Evolution of organellar proton-ATPases.

Proton ATPases function in biological energy conversion in every known living cell. Their ubiquity and antiquity make them a prime source for evolutionary studies. There are two related families of H(+)-ATPases; while the family of F-ATPases function in eubacteria chloroplasts and mitochondria, the family of V-ATPases are present in archaebacteria and the vacuolar system of eukaryotic cells. Sequence analysis of several subunits of V- and F-ATPases revealed several of the important steps in their evolution. Moreover, these studies shed light on the evolution of the various organelles of eukaryotes and suggested some events in the evolution of the three kingdoms of eubacteria, archaebacteria and eukaryotes.     

Subunit Composition, Structure, and Distribution of Bacterial V-Type ATPases

The overall structure of V-ATPase complexes resembles that of F-type ATPases, but the stalk region is different and more complex. Database searches followed by sequence analysis of the five water-soluble stalk region subunits C–G revealed that (i) to date V-ATPases are found in 16 bacterial species, (ii) bacterial V-ATPases are closer to archaeal A-ATPases than to eukaryotic V-ATPases, and (iii) different groups of bacterial V-ATPases exist. Inconsistencies in the nomenclature of types and subunits are addressed. Attempts to assign subunit positions in V-ATPases based on biochemical experiments, chemical cross-linking, and electron microscopy are discussed. A structural model for prokaryotic and eukaryotic V-ATPases is proposed. The prokaryotic V-ATPase is considered to have a central stalk between headpiece and membrane flanked by two peripheral stalks. The eukaryotic V-ATPases have one additional peripheral stalk.     

Three-dimensional structure of the intact Thermus thermophilus H+-ATPase/synthase by electron microscopy

ATPases are unique rotary motors that are essential to all living organisms because of their role in energy interconversion. A three-dimensional reconstruction of the intact H+-ATPase/synthase from Thermus thermophilus has revealed the presence of two interconnected peripheral stalks, a well-defined central stalk, and a hexagonally shaped hydrophobic domain. The peripheral stalks are each attached to the water soluble sector at a noncatalytic subunit interface and extend down toward the membrane where they interact with a strong elongated tube of density that runs parallel to the membrane and connects the two stalks. The central stalk is well resolved, especially with respect to its interaction with a single catalytic subunit giving rise to an asymmetry comparable to that identified in F-ATPases. The hexagonal shape of the membrane domain might suggest the presence of 12 proteolipids arranged as dimers, analogous to the proposed arrangement in the related eukaryotic V-ATPases.     

Detecting Changes in the Functional Constraints of Paralogous Genes

We describe a new procedure to determine whether regional alterations in the evolutionary constraints imposed on paralogous proteins have occurred. We used as models the A and B (alternatively called α and β) subunits of V/F/A-ATPases, originated by a gene duplication more than 3 billion years ago. Changes associated to three major splits (eubacteria versus Archaea-eukaryotes; Archaea versus eukaryotes; and among free-living bacteria and symbiotic mitochondria) were studied. Only in the first case, when we compared eubacterial or mitochondrial F-ATPases versus eukaryotic vacuolar V-ATPases or archaeal A-ATPases, constraint changes were observed. Modifications in the degree of regional constraining were not detected for the other two types of comparisons (V-ATPases versus A-ATPases and within F-ATPases, respectively). When the rates of evolution of the two subunits were compared, it was found that F-ATPases regulatory subunits evolved faster than catalytic subunits, but the opposite was true for A- and V-ATPases. Our results suggest that, even for universal and essential proteins, selective constraints may be occasionally altered. On the other hand, in some cases no changes were detected after periods of more than 2.2 billion years. Received: 24 February 2000 / Accepted: 12 August 2000     

Organellar proton-ATPases.

Proton pumps that belong to the families of F-ATPases and V-ATPases operate without the formation of a phosphorylated intermediate and contain several subunits grouped into distinct catalytic and membrane sectors. Recent studies on the structure and molecular biology of V-ATPases shed light not only on the structure-function relations between the two families, but also on their evolution in all organisms.     

