Probing the rotor subunit interface of the ATP synthase from Ilyobacter tartaricus

The interaction between the c(11)ring and the gammaepsilon complex, forming the rotor of the Ilyobacter tartaricus ATP synthase, was probed by surface plasmon resonance spectroscopy and in vitro reconstitution analysis. The results provide, for the first time, a direct and quantitative assessment of the stability of the rotor. The data indicated very tight binding between the c(11)ring and the gammaepsilon complex, with an apparent K(d) value of approximately 7.4nm. The rotor assembly was primarily dependent on the interaction of the cring with the gammasubunit, and binding of the cring to the free epsilon subunit was not observed. Mutagenesis of selected conserved amino acid residues of all three rotor components (cR45, cQ46, gammaE204, gammaF203 and epsilonH38) severely affected rotor assembly. The interaction kinetics between the gammaepsilon complex and c(11)ring mutants suggested that the assembly of the c(11)gammaepsiloncomplex was governed by interactions of low and high affinity. Low-affinity binding was observed between the polar loops of the cring subunits and the bottom part of the gamma subunit. High-affinity interactions, involving the two residues gammaE204 and epsilonH38, stabilized the holo-c(11)gammaepsilon complex. NMR experiments indicated the acquisition of conformational order in otherwise flexible C- and N-terminal regions of the gamma subunit on rotor assembly. The results of this study suggest that docking of the central stalk of the F(1)complex to the rotor ring of F(o) to form tight, but reversible, contacts provides an explanation for the relative ease of dissociation and reconstitution of F(1)F(o)complexes.     

Purification and Properties of the F1Fo ATPase of Ilyobacter tartaricus, a Sodium Ion Pump

The ATPase of Ilyobacter tartaricus was solubilized from the bacterial membranes and purified. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme revealed the usual subunit pattern of a bacterial F₁F_o ATPase. The polypeptides with apparent molecular masses of 56, 52, 35, 16.5, and 6.5 kDa were identified as the α, β, γ, , and c subunits, respectively, by N-terminal protein sequencing and comparison with the sequences of the corresponding subunits from the Na⁺-translocating ATPase of Propionigenium modestum. Two overlapping sequences were obtained for the polypeptides moving with an apparent molecular mass of 22 kDa (tentatively assigned as b and δ subunits). No sequence could be determined for the putative a subunit (apparent molecular mass, 25 kDa). The c subunits formed a strong aggregate with the apparent molecular mass of 50 kDa which required treatment with trichloroacetic acid for dissociation. The ATPase was inhibited by dicyclohexyl carbodiimide, and Na⁺ ions protected the enzyme from this inhibition. The ATPase was specifically activated by Na⁺ or Li⁺ ions, markedly at high pH. After reconstitution into proteoliposomes, the enzyme catalyzed the ATP-dependent transport of Na⁺, Li⁺, or H⁺. Proton transport was specifically inhibited by Na⁺ or Li⁺ ions, indicating a competition between these alkali ions and protons for binding and translocation across the membrane. These experiments characterize the I. tartaricus ATPase as a new member of the family of FS-ATPases, which use Na⁺ as the physiological coupling ion for ATP synthesis.     

Functional incorporation of chimeric b subunits into F1Fo ATP synthase

F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits.     

Bacillus subtilis F0F1 ATPase: DNA sequence of the atp operon and characterization of atp mutants.

