atp2, encoding the subunits of the F_OF₁ ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F_OF₁ ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5 end of atp1, coding for a putative gene product similar to uncI in Escherichia coli.A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.     

Photosynthetic ATPases: purification,properties, subunit isolation and function

Photosynthetic coupling factor ATPases (F₁-ATPases) generally censist of five subunits named , , , and in order of decreasing apparent molecular weight. The isolated enzyme has a molecular weight of between 390,000 to 400,000, with the five subunits probably occurring in a 3:3:1:1:1 ratio. Some photosynthetic F₁ ATPases are inactive as isolated and require treatment with protease, heat or detergent in order to elicit ATPase activity. This activity is sensitive to inhibition by free divalent cations and appears to be more specific for Ca²⁺ vs. Mg²⁺ as the metal ion substrate chelate. This preference for Ca²⁺ can be explained by the higher inhibition constant for inhibition of ATPase activity by free Ca²⁺. Methods for the assay of a Mg-dependent ATPase activity have recently been described. These depend on the presence of organic solvents or detergents in the reaction mixture for assay. The molecular mechanism behind the expression of either the Ca- or Mg-ATPase activities is unknown. F₁-ATPases function to couple proton efflux from thylakoid membranes or chromatophores to ATP synthesis. The isolated enzyme may thus also be assayed for the reconstitution of coupling activity to membranes depleted of coupling factor 1.The functions of the five subunits in the complex have been deduced from the results of chemical modification and reconstitution studies. The subunit is required for the functional binding of the F₁ to the F₀. The active site is probably contained in the (and ) subunit(s). The proposed functions for the and subunits are, however, still matters of controversy. Coupling factors from a wide variety of species including bacteria, algae, C₃ and C₄ plants, appear to be immunologically related. The subunits are the most strongly related, although the and subunits also show significant immunological cross-reactivity. DNA sequence analyses of the genes for the subunit of CF₁ have indicated that the primary sequence of this polypeptide is highly conserved. The genes for the polypeptides of CF₁ appear to be located in two cellular compartments. The , and subunits are coded for on chloroplast DNA, whereas the and subunits are probably nuclear encoded. Experiments involving protein synthesis by isolated chloroplasts or protein synthesis in the presence of inhibitors specific for one or the other set of ribosomes in the cell suggest the existence of pools of unassembled CF₁ subunits. These pools, if they do exist in vivo, probably make up no greater than 1% of the total CF₁ content of the cell.Abbreviations AMP-PNP adenylyl 5 imidodiphosphate - bchl bacteriochlorophyll - CF₁ chloroplast coupling factor 1 - CF₁-CF₀ the chloroplast ATP synthase complex - chl chlorophyll - CvF₁ F₁ from Chromatium vinosum - DCCD N, N-dicyclohexyl carbodiimide - EF₁ the coupling factor 1 isolated from membranes of Escherichia coli - F₀ the hydrophobic, integral membrane portion of the ATP synthase - F₁ coupling factor 1, the extrinsic membrane portion of the ATP synthase - FSBA 5-p-fluorosulfonylbenzoyladenosine - K_d dissociation constant - k_i inhibition constant - k_ii intercept inhibition constant - k_is slope inhibition constant - LS large subunit of ribulose bisphosphate carboxylase - MF₁ mitochondrial coupling factor 1 - M1F₁ F₁ from Mastigocladus laminosus - NBD-Cl 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole - PAGE polyacrylamide gel electrophoresis - RcF₁ F₁ from Rhodopseudomonas capsulata - RpF₁ F₁ from Rhodopseudomonas palustris - RrF₁ F₁ from Rhodospirillum rubrum - RsF₁ F₁ from Rhodopseudomonas sphaeroides - SDS sodium dodecyl sulfate - S1F₁ F₁ from Synechococcus lividus - SpF₁ F₁ from Spirulina platensis - TF₁ F₁ from the thermophilic bacterium, PS3 - tricine N-tris (hydroxymethyl) methyl glycine - tris tris (hydroxymethyl)-amino methane; and - Vmax maximal velocity or maximal activity     