Elucidation of the stator organization in the V-ATPase of Neurospora crassa

V-ATPases are membrane protein complexes that pump protons in the lumen of various subcellular compartments at the expense of ATP. Proton pumping is done by a rotary mechanism that requires a static connection between the membrane pumping domain (V(0)) and the extrinsic catalytic head (V(1)). This static connection is composed of several known subunits of the V-ATPase, but their location and topological relationships are still a matter of controversy. Here, we propose a model for the V-ATPase of Neurospora crassa on the basis of single-particle analysis by electron microscopy. Comparison of the resulting map to that of the A-ATPase from Thermus thermophilus allows the positioning of two subunits in the static connecting region that are unique to eukaryotic V-ATPases (C and H). These two subunits seem to be located on opposite sides of a semicircular arrangement of the peripheral connecting elements, suggesting a role in stabilizing the stator in V-ATPases.     

Dimeric core structure of modular stator subunit E of archaeal H+ -ATPase

Archaeal H(+)-ATPase (A-ATPase) is composed of an A(1) region that hydrolyzes ATP and an integral membrane part A(0) that conducts protons. Subunit E is a component of peripheral stator(s) that physically links A(1) and A(0) parts of the A-ATPase. Here we report the first crystal structure of subunit E of A-ATPase from Pyrococcus horikoshii OT3 at 1.85 A resolution. The protomer structure of subunit E represents a novel fold. The quaternary structure of subunit E is a homodimer, which may constitute the core part of the stator. To investigate the relationship with other stator subunit H, the complex of subunits EH was prepared and characterized using electrophoresis, mass spectrometry, N-terminal sequencing and circular dichroism spectroscopy, which revealed the polymeric and highly helical nature of the EH complex with equimolar stoichiometry of both the subunits. On the basis of the modular architecture of stator subunits, it is suggested that both cytoplasm and membrane sides of the EH complex may interact with other subunits to link A(1) and A(0) parts.     

The vacuolar (H+)-ATPase: subunit arrangement and in vivo regulation

The V-ATPases are responsible for acidification of intracellular compartments and proton transport across the plasma membrane. They play an important role in both normal processes, such as membrane traffic, protein degradation, urinary acidification, and bone resorption, as well as various disease processes, such as viral infection, toxin killing, osteoporosis, and tumor metastasis. V-ATPases contain a peripheral domain (V₁) that carries out ATP hydrolysis and an integral domain (V₀) responsible for proton transport. V-ATPases operate by a rotary mechanism involving both a central rotary stalk and a peripheral stalk that serves as a stator. Cysteine-mediated cross-linking has been used to localize subunits within the V-ATPase complex and to investigate the helical interactions between subunits within the integral V₀ domain. An essential property of the V-ATPases is the ability to regulate their activity in vivo. An important mechanism of regulating V-ATPase activity is reversible dissociation of the complex into its component V₁ and V₀ domains. The dependence of reversible dissociation on subunit isoforms and cellular environment has been investigated. Qi and Wang contributed equally to this work.     

Structural conservation and functional diversity of V-ATPases

The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H⁺-ATPase that was named V-ATPase. The structure and function of V-ATPases from various sources was extensively studied in the last few years. Several genes encoding subunits of the enzyme were cloned and sequenced. The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes. The two families of proton pumps share structural and functional similarity. They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors. Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium. The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase. They also shed light on the functional assembly of the enzyme in the vacuolar system.     

Structure, function and regulation of the vacuolar (H)-ATPases

The vacuolar (H⁺)-ATPases (or V-ATPases) function to acidify intracellular compartments in eukaryotic cells, playing an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, protein degradation and coupled transport. V-ATPases in the plasma membrane of specialized cells also function in renal acidification, bone resorption and cytosolic pH maintenance. The V-ATPases are composed of two domains. The V₁ domain is a 570-kDa peripheral complex composed of 8 subunits (subunits A–H) of molecular weight 70–13 kDa which is responsible for ATP hydrolysis. The V₀ domain is a 260-kDa integral complex composed of 5 subunits (subunits a–d) which is responsible for proton translocation. The V-ATPases are structurally related to the F-ATPases which function in ATP synthesis. Biochemical and mutational studies have begun to reveal the function of individual subunits and residues in V-ATPase activity. A central question in this field is the mechanism of regulation of vacuolar acidification in vivo. Evidence has been obtained suggesting a number of possible mechanisms of regulating V-ATPase activity, including reversible dissociation of V₁ and V₀ domains, disulfide bond formation at the catalytic site and differential targeting of V-ATPases. Control of anion conductance may also function to regulate vacuolar pH. Because of the diversity of functions of V-ATPases, cells most likely employ multiple mechanisms for controlling their activity.     