We cloned and sequenced an operon of nine genes coding for the subunits of the Bacillus subtilis F0F1 ATP synthase. The arrangement of these genes in the operon is identical to that of the atp operon from Escherichia coli and from three other Bacillus species. The deduced amino acid sequences of the nine subunits are very similar to their counterparts from other organisms. We constructed two B. subtilis strains from which different parts of the atp operon were deleted. These B. subtilis atp mutants were unable to grow with succinate as the sole carbon and energy source. ATP was synthesized in these strains only by substrate-level phosphorylation. The two mutants had a decreased growth yield (43 and 56% of the wild-type level) and a decreased growth rate (61 and 66% of the wild-type level), correlating with a twofold decrease of the intracellular ATP/ADP ratio. In the absence of oxidative phosphorylation, B. subtilis increased ATP synthesis through substrate-level phosphorylation, as shown by the twofold increase of by-product formation (mainly acetate). The increased turnover of glycolysis in the mutant strain presumably led to increased synthesis of NADH, which would account for the observed stimulation of the respiration rate associated with an increase in the expression of genes coding for respiratory enzymes. It therefore appears that B. subtilis and E. coli respond in similar ways to the absence of oxidative phosphorylation.     

Ecto-F1Fo ATP synthase/F1 ATPase: metabolic and immunological functions

PURPOSE OF REVIEW: Until recently, F1Fo ATP synthase expression was believed to be strictly confined to mitochondria where it generates most cellular ATP. This paper reviews the recent evidence for an extra-mitochondrial expression of its components by immunofluorescence, biochemistry and proteomics studies. It discusses its possible implications in an ecto-nucleotide metabolism and its pathophysiological role in normal and tumoral cells. RECENT FINDINGS: F1Fo ATP synthase components have been identified as cell-surface receptors for apparently unrelated ligands in the course of studies carried out on angiogenesis, lipoprotein metabolism, innate immunity, hypertension, or regulation of food intake. SUMMARY: F1Fo ATP synthase is expressed on endothelial cells where it binds angiostatin, regulates surface ATP levels, and modulates endothelial cell proliferation and differentiation. Through binding of apolipoprotein A-I, a similar complex, expressed on hepatocytes, regulates lipoprotein internalization. On tumors, it is recognized in association with apolipoprotein A-I by the antigen receptor of circulating cytotoxic lymphocytes of the gammadelta subtype and thus promotes an innate tumor cell recognition and lysis. It binds enterostatin on brain cells. Biochemistry and proteomics studies indicate an enrichment of F1Fo components in lipid rafts selectively with some other mitochondrial proteins, suggesting intracellular traffic connections between mitochondria and other membrane compartments. Finally, depending on cell type and environment, it can generate ATP or ADP which may transfer a downstream signal to purinergic receptors.     

Ultrafast purification and reconstitution of His-tagged cysteine-less Escherichia coli F1Fo ATP synthase

His-tagged cysteine-less F1Fo ATP synthase from Escherichia coli was purified using Ni-NTA affinity chromatography. During the purification procedure the loss of total ATPase activity did not exceed 50%, and the extent of purification was about 80-fold. The purified enzyme was essentially free of other proteins, was highly active in ATP hydrolysis (75 units/mg at pH 8 and 37 degrees C), and was sensitive to N,N'-dicyclohexylcarbodiimide (70%). Incorporation of F1Fo into soybean liposomes yielded well-coupled and highly active proteoliposomes. The entire procedure, from the disruption of cells by French press to the preparation of proteoliposomes, took only about 8 h. Some improvements in procedures for the estimation of rates of both ATP hydrolysis and ATP-dependent 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching are described.     

Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans

The F1 complex of the ATP synthase of Streptomyces lividans was isolated and purified. The procedure involved the solubilization of F1 from membranes with buffer of low ionic strength in the presence of EDTA, ion-exchange chromatography and gel filtration. The purified F1 complex from S. lividans (SLF1) consists of five subunits alpha, beta, gamma, delta and epsilon with molecular masses of 58,000, 50,000, 36,000, 28,000 and 13,000, respectively and exhibits immunological cross-reactivity with the F1 portion purified from Escherichia coli (ECF1). The enzymatic properties of SLF1 were determined by the use of microtiter-plate-based assay and compared with data obtained for ECF1. ATPase activity of SLF1 (specific activity: 20-30 U/mg) was only observed in the presence of high concentrations of Ca2+ (10mM). Stimulation of the ATPase activity by Mg2+ was not detectable; quite to the contrary, Mg2+ inhibited the Ca(2+)-stimulated activity of SLF1. SLF1 was re-bound to F1-stripped membranes of S. lividans, but not to F1-stripped membrane vesicles of E. coli. In contrast, ECF1 could be cross-reconstituted with F1-stripped membranes of S. lividans; however, a structural but not a functional reconstitution of the hybrid F1Fo complex was observed.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.     

