Rotation,Structure, and Classification of Prokaryotic V-ATPase

The prokaryotic V-type ATPase/synthases (prokaryotic V-ATPases) have simpler subunit compositions than eukaryotic V-ATPases, and thus are useful subjects for studying chemical, physical and structural properties of V-ATPase. In this review, we focus on the results of recent studies on the structure/function relationships in the V-ATPase from the eubacterium Thermus thermophilus. First, we describe single-molecule analyses of T. thermophilus V-ATPase. Using the single-molecule technique, it was established that the V-ATPase is a rotary motor. Second, we discuss arrangement of subunits in V-ATPase. Third, the crystal structure of the C-subunit (homolog of eukaryotic d-subunit) is described. This funnel-shape subunit appears to cap the proteolipid ring in the V₀ domain in order to accommodate the V₁ central stalk. This structure seems essential for the regulatory reversible association/dissociation of the V₁ and the V₀ domains. Last, we discuss classification of the V-ATPase family. We propose that the term prokaryotic V-ATPases should be used rather than the term archaeal-type ATPase (A-ATPase).     

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.     

Probing Subunit-Subunit Interactions in the Yeast Vacuolar ATPase by Peptide Arrays

Background

Vacuolar (H⁺)-ATPase (V-ATPase; V₁V_o-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V₁-ATPase - V_o-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains.

Methodology/Principal Findings

To gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit.

Conclusions

The subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.     

Structure and Assembly of the Yeast V-ATPase

The yeast V-ATPase belongs to a family of V-type ATPases present in all eucaryotic organisms. In Saccharomyces cerevisiae the V-ATPase is localized to the membrane of the vacuole as well as the Golgi complex and endosomes. The V-ATPase brings about the acidification of these organelles by the transport of protons coupled to the hydrolysis of ATP. In yeast, the V-ATPase is composed of 13 subunits consisting of a catalytic V₁ domain of peripherally associated proteins and a proton-translocating V₀ domain of integral membrane proteins. The regulatory subunit, Vma13p, was the first V-ATPase subunit to have its crystal structure determined. In addition to proteins forming the functional V-ATPase complex, three ER-localized proteins facilitate the assembly of the V₀ subunits following their translation and insertion into the membrane of the ER. Homologues of the Vma21p assembly factor have been identified in many higher eukaryotes supporting a ubiquitous assembly pathway for this important enzyme complex.     

V-ATPases in osteoclasts: structure, function and potential inhibitors of bone resorption

The vacuolar-type H(+)-ATPase (V-ATPase) proton pump is a macromolecular complex composed of at least 14 subunits organized into two functional domains, V(1) and V(0). The complex is located on the ruffled border plasma membrane of bone-resorbing osteoclasts, mediating extracellular acidification for bone demineralization during bone resorption. Genetic studies from mice to man implicate a critical role for V-ATPase subunits in osteoclast-related diseases including osteopetrosis and osteoporosis. Thus, the V-ATPase complex is a potential molecular target for the development of novel anti-resorptive agents useful for the treatment of osteolytic diseases. Here, we review the current structure and function of V-ATPase subunits, emphasizing their exquisite roles in osteoclastic function. In addition, we compare several distinct classes of V-ATPase inhibitors with specific inhibitory effects on osteoclasts. Understanding the structure-function relationship of the osteoclast V-ATPase may lead to the development of osteoclast-specific V-ATPase inhibitors that may serve as alternative therapies for the treatment of osteolytic diseases.     

Electron-microscopic structure of the V-ATPase from mung bean

The vacuolar H⁺-ATPase from mung bean (Vigna radiata L. cv. Wilczek) was purified to homogeneity. The purified complex contained all the reported subunits from mung bean, but also included a 40-kDa subunit, corresponding to the membrane-associated subunit d, which has not previously been observed. The structure of the V-ATPase from mung bean was studied by electron microscopy of negatively stained samples. An analysis of over 6,000 single-particle images obtained by electron microscopy of the purified complex revealed that the complex, similar to other V-ATPases, is organized into two major domains V₁ and V_o with overall dimensions of 25 nm×13.7 nm and a stalk region connecting the V₁ and V_o domains. Several individual areas of protein density were observed in the stalk region, indicating its complexity. The projections clearly showed that the complex contained one central stalk and at least two peripheral stalks. Subcomplexes containing subunits A, B and E, dissociated from the tonoplast membrane by KI, were purified. The structure of the subcomplex was also studied by electron microscopy followed by single-molecule analysis of 13,000 projections. Our preliminary results reveal an area of high protein density at the bottom of the subcomplex immediately below the cavity formed by the A and B subunits, indicating the position of subunit E.Abbreviations MSA Multivariate statistical analysis - 2D, 3D Two-, three-dimensional - V-ATPase Vacuolar H⁺-ATPase     

Assembly and Regulation of the Yeast Vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells. Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V₁) sector, which contains the ATP-binding sites, from the membrane (V₀) sector, which contains the proton pore. A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes. We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure. In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V₁ and V₀, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V₁ sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly.     