Structure,molecular genetics,and evolution of vacuolar H+-ATPases

Proton-ATPases can be divided into three classes denoted as P-, F-, and V-ATPases. The P-ATPases are evolutionarily distinct from the F- and V-type ATPases which have been shown to be related, probably evolved from a common ancestral enzyme. Like F-ATPases, V-ATPases are composed of two distinct structures: a catalytic sector that is hydrophilic in nature and a hydrophobic membrane sector which functions in proton conduction. Recent studies on the molecular biology of vacuolar H⁺-ATPases revealed surprising findings about the evolution of pronon pumps as well as important clues for the evolution of eukaryotic cells.     

Domain Architecture of the Stator Complex of the A1A0-ATP Synthase from Thermoplasma acidophilum

A key structural element in the ion translocating F-, A-, and V-ATPases is the peripheral stalk, an assembly of two polypeptides that provides a structural link between the ATPase and ion channel domains. Previously, we have characterized the peripheral stalk forming subunits E and H of the A-ATPase from Thermoplasma acidophilum and demonstrated that the two polypeptides interact to form a stable heterodimer with 1:1 stoichiometry (Kish-Trier, E., Briere, L. K., Dunn, S. D., and Wilkens, S. (2008) J. Mol. Biol. 375, 673–685). To define the domain architecture of the A-ATPase peripheral stalk, we have now generated truncated versions of the E and H subunits and analyzed their ability to bind each other. The data show that the N termini of the subunits form an α-helical coiled-coil, ∼80 residues in length, whereas the C-terminal residues interact to form a globular domain containingα- and β-structure. We find that the isolated C-terminal domain of the E subunit exists as a dimer in solution, consistent with a recent crystal structure of the related Pyrococcus horikoshii A-ATPase E subunit (Lokanath, N. K., Matsuura, Y., Kuroishi, C., Takahashi, N., and Kunishima, N. (2007) J. Mol. Biol. 366, 933–944). However, upon the addition of a peptide comprising the C-terminal 21 residues of the H subunit (or full-length H subunit), dimeric E subunit C-terminal domain dissociates to form a 1:1 heterodimer. NMR spectroscopy was used to show that H subunit C-terminal peptide binds to E subunit C-terminal domain via the terminal α-helices, with little involvement of the β-sheet region. Based on these data, we propose a structural model of the A-ATPase peripheral stalk.The archaeal ATP synthase (A₁A₀-ATPase),² along with the related F₁F₀- and V₁V₀-ATPases (proton pumping vacuolar ATPases), is a rotary molecular motor (1–4). The rotary ATPases are bilobular in overall architecture, with one lobe comprising the water-soluble A₁, F₁, or V₁ and the other comprising the membrane-bound A₀, F₀, or V₀ domain, respectively. The subunit composition of the A-ATPase is A₃B₃DE₂FH₂ for the A₁ and CIK_x for the A₀. In the A₁ domain, the three A and B subunits come together in an alternating fashion to form a hexamer with a hydrophobic inner cavity into which part of the D subunit is inserted. Subunits D and F comprise the central stalk connection to A₀, whereas two heterodimeric EH complexes are thought to form the peripheral stalk attachment to A₀ seen in electron microscopy reconstructions (5, 6). In the A₀ domain (subunits CIK_x), the K subunits (proteolipids) form a ring that is linked to the central stalk by the C subunit, whereas the cytoplasmic N-terminal domain of the I subunit probably mediates the binding of the EH peripheral stalks to A₀, as suggested for the bacterial A/V-type enzyme (7). Although closer in structure to the proton-pumping V-ATPase, the A-ATPase functions in vivo as an ATP synthase, coupling ion motive force to ATP synthesis, most likely via a similar rotary mechanism as demonstrated for the bacterial A/V- and the vacuolar type enzymes (8, 9). During catalysis, substrate binding occurs sequentially on the three catalytic sites, which are formed predominantly by the A subunits. This is accompanied by conformation changes in the A₃B₃ hexamer that are linked to the rotation of the embedded D subunit together with the rotor subunits F, C, and the proteolipid ring. Each copy of K contains a lipid-exposed carboxyl residue (Asp or Glu), which is transiently interfaced with the membrane-bound domain of I during rotation, thereby catalyzing ion translocation. The EH peripheral stalks function to stabilize the A₃B₃ hexamer against the torque generated during rotation of the central stalk. Much work has been accomplished to elucidate the architectural features of the rotational and catalytic domains, especially in the related F- and V-type enzymes. However, the peripheral stalk complexes in the A- and V-type enzymes remain an area open to question. Although the stoichiometry of the peripheral stalks in the A/V-type and the vacuolar type ATPases have recently been resolved to two and three, respectively (6, 10), the overall structure of the peripheral stalk, including the nature of attachment to the A₃B₃ hexamer and I subunit (called subunit a in the F- and V-ATPase), is not well understood. Some structural information exists in the form of the A-ATPase E subunit C-terminal domain (11), although isolation from its binding partner H may have influenced its conformation.Previously, our lab has characterized the Thermoplasma acidophilum A-ATPase E and H subunits individually and in complex (12). We found that despite their tendency to oligomerize when isolated separately, upon mixing, E and H form a tight heterodimer that was monodisperse and elongated in solution, which is consistent with its role as the peripheral stalk element in the A-ATPase. Here, we have expanded our study of the A-ATPase EH complex through the production of various N- and C-terminal truncation mutants of both binding partners. The data show that the EH complex is comprised of two distinct domains, one that contains both N termini interacting via a coiled-coil and a second that contains both C termini folded in a globular structure containing mixed secondary structure. Consistent with recent crystallographic data for the related A-ATPase from Pyrococcus horikoshii (11), we found that the isolated C-terminal domain of the E subunit exists as a stable homodimer in solution. However, the addition of subunit H or a peptide consisting of the 21 C-terminal residues of the subunit to the dimeric C-terminal domain of subunit E resulted in dissociation of the homodimer with concomitant formation of a 1:1 heterodimer containing the C termini of both polypeptides. This study delineates and characterizes the two domains of the EH complex and will aid in the further exploration of the nature of peripheral stalk attachment and function in the intact A₁A₀-ATPase.     