The nucleotide sequence of the atp genes coding for the F0 subunits a, b, c and the F1 subunit delta of the membrane bound ATP synthase of Escherichia coli

Summary The nucleotide sequence has been determined of a 2.500 base pair segment of the E. coli chromosome located between 3.75 and 6.25 kb counterclockwise of the origin of replication at 83.5 min. The sequence contains the atp genes coding for subunits a-, b-, c-, - and part of the -subunit of the membrane bound ATP synthase. The precise start positions of the atpE (c), atpF (b), atpH () and atpA () genes have been defined by comparison of the potential coding sequences with the known amino acid sequence of the c-subunit and the determined N-terminal amino acid sequences of the respective subunits. The genes are expressed in the counterclockwise direction. Their order (counterclockwise) is: atpB (a), atpE (c), atpF (b), atpH () and atpA (). The coding sequences for subunits b and yield polypeptides of 156 and 177 amino acids, respectively, in accordance with the established sizes of these subunits; the one for the c-subunit, the DCCD binding protein, fits perfectly with its known sequence of 79 amino acids. The a-subunit is comprised within a coding sequence yielding a polypeptide of 271 amino acids. It is suggested, however, that the a-subunit (atpB) contains only 201 amino acids, in accordance with its known size, starting from a translation initiation site within the larger coding sequence. The stoichiometry of the F₀ sector subunits is discussed and a model is proposed for the functioning of the highly charged b-subunit of the F₀ sector as the actual proton conductor.Abbreviations atp denotes genes coding for the ATP synthase subunits. This symbol has been proposed as a replacement of unc (von Meyenburg and Hansen 1980; Hansen et al. 1981 b); the alternative symbol pap has also been proposed (Kanazawa et al. 1981). For other genetic symbols see Bachmann and Low (1980) - bp basepair - kb kilobase (pair) - kD kilo Dalton - DCCD N N,N-Dicyclohexylcarbodiimide - SDS Sodium Dodecyl Sulphate     

Cell-free expression and assembly of ATP synthase

Cell-free (CF) expression technologies have emerged as promising methods for the production of individual membrane proteins of different types and origin. However, many membrane proteins need to be integrated in complex assemblies by interaction with soluble and membrane-integrated subunits in order to adopt stable and functionally folded structures. The production of complete molecular machines by CF expression as advancement of the production of only individual subunits would open a variety of new possibilities to study their assembly mechanisms, function, or composition. We demonstrate the successful CF formation of large molecular complexes consisting of both membrane-integrated and soluble subunits by expression of the atp operon from Caldalkalibacillus thermarum strain TA2.A1 using Escherichia coli extracts. The operon comprises nine open reading frames, and the 542-kDa F₁F_o-ATP synthase complex is composed of 9 soluble and 16 membrane-embedded proteins in the stoichiometry α₃β₃γδ?ab₂c₁₃. Complete assembly into the functional complex was accomplished in all three typically used CF expression modes by (i) solubilizing initial precipitates, (ii) cotranslational insertion into detergent micelles or (iii) cotranslational insertion into preformed liposomes. The presence of all eight subunits, as well as specific enzyme activity and inhibition of the complex, was confirmed by biochemical analyses, freeze-fracture electron microscopy, and immunogold labeling. Further, single-particle analysis demonstrates that the structure and subunit organization of the CF and the reference in vivo expressed ATP synthase complexes are identical. This work establishes the production of highly complex molecular machines in defined environments either as proteomicelles or as proteoliposomes as a new application of CF expression systems.     

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.     

