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Key Points The vacuolar (V-)ATPases are ATP-driven proton pumps that function to acidify intracellular compartments and transport protons across the plasma membrane. They function in various normal and disease processes, including membrane trafficking, protein degradation, bone resorption and tumour metastasis. V-ATPases are large, multisubunit complexes that are composed of two domains that operate by a rotary mechanism. ATP hydrolysis in the V 1 domain drives movement of a central rotary complex that, in turn, results in proton translocation through the integral V 0 domain. Assembly of V-ATPases requires a number of dedicated chaperones that reside in the endoplasmic reticulum and that assist in correct assembly of the integral V 0 domain. Targeting of V-ATPases to different cellular destinations is controlled by isoforms of subunit a, a 100-kDa subunit of V 0 . V-ATPase activity is regulated in vivo by a number of mechanisms, including reversible dissociation of the V 1 and V 0 domains. The assembly status of the V-ATPase is in turn dependent on various effectors, including RAVE (regulator of the ATPase of vacuolar and endosomal membranes), aldolase and the cellular environment. Various cells also control the density of V-ATPases in the plasma membrane as an important part of their physiology, including osteoclasts and renal intercalated cells. Insight into how this is accomplished has recently emerged from studies of epididymal cells in the male reproductive tract. The integral V 0 domain of the V-ATPase has been postulated to have a direct role in membrane fusion independently of its effects on acidification. Support for this hypothesis has recently emerged from studies in yeast, Drosophila melanogaster and Caenorhabditis elegans . The vacuolar ATPases are proton pumps that have a central role in maintaining the pH of intracellular compartments and in proton transport across the plasma membrane. Their activity is controlled at many different levels and, increasingly, their dysregulation is being linked to specific diseases. The acidity of intracellular compartments and the extracellular environment is crucial to various cellular processes, including membrane trafficking, protein degradation, bone resorption and sperm maturation. At the heart of regulating acidity are the vacuolar (V-)ATPases — large, multisubunit complexes that function as ATP-driven proton pumps. Their activity is controlled by regulating the assembly of the V-ATPase complex or by the dynamic regulation of V-ATPase expression on membrane surfaces. The V-ATPases have been implicated in a number of diseases and, coupled with their complex isoform composition, represent attractive and potentially highly specific drug targets.

Key Points The V-ATPases are composed of a peripheral domain (V 1 ), which is responsible for ATP hydrolysis, and an integral domain (V 0 ), which is responsible for proton translocation. Electron microscopy has shown the existence of multiple stalks that connect V 1 and V 0 . V-ATPases have an important role in various membrane-transport processes, including both endocytosis and intracellular transport. Moreover, the integral V 0 domain has recently been proposed to have a direct role in membrane fusion. V-ATPases in the plasma membrane of specialized cells function in processes such as renal acidification and bone resorption. Several genetic diseases have now been traced to defects in genes that encode V-ATPase subunits, including renal tubular acidosis and osteopetrosis. The V-ATPases resemble the F-ATPases, which normally function in ATP synthesis, and are believed to operate through a rotary mechanism. Information on subunit interactions and topology and the function of individual residues in activity has begun to emerge from studies using site-directed mutagenesis and covalent modification. The yeast V-ATPase requires a unique set of polypeptides for its assembly in the endoplasmic reticulum. Targeting of the V-ATPase seems to be controlled by signals that are located in the 100-kDa a subunit, although interaction with other cellular proteins, such as PDZ proteins, might be important. Several mechanisms have been proposed to regulate V-ATPase activity, including reversible dissociation, disulphide-bond formation and changes in coupling efficiency. A new ubiquitin-ligase component has recently been shown to have a role in regulated assembly of the V-ATPase. The pH of intracellular compartments in eukaryotic cells is a carefully controlled parameter that affects many cellular processes, including intracellular membrane transport, prohormone processing and transport of neurotransmitters, as well as the entry of many viruses into cells. The transporters responsible for controlling this crucial parameter in many intracellular compartments are the vacuolar (H + )-ATPases (V-ATPases). Recent advances in our understanding of the structure and regulation of the V-ATPases, together with the mapping of human genetic defects to genes that encode V-ATPase subunits, have led to tremendous excitement in this field.

The vacuolar (H + )-ATPases (or V-ATPases) function in the acidification of intracellular compartments in eukaryotic cells. The V-ATPases are multisubunit complexes composed of two functional domains. The peripheral V 1 domain, a 500-kDa complex responsible for ATP hydrolysis, contains at least eight different subunits of molecular weight 70-13 (subunits A-H). The integral V 0 domain, a 250-kDa complex, functions in proton translocation and contains at least five different subunits of molecular weight 100-17 (subunits a-d). Biochemical and genetic analysis has been used to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and coupling of these activities. Several mechanisms have been implicated in the regulation of vacuolar acidification in vivo, including control of pump density, regulation of assembly of V 1 and V 0 domains, disulfide bond formation, activator or inhibitor proteins, and regulation of counterion conductance. Recent information concerning targeting and regulation of V-ATPases has also been obtained.

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V0domain, which contains five different subunits (a, d, c, c′, and c″). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c′, and c″). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.