Assembly of the Yeast Vacuolar Proton-Translocating ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is the bestcharacterized member of the V-ATPase family. Biochemical and genetic screensled to the identification of a large number of genes in yeast, designatedVMA, encoding proteins required to assemble a functional V-ATPase. Atotal of thirteen genes encode subunits of the final enzyme complex. Inaddition to subunit-encoding genes, we have identified three genes that codefor proteins that are not part of the final V-ATPase complex yet required forits assembly. We refer to these nonsubunit Vma proteins as assembly factors,since their function is dedicated to assembling the V-ATPase. The assemblyfactors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmicreticulum (ER) and aid the assembly of newly synthesized V-ATPase subunitsthat are translocated into the ER membrane. At least two of these proteins,Vma12p and Vma22p, function together in an assembly complex and interactdirectly with nascent V-ATPase subunits.     

A Journey from Mammals to Yeast with Vacuolar H+-ATPase (V-ATPase)

The vacuolar H⁺-ATPase (V-ATPase) is one of the most fundamental enzymes in nature. It functions in almost every eukaryotic cell and energizes a wide variety of organelles and membranes. V-ATPase has a structure and mechanism of action similar to F-ATPase and several of their subunits probably evolved from common ancestors. In eukaryotic cells, F-ATPase is confined to the semiautonomous organelles, chloroplasts and mitochondria, which contain their own genes that encode some of the F-ATPase subunits. In contrast to F-ATPases, whose primary function in eukaryotic cells is to form ATP at the expense of the protonmotive force (pmf), V-ATPases function exclusively as ATP-dependent proton pumps. The pmf generated by V-ATPases in organelles and membranes of eukaryotic cells is utilized as a driving force for numerous secondary transport processes. It was the survival of the yeast mutant without the active enzyme and yeast genetics that allowed the identification of genuine subunits of the V-ATPase. It also revealed special properties of individual subunits, factors that are involved in the enzyme's biogenesis and assembly, as well as the involvement of V-ATPase in the secretory pathway, endocytosis, and respiration. It may be the insect V-ATPase that unconventionally resides in the plasma membrane of their midgut, that will give the first structure resolution of this complex.     

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.     

Structural and functional features of yeast V-ATPase subunit C

V-ATPase is a multi-subunit membrane protein complex, it translocates protons across biological membranes, generating electrical and pH gradients which are used for varieties of cellular processes. V-ATPase is composed of two distinct sub-complexes: a membrane bound V0 sub-complex, composed of 6 different subunits, which is responsible for proton transport and a soluble cytosolic facing V1 sub-complex, composed of 8 different subunits which hydrolyse ATP. The two sub-complexes are held together via a flexible stator. One of the main features of eukaryotic V-ATPase is its ability to reversibly dissociate to its sub-complexes in response to changing cellular conditions, which arrest both proton translocation and ATP hydrolysis, suggesting a regulation function. Subunit C (vma5p in yeast) was shown by several biochemical, genetic and recent structural data to function as a flexible stator holding the two sectors of the complex together and regulating the reversible association/dissociation of the complex, partly via association with F-actin filaments. Structural features of subunit C that allow smooth energy conversion and interaction with actin and nucleotides are discussed.     

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.     

Structure and Regulation of the V-ATPases

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 V₁ domain responsible for ATP hydrolysis and an integral V₀ 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 V₀ 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.     

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.     

Subcellular distribution of the V-ATPase complex in plant cells,and <Emphasis Type="Italic">in vivo</Emphasis> localisation of the 100 kDa subunit VHA-a within the complex

Background  

Vacuolar H⁺-ATPases are large protein complexes of more than 700 kDa that acidify endomembrane compartments and are part of the secretory system of eukaryotic cells. They are built from 14 different (VHA)-subunits. The paper addresses the question of sub-cellular localisation and subunit composition of plant V-ATPase in vivo and in vitro mainly by using colocalization and fluorescence resonance energy transfer techniques (FRET). Focus is placed on the examination and function of the 95 kDa membrane spanning subunit VHA-a. Showing similarities to the already described Vph1 and Stv1 vacuolar ATPase subunits from yeast, VHA-a revealed a bipartite structure with (i) a less conserved cytoplasmically orientated N-terminus and (ii) a membrane-spanning C-terminus with a higher extent of conservation including all amino acids shown to be essential for proton translocation in the yeast. On the basis of sequence data VHA-a appears to be an essential structural and functional element of V-ATPase, although previously a sole function in assembly has been proposed.     

Vma21p is a yeast membrane protein retained in the endoplasmic reticulum by a di-lysine motif and is required for the assembly of the vacuolar H(+)-ATPase complex.