The C-H Peripheral Stalk Base: A Novel Component in V1-ATPase Assembly

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V₁V_o-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V₁-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE₃G₃H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V₁-ATPase.     

The peripheral stalk of the mitochondrial ATP synthase

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.     

Inventing the dynamo machine: the evolution of the F-type and V-type ATPases

The rotary proton- and sodium-translocating ATPases are reversible molecular machines present in all cellular life forms that couple ion movement across membranes with ATP hydrolysis or synthesis. Sequence and structural comparisons of F- and V-type ATPases have revealed homology between their catalytic and membrane subunits, but not between the subunits of the central stalk that connects the catalytic and membrane components. Based on this pattern of homology, we propose that these ATPases originated from membrane protein translocases, which, themselves, evolved from RNA translocases. We suggest that in these ancestral translocases, the position of the central stalk was occupied by the translocated polymer.     

Evolution of proton pumping ATPases: Rooting the tree of life

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.     

Molecular cloning of cDNA encoding the C subunit of H(+)-ATPase from bovine chromaffin granules

A cDNA encoding subunit C of the V-ATPase from bovine chromaffin granules was cloned and sequenced. The gene encodes a hydrophilic protein of 382 amino acids with a calculated molecular weight of 43,989. Hydropathy plots revealed no apparent transmembrane segments and a rather high helix content was detected. A cDNA encoding most of the C subunit of the V-ATPase of human brain was also cloned and sequenced. The deduced amino acid sequence of this gene is almost identical to the bovine polypeptide with only one change of tyrosine 336 that was replaced by histidine in the human gene. Two polypeptide fragments derived from subunit E of V-ATPase from chromaffin granules were sequenced and found to be identical to the predicted amino acid sequence of this subunit from bovine kidney. These observations support the idea that the amino acid sequences of corresponding subunits from different V-ATPases are highly conserved. Unlike the A and B subunits of V-ATPases, that are homologous to the beta and alpha subunits of F-ATPases, subunits C and E showed no homology with analogous subunits of the F-ATPase family. It is proposed that the addition of the C and gamma subunits to the respective V- and F-ATPases during evolution defined them as two separate families of H(+)-ATPases.     