Eukaryotic cells have evolved a family of ATP-dependent proton pumps known as the vacuolar (H + )-ATPases (or V-ATPases) to regulate the pH of intracellular compartments, the extracellular space, and the cytoplasm. V-ATPases present within intracellular compartments are important for such normal cellular processes as receptor-mediated endocytosis and intracellular membrane traffic, protein processing and degradation and coupled transport of small molecules and ions. They also facilitate the entry of a number of envelope viruses and bacterial toxins, including influenza virus and anthrax toxin. V-ATPases present in the plasma membranes of cells are also important in normal physiology. They facilitate bone resorption by osteoclasts, acid secretion by intercalated cells of the kidney, pH homeostasis in macrophages and neutrophils, angiogenesis by endothelial cells, and sperm maturation and storage in the male reproductive tract. In the insect midgut, they establish a membrane potential used to drive K + secretion. Plasma membrane V-ATPases are especially important in human disease, with genetic defects in V-ATPases expressed in osteoclasts and intercalated cells leading to the diseases osteopetrosis and renal tubule acidosis, respectively. Plasma membrane V-ATPases have also been implicated in tumor cell invasion. V-ATPases are thus emerging as potential targets in the treatment of diseases such as osteoporosis and cancer.

The V-ATPases are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. This review focuses on the the V-ATPases from clathrin-coated vesicles and yeast vacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to that found in endosomes and synaptic vesicles, which function in receptor recycling, intracellular membrane traffic, and neurotransmitter uptake. The yeast vacuolar ATPase functions to acidify the central vacuole and to drive various coupled transport processes across the vacuolar membrane. The V-ATPases are composed of two functional domains. The V1 domain is a 570-kDa peripheral complex composed of eight subunits of molecular weight 70-14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V0 domain is a 260-kDa integral complex composed of five subunits of molecular weight 100-17 kDa (subunits a, d, c, c' and c\") that is responsible for proton translocation. Using chemical modification and site-directed mutagenesis, we have begun to identify residues that play a role in ATP hydrolysis and proton transport by the V-ATPases. A central question in the V-ATPase field is the mechanism by which cells regulate vacuolar acidification. Several mechanisms are described that may play a role in controlling vacuolar acidification in vivo. One mechanism involves disulfide bond formation between cysteine residues located at the catalytic nucleotide binding site on the 70-kDa A subunit, leading to reversible inhibition of V-ATPase activity. Other mechanisms include reversible assembly and dissociation of V1 and V0 domains, changes in coupling efficiency of proton transport and ATP hydrolysis, and regulation of the activity of intracellular chloride channels required for vacuolar acidification.

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.

The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and an integral V(0) domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V(0) domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.

The vacuolar (H+)-ATPases (or V-ATPases) are ATP-dependent proton pumps that function both to acidify intracellular compartments and to transport protons across the plasma membrane. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking, protein processing, and coupled transport. Plasma membrane V-ATPases function in renal acidification, bone resorption, pH homeostasis, and, possibly, tumor metastasis. This review will focus on work from our laboratories on the V-ATPases from mammalian clathrin-coated vesicles and from yeast. The V-ATPases are composed of two domains. The peripheral V1 domain has a molecular mass of 640 kDa and is composed of eight different subunits (subunits A-H) of molecular mass 70-13 kDa. The integral V0 domain, which has a molecular mass of 260 kDa, is composed of five different subunits (subunits a, d, c, c', and c'') of molecular mass 100-17 kDa. The V1 domain is responsible for ATP hydrolysis whereas the V0 domain is responsible for proton transport. Using a variety of techniques, including cysteine-mediated crosslinking and electron microscopy, we have defined both the overall shape of the V-ATPase and the V0 domain as well as the location of various subunits within the complex. We have employed site-directed and random mutagenesis to identify subunits and residues involved in nucleotide binding and hydrolysis, proton translocation, and the coupling of these two processes. We have also investigated the mechanism of regulation of the V-ATPase by reversible dissociation and the role of different subunits in this process.

Clathrin-coated vesicles isolated from calf brain contain an ATP-dependent proton pump. Proton movement was monitored by measuring [14C]methylamine distribution. Addition of Mg2+and ATP to coated vesicles equilibrated with [14C]methylamine resulted in the generation of a 4- to 5-fold concentration gradient, corresponding to a Δ pH of 0.6-0.7 units between the medium and the acidic inside of the coated vesicles. ATP-dependent [14C]methylamine uptake was abolished by the proton ionophore carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) and partially inhibited by the carboxyl reagent N, N′-dicyclohexylcarbodiimide but was unaffected by the Na+,K+-ATPase inhibitors strophanthidin (100 μ M) and vanadate (10 μ M) and the mitochondrial ATPase inhibitors oligomycin (10 μ g/ml) and aurovertin (1 μ g/ml). GTP, but not the nonhydrolyzable analog 5′-adenylylimidodiphosphate, could support [14C]methylamine uptake. Dissipation of the membrane potential with K+and valinomycin resulted in stimulation of [14C]methylamine uptake, whereas both FCCP and valinomycin stimulated the strophanthidin-resistant ATPase activity. These results are consistent with the existence of an electrogenic, ATP-dependent proton pump in clathrin-coated vesicles. This proton pump may play a role in the acidification events that are essential in receptor-mediated endocytosis.