Structure and regulation of the vacuolar ATPases

The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for both acidification of intracellular compartments and, for certain cell types, proton transport across the plasma membrane. Intracellular V-ATPases function in both endocytic and intracellular membrane traffic, processing and degradation of macromolecules in secretory and digestive compartments, coupled transport of small molecules such as neurotransmitters and ATP and in the entry of pathogenic agents, including envelope viruses and bacterial toxins. V-ATPases are present in the plasma membrane of renal cells, osteoclasts, macrophages, epididymal cells and certain tumor cells where they are important for urinary acidification, bone resorption, pH homeostasis, sperm maturation and tumor cell invasion, respectively. The V-ATPases are composed of a peripheral domain (V(1)) that carries out ATP hydrolysis and an integral domain (V(0)) responsible for proton transport. V(1) contains eight subunits (A-H) while V(0) contains six subunits (a, c, c', c', d and e). V-ATPases operate by a rotary mechanism in which ATP hydrolysis within V(1) drives rotation of a central rotary domain, that includes a ring of proteolipid subunits (c, c' and c'), relative to the remainder of the complex. Rotation of the proteolipid ring relative to subunit a within V(0) drives active transport of protons across the membrane. Two important mechanisms of regulating V-ATPase activity in vivo are reversible dissociation of the V(1) and V(0) domains and changes in coupling efficiency of proton transport and ATP hydrolysis. This review focuses on recent advances in our lab in understanding the structure and regulation of the V-ATPases.     

Structure,subunit function and regulation of the coated vesicle and yeast vacuolar (H(+))-ATPases

The vacuolar (H(+))-ATPases (or V-ATPases) are ATP-dependent proton pumps that function to acidify intracellular compartments in eukaryotic cells. This acidification is essential for such processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation and the coupled transport of small molecules. V-ATPases in the plasma membrane of specialized cells also function in such processes as renal acidification, bone resorption and pH homeostasis. Work from our laboratory has focused on the V-ATPases from clathrin-coated vesicles and yeast vacuoles.Structurally, the V-ATPases are composed of two domains: a peripheral complex (V(1)) composed of eight different subunits (A-H) that is responsible for ATP hydrolysis and an integral complex (V(0)) composed of five different subunits (a, d, c, c' and c") that is responsible for proton translocation. Electron microscopy has revealed the presence of multiple stalks connecting the V(1) and V(0) domains, and crosslinking has been used to address the arrangement of subunits in the complex. Site-directed mutagenesis has been employed to identify residues involved in ATP hydrolysis and proton translocation and to study the topology of the 100 kDa a subunit. This subunit has been shown to control intracellular targeting of the V-ATPase and to influence reversible dissociation and coupling of proton transport and ATP hydrolysis.     

V-ATPases的功能及其抑制剂研究进展

V-ATPases作为一类酶,在真核细胞中广泛存在。V-ATPases是一个由多个亚基组成的复合物,主要有两个结构域,分别是位于外周的V1结构域和跨膜的V0结构域。V1结构域可以通过水解ATP供能;而V0结构域是质子的通道。它们发挥作用主要是通过水解ATP供能,泵运H＋进入囊泡腔中或泵H＋出细胞外。V-ATPases定位于细胞器膜及某些特殊细胞的细胞质膜,参与骨吸收、肿瘤的侵袭及耐药等生理及病理过程,因而V-ATPases是治疗骨质疏松、糖尿病及肿瘤等人类疾病的候选分子靶标。目前有许多研究致力于发现新的潜在的特异的V-ATPase抑制剂。     

Subunit H of the vacuolar (H+) ATPase inhibits ATP hydrolysis by the free V1 domain by interaction with the rotary subunit F

The vacuolar (H+) ATPases (V-ATPases) are large, multimeric proton pumps that, like the related family of F1F0 ATP synthases, employ a rotary mechanism. ATP hydrolysis by the peripheral V1 domain drives rotation of a rotary complex (the rotor) relative to the stationary part of the enzyme (the stator), leading to proton translocation through the integral V0 domain. One mechanism of regulating V-ATPase activity in vivo involves reversible dissociation of the V1 and V0 domains. Unlike the corresponding domains in F1F0, the dissociated V1 domain does not hydrolyze ATP, and the free V0 domain does not passively conduct protons. These properties are important to avoid generation of an uncoupled ATPase activity or an unregulated proton conductance upon dissociation of the complex in vivo. Previous results (Parra, K. J., Keenan, K. L., and Kane, P. M. (2000) J. Biol. Chem. 275, 21761-21767) showed that subunit H (part of the stator) inhibits ATP hydrolysis by free V1. To test the hypothesis that subunit H accomplishes this by bridging rotor and stator in free V1, cysteine-mediated cross-linking studies were performed. Unique cysteine residues were introduced over the surface of subunit H from yeast by site-directed mutagenesis and used as the site of attachment of the photo-activated cross-linking reagent maleimido benzophenone. After UV-activated cross-linking, cross-linked products were identified by Western blot using subunit-specific antibodies. The results indicate that the subunit H mutant S381C shows cross-linking between subunit H and subunit F (a rotor subunit) in the free V1 domain but not in the intact V1V0 complex. These results indicate that subunits H and F are proximal in free V1, supporting the hypothesis that subunit H inhibits free V1 by bridging the rotary and stator domains.     

