Features of V-ATPases that distinguish them from F-ATPases

The general structure of F- and V-ATPases is quite similar and they may share a common mechanism of action that involves mechanochemical energy transduction. Both holoenzymes are composed of catalytic sectors, F1 and V1 respectively, and membrane sectors, F(o) and V(o) respectively. Although we assume that a similar mechanism underlies ATP-dependent proton pumping by F- and V-ATPases in eukaryotic cells, the latter cannot catalyze pmf-driven ATP synthesis. The loss of this ability is probably due to a proton slip that is a consequence of alterations in its membrane sector. The major events include gene duplication of the proteolipids and the presence of three distinct proteolipids in each complex.     

Introduction: A Close Look at the Vacuolar ATPase

The vacuolar ATPases (V-type ATPases) are a family of ATP-dependent ion pumps and found in two principal locations, in endomembranes and in plasma membranes. This family of ATPases is responsible for acidification of intracellulare compartments and, in certain cases, ion transport across the plasma membrane of eucaryotic cells. V-ATPases are composed of two distinct domains: a catalytic V₁ sector, in which ATP hydrolysis takes place, and the membrane-embedded sector, V₀, which functions in ion conduction. In the past decade impressive progress has been made in elucidating the properties structure, function and moleculare biology. These knowledge sheds light also on the evolution of V-ATPases and their related families of A-(A₁A₀-ATPase) and F-type (F₁F₀-ATPases)ATPases.     

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.     

Interactions Between Vacuolar H<Superscript>+</Superscript>-ATPases and Microfilaments in Osteoclasts

Vacuolar H⁺-ATPases (V-ATPases) are transported from cytosolic compartments to the ruffled plasma membrane of osteoclasts as they activate to resorb bone. Transport of V-ATPases is essential for bone resorption, and is associated with binding interactions between V-ATPases and microfilaments that are mediated by an actin-binding site in subunit B. This site is contained within 44 amino acids in the amino terminal domain, and requires a sequence motif that resembles an actin-binding motif found in mammalian profilin 1. Small alterations in the profilin-like sequence disrupt the actin-binding activity of subunit B. The interaction between V-ATPases and microfilaments in osteoclasts is regulated in response to changes in phosphatidylinositol-3 kinase activity. During internalization of V-ATPases from the plasma membrane of osteoclasts after a cycle of resorption, V-ATPases bind microfilaments that are in podosomes, dynamic actin-based structures, also present in metastatic cancer cells. Studies are ongoing to establish the physiological role of the microfilament-binding activity of subunit B in osteoclasts and in other cells.     

New insights into structure-function relationships between archeal ATP synthase (A1A0) and vacuolar type ATPase (V1V0)

Adenosine triphosphate, ATP, is the energy currency of living cells. While ATP synthases of archae and ATP synthases of pro- and eukaryotic organisms operate as energy producers by synthesizing ATP, the eukaryotic V-ATPase hydrolyzes ATP and thus functions as energy transducer. These enzymes share features like the hydrophilic catalytic- and the membrane-embedded ion-translocating sector, allowing them to operate as nano-motors and to transform the transmembrane electrochemical ion gradient into ATP or vice versa. Since archaea are rooted close to the origin of life, the A-ATP synthase is probably more similar in its composition and function to the "original" enzyme, invented by Nature billion years ago. On the contrary, the V-ATPases have acquired specific structural, functional and regulatory features during evolution. This review will summarize the current knowledge on the structure, mechanism and regulation of A-ATP synthases and V-ATPases. The importance of V-ATPase in pathophysiology of diseases will be discussed.     

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.     

Subunit Structure, Function, and Arrangement in the Yeast and Coated Vesicle V-ATPases

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.     

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V₀) and a peripherally associated catalytic sector (V₁). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V₀ sector as well as for V₁ attachment. Vma6p has been characterized as a nonintegrally associated V₀ subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.     

