Electric Forces and ATP Synthesis.

The stoichiometry of yeast V(1)-ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V(1)-ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V(1)-ATPase purified from yeast grown on (14)N-containing medium were titrated with defined amounts of (15)N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, (14)N- and (15)N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V(1)-ATPase is A(3)B(3)DE(3)FG(3)H.

The vacuolar membrane ATPase of Neurospora crassa closely resembles the mitochondrial ATPase in its substrate specificity, substrate affinity, and sensitivity to the inhibitor N,N'-dicyclohexylcarbodiimide. Three different mutants with altered mitochondrial ATPase activity, exhibited as 1) resistance to N,N'-dicyclohexylcarbodiimide, 2) enhanced sensitivity to N,N'-dicyclohexylcarbodiimide, and 3) very low specific activity, were found to be unaltered in the vacuolar membrane ATPase. The vacuolar membrane ATPase was similar to the mitochondrial ATPase and approximately 10-fold more sensitive than the plasma membrane ATPase in its sensitivity to the inhibitors 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, 2',3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, and 5'-adenylylimidodiphosphate. By contrast, the vacuolar ATPase resembled the plasma membrane ATPase in its response to quercetin (both 10-fold more sensitive than the mitochondrial ATPase); it was unique in its sensitivity to KNO3. A N,N'-dicyclohexylcarbodiimide-binding protein, migrating between molecular weight markers of 14,400 and 21,500, was identified as a putative component of the vacuolar membrane ATPase. Taken together, these findings support the argument that the vacuolar membrane ATPase is a distinct enzyme, more like the mitochondrial F0F1 ATPase than the plasma membrane ATPase.

ATP-dependent, azide-sensitive rotation of the gamma subunit relative to the alpha3beta3 hexagonal ring of ATP synthase was observed with a single molecule imaging system. Thus, ATP synthase is a rotary motor enzyme, the first ever found.

The H+-translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of Mr approximately equal to 70,000 and approximately equal to 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[14C]ethylmaleimide and 7-chloro-4-nitro[14C]benzo-2-oxa-1,3-diazole, labeled the Mr approximately equal to 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[14C]dicyclohexylcarbodiimide labeled a polypeptide of Mr approximately equal to 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of Mr 5.2 X 10(5), 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochondrial ATPase but strongly crossreacted with antiserum against a polypeptide of Mr approximately equal to 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F0F1 ATPases.

Structure of dimeric ATP synthase from mitochondria: An angular association of monomers induces the strong curvature of the inner membrane

A Model for the Proteolipid Ring and Bafilomycin/Concanamycin-binding Site in the Vacuolar ATPase of Neurospora crassa

The Rotary Motor of Life: Single-Molecule Imaging and Molecular Dynamics Simulation of F1-ATPase

My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

Using vacuolar membranes from Neurospora crassa, we observed that sulfite prevented the loss of vacuolar ATPase activity that otherwise occurred during 36 h at room temperature. Sulfite neither activated nor changed the kinetic behavior of the enzyme. Further, in the presence of sulfite, the vacuolar ATPase was not inhibited by nitrate. We tested the hypothesis that sulfite acts as a reducing agent to stabilize the enzyme, while nitrate acts as an oxidizing agent, inhibiting the enzyme by promoting the formation of disulfide bonds. All reducing agents tested, dithionite, selenite, thiophosphate, dithiothreitol and glutathione, prevented the loss of ATPase activity. On the other hand, all oxidizing agents tested, bromate, iodate, arsenite, perchlorate, and hydrogen peroxide, were potent inhibitors of ATPase activity. The inhibitory effect of the oxidizing agents was specific for the vacuolar ATPase. The mitochondrial ATPase, assayed under identical conditions, was not inhibited by any of the oxidizing agents. Analysis of proteins with two-dimensional gel electrophoresis indicated that nitrate can promote the formation of disufide bonds between proteins in the vacuolar membrane. These data suggest a mechanism to explain why nitrate specifically inhibits vacuolar ATPases, and they support the proposal by Feng and Forgac (Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 13244-13230) that oxidation and reduction of critical cysteine residues may regulate the activity of vacuolar ATPases in vivo.

V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μM at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μM ATP (<<Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 10(6) M(-1) s(-1). At 40 mM ATP (>>Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.

F-type and V-type ATPases couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes and have similarities in structure and mechanism. In both types of enzymes three main parts can be distinguished: headpiece, membrane-bound piece and stalk region. We report on structural details of the membrane sector and stalk region, including the stator, of V-type ATPase from Clostridium fervidus, as determined by electron microscopy. Besides visualization of the stator structure, one of the main findings is that in certain projections the central stalk connecting V1 and V0 makes an angle of about 70° with the membrane. Implications for the subunit arrangement in V-type and F-type ATPase are discussed.

