The vacuolar (H + )-ATPases are ATP-dependent proton pumps that function to acidify intracellular compartments and, in some cases, transport protons across the plasma membrane of eukaryotic cells. Intracellular V-ATPases play an important role in such normal physiological processes as receptor-mediated endocytosis, intracellular membrane traffic, pro-hormone processing, protein degradation and the coupled uptake of small molecules, such as neurotransmitters. They also function in the entry of various pathogenic agents, including many envelope viruses, like influenza virus, and toxins, like anthrax toxin. Plasma membrane V-ATPases function in renal pH homeostasis, bone resorption and sperm maturation, as well as in various disease processes, including renal tubular acidosis, osteopetrosis and tumor metastasis. V-ATPases are composed of a peripheral V 1 domain containing eight different subunits that is responsible for ATP hydrolysis and an integral V 0 domain containing six different subunits that translocates protons. In mammalian cells most of the V-ATPase subunits exist in multiple isoforms which are often expressed in a tissue specific manner. Isoforms of one of the V 0 subunits (subunit a) have been shown to possess information that targets the V-ATPase to distinct cellular destinations. Mutations in isoforms of subunit a lead to the human diseases osteopetrosis and renal tubular acidosis. A number of mechanisms are employed to regulate V-ATPase activity in vivo, including reversible dissociation of the V 1 and V 0 domains, control of the tightness of coupling of proton transport and ATP hydrolysis and selective targeting of V-ATPases to distinct cellular membranes. Isoforms of subunit a are involved in regulation both by control of coupling and by selective targeting. This review will begin with a brief introduction to the function, structure and mechanism of the VATPases followed by a discussion of the role of V-ATPase subunit isoforms and the mechanisms involved in regulation of V-ATPase activity. V-ATPase FunctionThe vacuolar (H + )-ATPases (V-ATPases) are a family of ATP-dependent proton pumps localized to a variety of cellular membranes of eukaryotic cells, including endosomes, lysosomes, Golgi-derived vesicles, secretory vesicles and, for some cells, the plasma membrane (1-3). V-ATPases within endosomes function to dissociate internalized ligands, such as low density lipoprotein (LDL), from their receptors, thus facilitating recycling of the receptors to the plasma membrane (4). They are also required for budding of endosomal carrier vesicles that carry internalized ligands from early to late endosomes (5) and for dissociation of lysosomal enzymes from the mannose-6-phosphate receptors that target them from the Golgi to the lysosome (6). Acidic endosomal compartments also provide the entry point for the cytotoxic portions of many envelope viruses, including influenza and Ebola virus, and toxins, such as diphtheria and anthrax toxin (7). The ability of these pathogenic agents to infect or kill ...

Vacuolar-type HThe slower k on ATP than k on ADP and strong Mg-ADP inhibition may contribute to prevent wasteful consumption of ATP under in vivo conditions when the proton motive force collapses. Vacuolar-type Hϩ -ATPases (V-ATPases) 2 are found in a wide range of organisms. V-ATPase in eukaryotes functions as an ATP hydrolysis-driven proton pump that carries out acidification of cellular compartments, such as lysosomes, and extracellular fluid in the case of renal acidification, bone resorption, and tumor metastasis (1). A family of V-ATPases is also found in archaea and some eubacteria (the prokaryotic V-ATPase family) (2-7).3 A major role of the V-ATPase in prokaryotes is to produce ATP, a function performed by F 0 F 1 in eukaryotes and most eubacteria. V-ATPase and F 0 F 1 function by a rotary ATP synthase/ATPase mechanism (1). The hydrophilic domain of both V-ATPase and F 0 F 1 (called V 1 and F 1 , respectively) is responsible for ATP synthesis/hydrolysis and is connected via the central rotor and peripheral stator stalks to the transmembrane domain (V 0 and F 0 , respectively), which functions as an ion channel (1, 8). Although composition and arrangement of subunits differ considerably between V-ATPase and F 0 F 1 , they seem to share a common rotary catalysis mechanism, catalyzing the interconversion of the energy from proton translocation across membranes and the energy of ATP synthesis/hydrolysis through rotation of the central rotor subunits (8). It is thought that rotary catalysis is basically reversible (8, 9). When the transmembrane electrochemical gradient of protons (proton motive force (pmf)) is of sufficient