Biosynthesis and Regulation of the Yeast Vacuolar H+-ATPase

The yeast V-ATPase is highly similar to V-ATPases of higher organismsand has proved to be a biochemically and genetically accessible model formany aspects of V-ATPase function. Like other V-ATPases, the yeast enzymeconsists of a complex of peripheral membrane proteins, the V₁sector, attached to a complex of integral membrane subunits, theV₀ sector. Multiple pathways for biosynthetic assembly of theenzyme appear to be available to cells containing a full complement ofsubunits and enzyme activity may be further controlled during biosynthesis bya protease activity localized to the late Golgi apparatus. Surprisingly, theassembled V-ATPase is not a static structure. Instead, fully assembledV₁V₀ complexes appear to exist in a dynamic equilibriumwith inactive cytosolic V₁ and membrane-bound V₀complexes and this equilibrium can be rapidly shifted in response to changesin carbon source. The reversible disassembly of the yeast V-ATPase may be anovel regulatory mechanism, common to V-ATPases, that works in vivoin coordination with many other regulatory mechanisms.     

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.     

Iejimalides Show Anti-Osteoclast Activity via V-ATPase Inhibition

Iejimalides (IEJLs), 24-membered macrolides, are potent antitumor compounds, but their molecular targets remain to be revealed. In the course of screening, we identified IEJLs as potent osteoclast inhibitors. Since it is known that osteoclasts are sensitive to vacuolar H⁺-ATPase (V-ATPase) inhibitor, we investigated the effect of IEJLs on V-ATPases. IEJLs inhibited the V-ATPases of both mammalian and yeast cells in situ, and of yeast V-ATPases in vitro. A bafilomycin-resistant yeast mutant conferred IEJL resistance, suggesting that IEJLs bind a site similar to the bafilomycins/concanamycins-binding site. These results indicate that IEJLs are novel V-ATPase inhibitors, and that antitumor and antiosteporotic activities are exerted via V-ATPase inhibition.     

Mechanism of activation and spectral shift of the F-695 emission band in chloroplasts as induced by 1,10-phenanthroline

1. Changes in the fluorescence emission spectrum of chloroplast, at 77 °K, induced by chaotropic reagents and 1,10-phenanthroline, were analyzed.2. Fourth-derivative analysis of the emission spectra identified the exact location of a new band (referred to as “F-700”) at 700 nm and showed that the conversion of F-695 into F-700 does not occur by a gradual red-shift of the F-695 band, but by the appearance of a new band at 700 nm at the expense of an intensity decrease in the F-695 emission.3. F-700 shows two distinct fluorescence characteristics, namely the wavelength of its emission maximum and its intensity, but still retains the principal properties of F-695 such as steep temperature dependence at low temperatures, transient phenomena at 77 °K, and an excitation spectrum of the Photosystem II type. Thus F-700 is concluded to be a modified state of F-695.4. In addition to the compounds of the urea-guanidine class, inorganic anions such as SCN^?, I^? and ClO₄^? were active in the transformation. The specificity and theorder of effectiveness of these reagents indicated that their action is that of chaotropic reagents. Transformation was inhibited by the presence of compounds such as sugars, salts, alcohols and dimethylsulfoxide which seem to affect the activity of water.5. 5-Methyl-1,10-phenanthroline partly substituted for the action of 1,10-phenanthroline, while the other six different derivatives of 1,10-phenanthroline and a few other bifunctional ligands were inactive. The structure-activity relations and the effective concentrations in the transformation differed greatly from those of the inhibition of the electron transport chain, suggesting that the action of 1,10-phenanthroline in the transformation is a yet unrecognized action of this reagent on Photosystem II.6. Transformation was generally observed in chloroplast preparations from 11 different higher plants and two species of algae tested. In Lolium sp. the transformation was partly attained by 1,10-phenanthroline alone.7. From these results, the state of F-695 in chloroplast membranes and the mechanism of transformation into F-700 are discussed.     

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.     

Animal plasma membrane energization by proton-motive V-ATPases

Proton-translocating, vacuolar-type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na⁺ gradients, imposed by Na⁺/K⁺ ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H⁺ V-ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V-ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V-ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na⁺ uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V-ATPases. Awareness of plasma membrane energization by V-ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture. BioEssays 21:637–648, 1999. © 1999 John Wiley & Sons, Inc.     