BackgroundThe F- and V-type ATPases are rotary molecular machines that couple translocation of protons or sodium ions across the membrane to the synthesis or hydrolysis of ATP. Both the F-type (found in most bacteria and eukaryotic mitochondria and chloroplasts) and V-type (found in archaea, some bacteria, and eukaryotic vacuoles) ATPases can translocate either protons or sodium ions. The prevalent proton-dependent ATPases are generally viewed as the primary form of the enzyme whereas the sodium-translocating ATPases of some prokaryotes are usually construed as an exotic adaptation to survival in extreme environments.ResultsWe combine structural and phylogenetic analyses to clarify the evolutionary relation between the proton- and sodium-translocating ATPases. A comparison of the structures of the membrane-embedded oligomeric proteolipid rings of sodium-dependent F- and V-ATPases reveals nearly identical sets of amino acids involved in sodium binding. We show that the sodium-dependent ATPases are scattered among proton-dependent ATPases in both the F- and the V-branches of the phylogenetic tree.ConclusionBarring convergent emergence of the same set of ligands in several lineages, these findings indicate that the use of sodium gradient for ATP synthesis is the ancestral modality of membrane bioenergetics. Thus, a primitive, sodium-impermeable but proton-permeable cell membrane that harboured a set of sodium-transporting enzymes appears to have been the evolutionary predecessor of the more structurally demanding proton-tight membranes. The use of proton as the coupling ion appears to be a later innovation that emerged on several independent occasions.ReviewersThis article was reviewed by J. Peter Gogarten, Martijn A. Huynen, and Igor B. Zhulin. For the full reviews, please go to the Reviewers' comments section.

F <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> -ATPase, a rotary motor enzyme, can catalyse ATP hydrolysis in which the central γ-subunit ratates inside the α <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> β <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sub> cylinder. Here, a four-state catalytic model of F <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> -ATPase is studied in which we think that the ATP hydrolysis and synthesis are ATP-dependent and ADP/Pi-dependent, respectively. The results show that the catalytic ratation mechanism of F <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> -ATPase is affected distinctly by the ATP/ADP/Pi concentrations. The model accords well with the expermental observations. Moreover, when the external load exists, the mean rotation rate of F <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> -ATPase is also affected apparently, and the external torque which decreases the mean ratation rate of the F <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">1</sub> motor to zero equals to the constant one which is produced during the ratation of the motor.

&amp;lt;p&amp;gt;The membrane rotary energy-yielding ATPases represent the cornerstone of cellular bioenergetics for all three domains of life. The archaeal ATPases (A-type ATPases) are functionally similar to the eukaryotic and bacterial F-type ATPases that catalyze ATP synthesis using a PMF. However, they are structurally more similar to the vacuolar-type (V-type) ATPases of eukaryotes and some bacteria that function as proton pumps driven by ATP hydrolysis. Significant variation in subunit composition, structure, and mechanism of the archaeal ATPases is thought to confer adaptive advantage in the variety of extreme environments that archaea inhabit.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;The ammonia-oxidizing archaea are recognized to exert primary control of nitrification in the marine environment, are major contributors to soil nitrification, and have a habitat range extending from geothermal systems, to acidic soils and the oceanic abyss. The basis for their remarkable adaptive radiation is obscured by a relatively simple metabolism &amp;amp;#8211; autotrophic growth using ammonia for energy and nitrogen. In this study, we find that their adaptation to acidic habitats and the extreme pressures of the hadal zone of the ocean at depths below 6000 meters is correlated with horizontal transfer of a variant of the energy-yielding ATPase (atp) operon. Whereas the ATPase genealogy of neutrophilic soil and upper ocean pelagic AOA is congruent with their organismal genealogy inferred from concatenated conserved proteins, a common clade of V-type ATPases unites phylogenetically disparate clades of acidophilic and piezophilic species.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;A function of the so-acquired V-ATPases in pumping excessive cytoplasmic protons at low pH is consistent with its increased expression by acid-tolerant AOA with decreasing pH. Consistently, heterologous expression of the thaumarchaeotal V-ATPase significantly increased the growth rate of E.coli at low pH. Additional support for adaptive significance derives from our observation that horizontal transfer is also associated with the adaptive radiation of Micrarchaeota, Parvarchaeota and Marsarchaeota into acidic environments. Their ATPases are affiliated with the acidophilic lineage ATPases of Thermoplasmatales and phylogenetically divergent from the corresponding species tree.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;Another notable finding is that single hadopelagic AOA species contain both A- and V-type ATPases, suggesting that extensive horizontal transfer of atp operons is a&amp;amp;#160;highly active and ongoing process within AOA. The presence of an additional V-type ATPase in hadopelagic AOA may provide fitness advantages in the deep ocean with elevated hydrostatic pressure, as the proposed function of V-ATPase in pumping excessive cytoplasmic protons at high pressure may serve to maintain the cytosolic pH homeostasis in marine AOA.&amp;lt;/p&amp;gt;&amp;lt;p&amp;gt;Taken together, our study provides the first clear evidence of a significant role of horizontal transfer of atp operon in the adaptive radiation of AOA, one of the most successful organisms on Earth, and other archaeal species, spanning the TACK and DPANN superphyla as well as Euryarchaeota phylum.&amp;lt;/p&amp;gt;

Nucleotide Binding States of Subunit A of the A-ATP Synthase and the Implication of P-Loop Switch in Evolution