The stator complex of the A1A0-ATP synthase--structural characterization of the E and H subunits

Archaeal ATP synthase (A-ATPase) is the functional homolog to the ATP synthase found in bacteria, mitochondria and chloroplasts, but the enzyme is structurally more related to the proton-pumping vacuolar ATPase found in the endomembrane system of eukaryotes. We have cloned, overexpressed and characterized the stator-forming subunits E and H of the A-ATPase from the thermoacidophilic Archaeon, Thermoplasma acidophilum. Size exclusion chromatography, CD, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and NMR spectroscopic experiments indicate that both polypeptides have a tendency to form dimers and higher oligomers in solution. However, when expressed together or reconstituted, the two individual polypeptides interact with high affinity to form a stable heterodimer. Analyses by gel filtration chromatography and analytical ultracentrifugation show the heterodimer to have an elongated shape, and the preparation to be monodisperse. Thermal denaturation analyses by CD and differential scanning calorimetry revealed the more cooperative unfolding transitions of the heterodimer in comparison to those of the individual polypeptides. The data are consistent with the EH heterodimer forming the peripheral stalk(s) in the A-ATPase in a fashion analogous to that of the related vacuolar ATPase.     

Structural divergence of the rotary ATPases

The rotary ATPase family of membrane protein complexes may have only three members, but each one plays a fundamental role in biological energy conversion. The F?F(o)-ATPase (F-ATPase) couples ATP synthesis to the electrochemical membrane potential in bacteria, mitochondria and chloroplasts, while the vacuolar H?-ATPase (V-ATPase) operates as an ATP-driven proton pump in eukaryotic membranes. In different species of archaea and bacteria, the A?A(o)-ATPase (A-ATPase) can function as either an ATP synthase or an ion pump. All three of these multi-subunit complexes are rotary molecular motors, sharing a fundamentally similar mechanism in which rotational movement drives the energy conversion process. By analogy to macroscopic systems, individual subunits can be assigned to rotor, axle or stator functions. Recently, three-dimensional reconstructions from electron microscopy and single particle image processing have led to a significant step forward in understanding of the overall architecture of all three forms of these complexes and have allowed the organisation of subunits within the rotor and stator parts of the motors to be more clearly mapped out. This review describes the emerging consensus regarding the organisation of the rotor and stator components of V-, A- and F-ATPases, examining core similarities that point to a common evolutionary origin, and highlighting key differences. In particular, it discusses how newly revealed variation in the complexity of the inter-domain connections may impact on the mechanics and regulation of these molecular machines.     

The membrane domain of the Na+-motive V-ATPase from Enterococcus hirae contains a heptameric rotor

In F-ATPases, ATP hydrolysis is coupled to translocation of ions through membranes by rotation of a ring of c subunits in the membrane. The ring is attached to a central shaft that penetrates the catalytic domain, which has pseudo-3-fold symmetry. The ion translocation pathway lies between the external circumference of the ring and another hydrophobic protein. The H+ or Na+:ATP ratio depends upon the number of ring protomers, each of which has an essential carboxylate involved directly in ion translocation. This number and the ratio differ according to the source, and 10, 11, and 14 protomers have been found in various enzymes, with corresponding calculated H+ or Na+:ATP ratios of 3.3, 3.7, and 4.7. V-ATPases are related in structure and function to F-ATPases. Oligomers of subunit K from the Na+-motive V-ATPase of Enterococcus hirae also form membrane rings but, as reported here, with 7-fold symmetry. Each protomer has one essential carboxylate. Thus, hydrolysis of one ATP provides energy to extrude 2.3 sodium ions. Symmetry mismatch between the catalytic and membrane domains appears to be an intrinsic feature of both V- and F-ATPases.