strength, pmf drives rotation of a central rotor shaft to synthesize ATP. In contrast, when pmf is weak, the enzymes become an ATPdriven proton pump that rotates in the opposite direction driven by the energy released by ATP hydrolysis. Indeed, it has been shown that yeast V-ATPase, which functions as a proton pump in vivo, is able to catalyze ATP synthesis when exposed to an electrochemical gradient in vitro (10). In addition the F 1 portion of F 0 F 1 can synthesize ATP when the rotor shaft is forced to rotate in a direction opposite that of ATP hydrolysis (11,12). It is well known that the ATP hydrolysis reaction catalyzed by both V-ATPase and F 0 F 1 is highly regulated by a number of different mechanisms to prevent wasteful ATP consumption (1, 13). One such mechanism is Mg-ADP inhibition, whereby Mg-ADP binds into the catalytic site of the V 1 and F 1 domains and thus inhibits ATP hydrolysis (14 -17). Some enzymes appear to be inhibited irreversibly by these mechanisms (18,19).V-ATPase from the thermophilic bacterium, Thermus thermophilus, has been extensively investigated by biochemical and biophysical methods. Subunit rotation coupled to ATP hydrolysis of the V 1 portion has been visualized using a single mole-

Background: Subunit a of the V-ATPase is thought to contribute to proton-conducting hemichannels within the integral V 0 domain. Results:We have identified transport-important residues and further defined the topology of subunit a. Conclusion: We propose a model for the proton-conducting hemichannels in V 0 . Significance: This represents the first proposed mechanism for proton transport through the V-ATPase.

ATP synthesis by V-ATPase from the thermophilic bacterium Thermus thermophilus driven by the acid-base transition was investigated. The rate of ATP synthesis increased in parallel with the increase in proton motive force (PMF) >110 mV, which is composed of a difference in proton concentration (⌬pH) and the electrical potential differences (⌬⌿) across membranes. The optimum rate of synthesis reached 85 s ؊1 , and the H ؉ /ATP ratio of 4.0 ؎ 0.1 was obtained. ATP was synthesized at a considerable rate solely by ⌬pH, indicating ⌬⌿ was not absolutely required for synthesis. Consistent with the H ؉ /ATP ratio, cryoelectron micrograph images of 2D crystals of the membrane-bound rotor ring of the V-ATPase at 7.0-Å resolution showed the presence of 12 Vo-c subunits, each composed of two transmembrane helices. These results indicate that symmetry mismatch between the rotor and catalytic domains is not obligatory for rotary ATPases/synthases. ATP synthase ͉ rotary motor ͉ membrane protein ͉ bioenergetics ͉ two-dimensional crystal M embers of the F o F 1 and V-ATPase superfamily (rotary ATPase/synthase) use a rotary catalytic mechanism to perform their specific function (1, 2). The F o F 1 mainly catalyzes ATP synthesis in mitochondria, chloroplasts, and aerobic bacteria (3, 4). In contrast, V-ATPases exist in the endomembranes of all eukaryotic cells and in the plasma membrane of some specific eukaryotic cells functioning as a proton pump with a variety of cellular functions (2). The homologues of eukaryotic V-ATPases are also found in the plasma membrane of some bacteria (5, 6). Like the F o F 1 , V-ATPase from the thermophilic eubacterium Thermus thermophilus catalyzes ATP synthesis (7,8). In addition, it has the simplest known subunit structure (Fig. 1a) and is thus an excellent model for studying the mechanism of action of these important molecules. Subunits A and B of V-ATPase are the counterparts of subunits ␤ and ␣ of F o F 1 ATPase. Three copies of each subunit are arranged around the central rotor, which is made of single copies of subunits D and F. The A 3 B 3 DF moiety, termed V 1 , is responsible for the ATP hydrolysis or ATP synthesis reaction. The remaining subunits, V o -a (sometimes referred to as subunit I), V o -c (sometimes referred to as subunit L), and V o -d, E, and G form the V o domain of T. thermophilus V-ATPase (9). The V o -c subunits, which are folded into two transmembrane helices, constitute a membraneembedded oligomeric ring structure (10). The V o -c rotor ring and subunit V o -a form a proton channel, as seen in the c rotor ring of the F o -a subunit, despite low sequence similarity between the proteins.The basic mechanism of ATP synthesis for F o F 1 is well understood, as described below. Briefly, the ring of the F o -c subunit oligomer and ␥-subunits of F 1 comprise the central rotor, and together these rotate as a single body (11). The transmembrane electrochemical potential gradient of proton [⌬ H ϩ ϭ PMF ϫ F (PMF, proton motive force; F, Faraday constant)] drives rotation of the roto...