The yeast vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit complex comprised of peripheral membrane subunits involved in ATP hydrolysis and integral membrane subunits involved in proton pumping. The yeast vma21 mutant was isolated from a screen to identify mutants defective in V-ATPase function. vma21 mutants fail to assemble the V-ATPase complex onto the vacuolar membrane: peripheral subunits accumulate in the cytosol and the 100-kDa integral membrane subunit is rapidly degraded. The product of the VMA21 gene (Vma21p) is an 8.5-kDa integral membrane protein that is not a subunit of the purified V-ATPase complex but instead resides in the endoplasmic reticulum. Vma21p contains a dilysine motif at the carboxy terminus, and mutation of these lysine residues abolishes retention in the endoplasmic reticulum and results in delivery of Vma21p to the vacuole, the default compartment for yeast membrane proteins. Our findings suggest that Vma21p is required for assembly of the integral membrane sector of the V-ATPase in the endoplasmic reticulum and that the unassembled 100-kDa integral membrane subunit present in delta vma21 cells is rapidly degraded by nonvacuolar proteases.     

Neurotransmitter release: the dark side of the vacuolar-H+ATPase

Vacuolar-H⁺ATPase (V-ATPase) is a complex enzyme with numerous subunits organized in two domains. The membrane domain V0 contains a proteolipid hexameric ring that translocates protons when ATP is hydrolysed by the catalytic cytoplasmic sector (V1). In nerve terminals, V-ATPase generates an electrochemical proton gradient that is acid and positive inside synaptic vesicles. It is used by specific neurotransmitter-proton antiporters to accumulate neurotransmitters inside their storage organelles. During synaptic activity, neurotransmitters are released from synaptic vesicles docked at specialized portions of the presynaptic plasma membrane, the active zones. A fusion pore opens that allows the neurotransmitter to be released from the synaptic vesicle lumen into the synaptic cleft. We briefly review experimental data suggesting that the membrane domain of V-ATPase could be such a fusion pore.We also discuss the functional implications for quantal neurotransmitter release of the sequential use of the same V-ATPase membrane domain in two different events, neurotransmitter accumulation in synaptic vesicles first, and then release from these organelles during synaptic activity.     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump     

Structural bases of physiological functions and roles of the vacuolar H(+)-ATPase

Vacuolar-type H⁺-ATPases (V-ATPases) is a large multi-protein complex containing at least 14 different subunits, in which subunits A, B, C, D, E, F, G, and H compose the peripheral 500-kDa V₁ responsible for ATP hydrolysis, and subunits a, c, c′, c″, and d assembly the 250-kDa membrane-integral V₀ harboring the rotary mechanism to transport protons across the membrane. The assembly of V-ATPases requires the presence of all V₁ and V₀ subunits, in which the V₁ must be completely assembled prior to association with the V₀, accordingly the V₀ failing to assemble cannot provide a membrane anchor for the V₁, thereby prohibiting membrane association of the V-ATPase subunits. The V-ATPase mediates acidification of intracellular compartments and regulates diverse critical physiological processes of cell for functions of its numerous functional subunits. The core catalytic mechanism of the V-ATPase is a rotational catalytic mechanism. The V-ATPase holoenzyme activity is regulated by the reversible assembly/disassembly of the V₁ and V₀, the targeting and recycling of V-ATPase-containing vesicles to and from the plasma membrane, the coupling ratio between ATP hydrolysis and proton pumping, ATP, Ca²⁺, and its inhibitors and activators.     

The cellular energization state affects peripheral stalk stability of plant vacuolar H+-ATPase and impairs vacuolar acidification

The plant vacuolar H(+)-ATPase takes part in acidifying compartments of the endomembrane system including the secretory pathway and the vacuoles. The structural variability of the V-ATPase complex as well as its presence in different compartments and tissues involves multiple isoforms of V-ATPase subunits. Furthermore, a versatile regulation is essential to allow for organelle- and tissue-specific fine tuning. In this study, results from V-ATPase complex disassembly with a chaotropic reagent, immunodetection and in vivo fluorescence resonance energy transfer (FRET) analyses point to a regulatory mechanism in plants, which depends on energization and involves the stability of the peripheral stalks as well. Lowering of cellular ATP by feeding 2-deoxyglucose resulted in structural alterations within the V-ATPase, as monitored by changes in FRET efficiency between subunits VHA-E and VHA-C. Potassium iodide-mediated disassembly revealed a reduced stability of V-ATPase after 2-deoxyglucose treatment of the cells, but neither the complete V(1)-sector nor VHA-C was released from the membrane in response to 2-deoxyglucose treatment, precluding a reversible dissociation mechanism like in yeast. These data suggest the existence of a regulatory mechanism of plant V-ATPase by modification of the peripheral stator structure that is linked to the cellular energization state. This mechanism is distinct from reversible dissociation as reported for the yeast V-ATPase, but might represent an evolutionary precursor of reversible dissociation.