The mechanochemistry of V-ATPase proton pumps

The vacuolar H(+)-ATPases (V-ATPases) are a universal class of proton pumps that are structurally similar to the F-ATPases. Both protein families are characterized by a membrane-bound segment (V(o), F(o)) responsible for the translocation of protons, and a soluble portion, (V(1), F(1)), which supplies the energy for translocation by hydrolyzing ATP. Here we present a mechanochemical model for the functioning of the V(o) ion pump that is consistent with the known structural features and biochemistry. The model reproduces a variety of experimental measurements of performance and provides a unified view of the many mechanisms of intracellular pH regulation.     

Characterization of vacuolar-ATPase and selective inhibition of vacuolar-H(+)-ATPase in osteoclasts

V-ATPase plays important roles in controlling the extra- and intra-cellular pH in eukaryotic cell, which is most crucial for cellular processes. V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and integral V(0) domain responsible for proton translocation. Osteoclasts are multinucleated cells responsible for bone resorption and relate to many common lytic bone disorders such as osteoporosis, bone aseptic loosening, and tumor-induced bone loss. This review summarizes the structure and function of V-ATPase and its subunit, the role of V-ATPase subunits in osteoclast function, V-ATPase inhibitors for osteoclast function, and highlights the importance of V-ATPase as a potential prime target for anti-resorptive agents.     

Molecular characterization of the yeast vacuolar H+-ATPase proton pore

The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.     

Function, structure and regulation of the vacuolar (H+)-ATPases

The vacuolar ATPases (or V-ATPases) are ATP-driven proton pumps that function to both acidify intracellular compartments and to transport protons across the plasma membrane. Intracellular V-ATPases function in such normal cellular processes as receptor-mediated endocytosis, intracellular membrane traffic, prohormone processing, protein degradation and neurotransmitter uptake, as well as in disease processes, including infection by influenza and other viruses and killing of cells by anthrax and diphtheria toxin. Plasma membrane V-ATPases are important in such physiological processes as urinary acidification, bone resorption and sperm maturation as well as in human diseases, including osteopetrosis, renal tubular acidosis and tumor metastasis. V-ATPases are large multi-subunit complexes composed of a peripheral domain (V₁) responsible for hydrolysis of ATP and an integral domain (V₀) that carries out proton transport. Proton transport is coupled to ATP hydrolysis by a rotary mechanism. V-ATPase activity is regulated in vivo using a number of mechanisms, including reversible dissociation of the V₁ and V₀ domains, changes in coupling efficiency of proton transport and ATP hydrolysis and changes in pump density through reversible fusion of V-ATPase containing vesicles. V-ATPases are emerging as potential drug targets in treating a number of human diseases including osteoporosis and cancer.     

Structure and Properties of the Clathrin-Coated Vesicle and Yeast Vacuolar V-ATPases

The V-ATPases are a family of ATP-dependent proton pumps responsible foracidification of intracellular compartments in eukaryotic cells. This reviewfocuses on the the V-ATPases from clathrin-coated vesicles and yeastvacuoles. The V-ATPase of clathrin-coated vesicles is a precursor to thatfound in endosomes and synaptic vesicles, which function in receptorrecycling, intracellular membrane traffic, and neurotransmitter uptake. Theyeast vacuolar ATPase functions to acidify the central vacuole and to drivevarious coupled transport processes across the vacuolar membrane. TheV-ATPases are composed of two functional domains. The V₁ domain isa 570-kDa peripheral complex composed of eight subunits of molecular weight70—14 kDa (subunits A—H) that is responsible for ATP hydrolysis.The V₀ domain is a 260-kDa integral complex composed of fivesubunits of molecular weight 100—17 kDa (subunits a, d, c, c8 and c9)that is responsible for proton translocation. Using chemical modification andsite-directed mutagenesis, we have begun to identify residues that play arole in ATP hydrolysis and proton transport by the V-ATPases. A centralquestion in the V-ATPase field is the mechanism by which cells regulatevacuolar acidification. Several mechanisms are described that may play a rolein controlling vacuolar acidification in vivo. One mechanisminvolves disulfide bond formation between cysteine residues located at thecatalytic nucleotide binding site on the 70-kDa A subunit, leading toreversible inhibition of V-ATPase activity. Other mechanisms includereversible assembly and dissociation of V₁ and V₀domains, changes in coupling efficiency of proton transport and ATPhydrolysis, and regulation of the activity of intracellular chloride channelsrequired for vacuolar acidification.     