Structure and function of V-ATPases in osteoclasts: potential therapeutic targets for the treatment of osteolysis

Excessive activity of osteoclasts becomes manifest in many common lytic bone disorders such as osteoporosis, Paget's disease, bone aseptic loosening and tumor-induced bone destruction. Vacuolar proton pump H+-adenosine triphosphatases (V-ATPases), located on the bone-apposed plasma membrane of the osteoclast, are imperative for the function of osteoclasts, and thus are a potential molecular target for the development of novel anti-resorptive agents. To date, the V-ATPases core structure has been well modeled and consists of two distinct functional domains, the V1 (A, B1, B2, C1, C2, D, E1, E2, F, G1, G2, G3, and H subunits) and V0 (a1, a2, a3, a4, d1, d2, c, c' e1, e2 subunits) as well as the accessory subunits ac45 and M8-9. However, the exact configuration of osteoclast specific V-ATPases remains to be established. Inactivation of subunit a3 leads to osteopetrosis in both mice and man because of non-functional osteoclasts that are capable of acidifying the extracellular resorption lacuna. On the other hand, inactivation of subunits c, d1 and ac45 results in early embryonic lethality, indicating that certain subunits, such as a3, are more specific to osteoclast function than others. In osteoclasts, V-ATPases also cooperate with chloride channel protein CLC-7 to acidify the resorption lacuna. In addition, development of V-ATPases inhibitors such as bafilomycin A1, SB 242784 and FR167356 that selectively target osteoclast specific V-ATPases remains a challenge. Understanding the molecular and cellular mechanisms by which specific subunits of V-ATPase regulate osteoclast function might facilitate the development of novel and selective inhibitors for the treatment of lytic bone disorders. This review summarizes recent research developments in V-ATPases with particular emphasis on osteoclast biology.     

Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.     

Saccharomyces cerevisiae Vacuolar H+-ATPase Regulation by Disassembly and Reassembly: One Structure and Multiple Signals

Vacuolar H⁺-ATPases (V-ATPases) are highly conserved ATP-driven proton pumps responsible for acidification of intracellular compartments. V-ATPase proton transport energizes secondary transport systems and is essential for lysosomal/vacuolar and endosomal functions. These dynamic molecular motors are composed of multiple subunits regulated in part by reversible disassembly, which reversibly inactivates them. Reversible disassembly is intertwined with glycolysis, the RAS/cyclic AMP (cAMP)/protein kinase A (PKA) pathway, and phosphoinositides, but the mechanisms involved are elusive. The atomic- and pseudo-atomic-resolution structures of the V-ATPases are shedding light on the molecular dynamics that regulate V-ATPase assembly. Although all eukaryotic V-ATPases may be built with an inherent capacity to reversibly disassemble, not all do so. V-ATPase subunit isoforms and their interactions with membrane lipids and a V-ATPase-exclusive chaperone influence V-ATPase assembly. This minireview reports on the mechanisms governing reversible disassembly in the yeast Saccharomyces cerevisiae, keeping in perspective our present understanding of the V-ATPase architecture and its alignment with the cellular processes and signals involved.     

Subunit C of the Vacuolar-Type ATPase from the Vanadium-Rich Ascidian Ascidia sydneiensis samea Rescued the pH Sensitivity of Yeast vma5 Mutants

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H⁺-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A₁, neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells. Received August 11, 2000; accepted March 5, 2001.     

Plasmalemmal vacuolar-type H+-ATPase in cancer biology

Vacuolar-type H⁺-adenosine triphosphatase (V-ATPase) is one of the most fundamental enzymes in nature. V-ATPases are responsible for the regulation of proton concentration in the intracellular acidic compartments. It has similar structure with the mitochondrial F₀F₁-ATP synthase (F-ATPase).^† The V-ATPases are composed of multiple subunits and have various physiological functions, including membrane and organelle protein sorting, neurotransmitter uptake, cellular degradative processes, and cytosolic pH regulation. The V-ATPases have been involved in multidrug resistance. Recently, plasma membrane V-ATPases have been involved in regulation of extracellular acidity, essential for cellular invasiveness and proliferation in tumor metastasis. The current knowledge regarding the structure and function of V-ATPase and its role in cancer biology is discussed. F in F₀F₁ ATPase is the coupling energy factor.     

Vacuolar H(+)-ATPases: intra- and intermolecular interactions

V-ATPases in eukaryotes are heteromultimeric, H(+)-transporting proteins. They are localized in a multitude of different membranes and energize many different transport processes. Unique features of V-ATPases are, on the one hand, their ability to regulate enzymatic and ion transporting activity by the reversible dissociation of the catalytic V(1) complex from the membrane bound proton translocating V(0) complex and, on the other hand, their high sensitivity to specific macrolides such as bafilomycin and concanamycin from streptomycetes or archazolid and apicularen from myxomycetes. Both features require distinct intramolecular as well as intermolecular interactions. Here we will summarize our own results together with newer developments in both of these research areas.