Algae cells with deletion of the segment D210-R226 in γ subunit from chloroplast ATP synthase have lower transmembrane proton gradient and grow slowly

The γ-subunits of chloroplast ATP synthases are about 30 amino acids longer than the bacterial or mitochondrial homologous proteins. This additional sequence is located in the mean part of the polypeptide chain and includes in green algae and higher plants two cysteines (Cys198 and Cys204 in Chlamydomonas reinhardtii) responsible for thiol regulation. In order to investigate its functional significance, a segment ranging from Asp-D210 to Arg-226 in the γ-subunit of chloroplast ATP synthase from C. reinhardtii was deleted. This deletion mutant called T2 grows photoautotrophically, but slowly than the parental strain. The chloroplast ATP synthase complex with the mutated γ is assembled, membrane bound, and as CF₀CF₁ displays normal ATPase activity, but photophosphorylation is inhibited by about 20 %. This inhibition is referred to lower light-induced transmembrane proton gradient. Reduction of the proton gradient is apparently caused by a disturbed functional connection between CF₁ and CF₀ effecting a partially leaky ATP synthase complex.     

The b Subunits in the Peripheral Stalk of F1F0 ATP Synthase Preferentially Adopt an Offset Relationship

The peripheral stalk of F₁F₀ ATP synthase is essential for the binding of F₁ to F_O and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b′ subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463–5471). The spatial arrangement of the chimeric b and b′ subunits, abbreviated Tb and Tb′, has been investigated by Cu²⁺-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F₁F₀ ATP synthase complexes. In both cases, disulfides were preferentially formed between Tb_A83C and Tb′_A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.F₁F₀ ATP synthases are found in the inner mitochondrial membrane, the thylakoid membrane of chloroplasts, and the cytoplasmic membrane of bacteria (1–4). These enzymes are responsible for harnessing an electrochemical gradient of protons across the membranes for the synthesis of ATP. In Escherichia coli F₁F₀ ATP synthase, the membrane-embedded F₀ sector is composed of subunits ab₂c₁₀ and a soluble F₁ portion composed of subunits α₃β₃γδϵ. The F₀ sector houses a proton channel located principally in the a subunit, and the flow of protons through F₀ generates torque used to rotate the c₁₀ subunit ring relative to the ab₂ subunits. The F₁ γ and ϵ subunits are bound to the c₁₀ ring and form the central or rotor stalk. Catalytic sites are located at the interfaces of each αβ pair in F₁. The γ subunit extends into the center of the α₃β₃ hexamer, creating an asymmetry in the conformations of the αβ pairs (5). It is the rotation of the γ subunit and the resulting sequential conformational changes in each αβ pair that provides the driving force for the synthesis of ATP at the catalytic sites. The α₃β₃ hexamer is held stationary relative to the rotary stalk by the peripheral stalk consisting of the b₂δ subunits.The peripheral stalk is essential for binding F₁ to F₀ and for coupling proton translocation to catalytic activity (6–8). In the E. coli enzyme, the peripheral stalk is a dimer of identical b subunits. The stalk has been conceptually divided into functional domains called the membrane domain (b_M1-I33), the tether domain (b_E34-A61), the dimerization domain (b_T62-K122), and the F₁-binding domain (b_Q123-L156) (9). Although there is ample evidence of direct protein-protein interactions between b subunits within the membrane, dimerization, and F₁-binding domains, there is remarkably little evidence of tight packing between the b subunits in the tether domain. In fact, electron spin resonance studies suggested that the tether domains of the two b subunits may be separated by more than 20 Å in the F₁F₀ complex (10, 11). Much of what is known about the structure of the stalk has been inferred from analysis of the properties of polypeptides modeling segments of the b subunit. The structure of a peptide modeling the membrane domain, b_M1-E34, has been determined by NMR (12), and a peptide based on the dimerization domain, b_T62-K122, has been determined by x-ray diffraction (13). Both polypeptides assumed α-helical conformations, but neither structure directly revealed b subunit dimerization interactions. Recently, Priya et al. (14) reported a low resolution structure of a b_M22-L156 dimer, but the extended conformation appears to be slightly too long to accurately reflect the dimensions of the peripheral stalk within the F₁F₀ complex.Molecular modeling efforts supported by a variety of biochemical and biophysical experiments have yielded competing