Ectopic F0F1 ATP synthase contains both nuclear and mitochondrially-encoded subunits

Over the past few years, several reports have described the presence of F₀F₁ ATP synthase subunits at the surface of hepatocytes, where the hydrolytic activity of F₁ sector faces outside and triggers HDL endocytosis. An intriguing question is whether the ectopic enzyme has same subunit composition and molecular mass as that of the mitochondrial ATP synthase. Also due to the polar nature of hepatocytes, the enzyme may be localized to a particular cell boundary. Using different methods to prepare rat liver plasma membranes, which have been subjected to digitonin extraction, hr CN PAGE, immunoblotting, and mass spectrometry analysis, we demonstrate the presence of ecto-F₀F₁ complexes which have a similar molecular weight to the monomeric form of the mitochondrial complexes, containing both nuclear and mitochondrially-encoded subunits. This finding makes it unlikely that the enzyme assembles on the plasma membranes, but suggest it to be transported whole after being assembled in mitochondria by still unknown pathways. Moreover, the plasma membrane preparation enriched in basolateral proteins contains much higher amounts of complete and active F₀F₁ complexes, consistent with their specific function to modulate the HDL uptake on hepatocyte surface.     

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     

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

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

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.     

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

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

The Fo subunits of the Escherichia coli F1Fo-ATP synthase are sufficient to form a functional proton pore

The assembly of the Fo sector of the Escherichia coli ATP synthase has been studied using both structural and functional criteria for assembly. Cross-linking E. coli minicell membranes containing only the Fo subunits a, b, and c with dithiobis(succinimidyl propionate) (DSP) produces b2 and c2 dimers that are generated by cross-linking membranes containing the assembled holoenzyme. Five plasmids carrying the genes specifying the Fo polypeptides in a bacterial strain lacking all of the unc (ATP synthase) genes show a good correlation between Fo function and the amount of the membrane-bound Fo polypeptides. In this report we revise a conclusion reached previously (Klionsky, D.J., Brusilow, W.S.A., and Simoni, R.D. (1983) J. Biol. Chem. 258, 10136-10143) and present evidence that the Fo subunits alone are sufficient to assemble a functional proton pore.     

Reconstitution of Fo of the sodium ion translocating ATP synthase of Propionigenium modestum from its heterologously expressed and purified subunits.

The atpB and atpF genes of Propionigenium modestum were cloned as His-tag fusion constructs and expressed in Escherichia coli. Both recombinant subunits a and b were purified via Ni(2+) chelate affinity chromatography. A functionally active Fo complex was reassembled in vitro from subunits a, b and c, and incorporated into liposomes. The F(o) liposomes catalysed (22)Na(+) uptake in response to an inside negative potassium diffusion potential, and the uptake was prevented by modification of the c subunits with N,N'-dicyclohexylcarbodiimide (DCCD). In the absence of a membrane potential the Fo complexes catalysed (22)Na(+)(out)/Na(+)(in)-exchange. After F(1) addition the F(1)F(o) complex was formed and the holoenzyme catalysed ATP synthesis, ATP dependent Na(+) pumping, and ATP hydrolysis, which was inhibited by DCCD. Functional F(o) hybrids were reconstituted with recombinant subunits a and b from P. modestum and c(11) from Ilyobacter tartaricus. These Fo hybrids had Na(+) translocation activities that were not distinguishable from that of P. modestum F(o).