The integral V 0 domain of the vacuolar (H ؉ )-ATPases (VATPases) provides the pathway by which protons are transported across the membrane. Subunit a is a 100-kDa integral subunit of V 0 that plays an essential role in proton translocation. To better define the membrane topology of subunit a, unique cysteine residues were introduced into a Cys-less form of the yeast subunit a (Vph1p) and the accessibility of these cysteine residues to modification by the membrane permeant reagent N-ethylmaleimide (NEM) and the membrane impermeant reagent polyethyleneglycol maleimide (PEG-mal) in the presence and absence of the protein denaturant SDS was assessed. Thirty Vph1p mutants containing unique cysteine residues were constructed and analyzed. Cysteines introduced between residues 670 and 710 and between 807 and 840 were modified by PEGmal in the absence of SDS, indicating a cytoplasmic orientation. Cysteines introduced between residues 602 and 620 and between residues 744 and 761 were modified by NEM but not PEG-mal in the absence of SDS, suggesting a lumenal orientation. Finally, cysteines introduced at residues 638, 645, 648, 723, 726, 734, and at nine positions between residue 766 and 804 were modified by NEM and PEG-mal only in the presence of SDS, consistent with their presence within the membrane or at a protein-protein interface. The results support an eight transmembrane helix (TM) model of subunit a in which the C terminus is located on the cytoplasmic side of the membrane and provide information on the location of hydrophilic loops separating TM6, 7, and 8.2 are ATP-dependent proton pumps present in a variety of intracellular compartments, including endosomes, lysosomes, Golgi-derived vesicles, and secretory vesicles (1-3). Acidification of intracellular compartments is important for membrane traffic processes, protein degradation and processing, coupled transport of small molecules, and the entry of various pathogens, including envelope viruses like influenza virus and bacterial toxins like anthrax toxin (4). V-ATPases are also present at the plasma membrane of a variety of cell types, including renal-intercalated cells, osteoclasts, macrophages, and neutrophils, epididymal clear cells, insect goblet cells, and certain tumor cells (5-10). Plasma membrane V-ATPases play a critical role in processes such as urinary acidification, bone resorption, sperm maturation, pH homeostasis, coupled transport, and tumor metastasis.The V-ATPases are multisubunit complexes containing two domains (1-3). The V 1 domain is peripheral to the membrane, contains eight different polypeptides (subunits A-H) and carries out ATP hydrolysis. The V 0 domain is membrane-integral, contains subunits a, c, cЈ, cЉ, d, and e (in yeast), and is responsible for proton transport. Both the proteolipid subunits (c, cЈ, and cЉ) and subunit a contain residues that are essential for proton translocation (11, 12). The proteolipid subunits form a ring containing buried glutamic acid residues (13, 14) that are thought to undergo reversible protonation du...