right-handed coiled coil (15, 16) and left-handed coiled coil (17, 18) models for the peripheral stalk. The parallel two-stranded left-handed coiled coli is a well known structure characterized by knobs-into-holes packing of the side chains of the two helices that are aligned in-register. An in-register conformation implies that any specific amino acid on the b subunit-subunit interface would occupy a position immediately adjacent to its counterpart in the other b subunit. In contrast, del Rizzo et al. (16) proposed a novel parallel right-handed coiled coil with the helices of the two b subunits offset by approximately one and a half turns of an α helix. This staggered model positions the two identical residues contributed by each of the b subunits in a homodimer into differing environments and at considerable distance from one another. Sequence analyses have been offered in support of both models (16, 18). In terms of experimental support, cross-linking studies of polypeptides have provided evidence that dimer packing could be in-register at many sites starting from residue Ala⁵⁹ and continuing to the carboxyl termini in model b_Y24–L156 dimers and in b_D53–K122 dimers (9, 16). Cross-linking at a few of the carboxyl-proximal positions has been confirmed within intact F₁F₀ ATP synthase complexes (19, 20). Recent electron spin resonance distance measurements on b_24–156 have also been interpreted as support for the in-register arrangement (17, 18). Conversely, work with polypeptides modeling the b subunit has generated evidence favoring a staggered conformation in this section of the dimer (15, 16, 21). In mixtures of dimerization domain polypeptides with cysteines incorporated at different sites, disulfides preferentially formed between positions that were 4–7 residues apart. For example, b_D53–L156 dimers were covalently locked into the offset conformation by the formation of disulfide bridges between cysteines introduced at positions b_A79 and b_R83 as well as b_R83 and b_A90 (15). These staggered dimers were more stable, melted with higher cooperativity, and bound soluble F₁ with higher affinity than b_D53–L156 dimers fixed in the in-register arrangement. Moreover, active and coupled F₁F₀ complexes were assembled with heterodimeric peripheral stalks using b subunits with tether domains varying in length by as many as 14 amino acids (22). These F₁F₀ complexes had peripheral stalks that were by definition out of register, at least within the tether domain.In contrast to the homodimer of identical b subunits observed in the peripheral stalk of E. coli, photosynthetic organisms express two b-like subunits, b and b′, that are thought to form heterodimeric peripheral stalks in F₁F₀ ATP synthase. Previously, we generated heterodimeric peripheral stalks within the E. coli F₁F₀ by constructing chimeric b subunits (23). Segments of the tether and dimerization domains of the E. coli b subunit were replaced with the homologous regions of the Thermosynechococcus elongatus b and b′ subunits. The chimeric subunits formed heterodimeric peripheral stalks that were incorporated into intact, functional F₁F₀ ATP synthase complexes. The most active chimeric enzymes had T. elongatus primary sequences replacing residues b_E39–I86 of the E. coli b subunit. For simplicity, these chimeric subunits will be referred to here as Tb and Tb′.The ability to generate F₁F₀ ATP synthases with Tb/Tb′ heterodimeric peripheral stalks provided a means to investigate the positions of the two subunits in the peripheral stalk. In the present work, we show that the Tb and Tb′ subunits assumed preferred positions relative to one another within the F₁F₀ complex. The staggered conformation appears to be a favored and functional conformation for the peripheral stalk. However, within a population of F₁F₀ complexes, some complexes with peripheral stalks in the in-register conformation are likely to exist.     

Positioning of protein-coding genes on the soybean chloroplast genome

Seven major plastid protein encoding genes were positioned on the soybean chloroplast DNA by heterologous hybridization. These include the genes for the alpha, beta and epsilon subunits of the CF₁ component of ATP synthase (atpA, atpB and atpE respectively), for subunit III of the CF₀ component of ATP synthase (atpH), for the cytochrome f (cytF), for the ‘32 Kd’ thylakoid protein (psbA), and for the large subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcL), all of which map in the large single copy region. The atpB, atpE and rbcL genes are located in the region adjacent to one of the segments of the inverted repeat. The genetic organization of the soybean chloroplast DNA is compared to that of other plastid genomes.