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.     

Function and subunit interactions of the N-terminal domain of subunit a (Vph1p) of the yeast V-ATPase

The vacuolar (H+)-ATPases (V-ATPases) are ATP-dependent proton pumps that operate by a rotary mechanism in which ATP hydrolysis drives rotation of a ring of proteolipid subunits relative to subunit a within the integral V(0) domain. In vivo dissociation of the V-ATPase (an important regulatory mechanism) generates a V(0) domain that does not passively conduct protons. EM analysis indicates that the N-terminal domain of subunit a approaches the rotary subunits in free V(0), suggesting a possible mechanism of silencing passive proton transport. To test the hypothesis that the N-terminal domain inhibits passive proton flux by preventing rotation of the proteolipid ring in free V(0), factor Xa cleavage sites were introduced between the N- and C-terminal domains of subunit a (the Vph1p isoform in yeast) to allow its removal in vitro after isolation of vacuolar membranes. The mutant Vph1p gave rise to a partially uncoupled V-ATPase complex. Cleavage with factor Xa led to further loss of coupling of proton transport and ATP hydrolysis. Removal of the N-terminal domain by cleavage with factor Xa and treatment with KNO3 and MgATP did not, however, lead to an increase in passive proton conductance by free V(0), suggesting that removal of the N-terminal domain is not sufficient to facilitate passive proton conductance through V(0). Photoactivated cross-linking using the cysteine reagent maleimido benzophenone and single cysteine mutants of subunit a demonstrated the proximity of specific sites within the N-terminal domain and subunits E and G of the peripheral stalk. These results suggest that a localized region of the N-terminal domain (residues 347-369) is important in anchoring the peripheral stator in V1V0.     

Evidence that the NH2 terminus of vph1p, an integral subunit of the V0 sector of the yeast V-ATPase, interacts directly with the Vma1p and Vma13p subunits of the V1 sector

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.     

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.     

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.     

Deltapsi and DeltapH are equivalent driving forces for proton transport through isolated F(0) complexes of ATP synthases

The membrane-embedded F(0) part of ATP synthases is responsible for ion translocation during ATP synthesis and hydrolysis. Here, we describe an in vitro system for measuring proton fluxes through F(0) complexes by fluorescence changes of the entrapped fluorophore pyranine. Starting from purified enzyme, the F(0) part was incorporated unidirectionally into phospholipid vesicles. This allowed analysis of proton transport in either synthesis or hydrolysis direction with Deltapsi or DeltapH as driving forces. The system displayed a high signal-to-noise ratio and can be accurately quantified. In contrast to ATP synthesis in the Escherichia coli F(1)F(0) holoenzyme, no significant difference was observed in the efficiency of DeltapH or Deltapsi as driving forces for H(+)-transport through F(0). Transport rates showed linear dependency on the driving force. Proton transport in hydrolysis direction was about 2400 H(+)/(s x F(0)) at Deltapsi of 120 mV, which is approximately twice as fast as in synthesis direction. The chloroplast enzyme was faster and catalyzed H(+)-transport at initial rates of 6300 H(+)/(s x F(0)) under similar conditions. The new method is an ideal tool for detailed kinetic investigations of the ion transport mechanism of ATP synthases from various organisms.     

Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain

The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.     

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.     

Cytoplasmic terminus of vacuolar type proton pump accessory subunit Ac45 is required for proper interaction with V(0) domain subunits and efficient osteoclastic bone resorption

Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H+)-ATPases (V-ATPases) polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V(1) and V(0). The peripheral cytoplasmically oriented V(1) domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V(0) domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V(0) domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c', and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c', and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V(0) domain and efficient osteoclastic bone resorption.     

The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis

A study is presented on the effect of diamide-induced disulfide cross-linking of F(1)-gamma and F(0)I-PVP(b) subunits on proton translocation in the mitochondrial ATP synthase. The results show that, upon cross-linking of these subunits, whilst proton translocation from the A side to the B F(1) side is markedly accelerated with decoupling of oxidative phosphorylation, proton translocation in the reverse direction, driven by either ATP hydrolysis or a diffusion potential, is unaffected. These observations reveal further peculiarities of the mechanism of energy transfer in the ATP synthase of coupling membranes.     

Peripheral stator of the yeast V-ATPase: stoichiometry and specificity of interaction between the EG complex and subunits C and H

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.