A pH-sensitive luminal His-cluster promotes interaction of PAM with V-ATPase along the secretory and endocytic pathways of peptidergic cells

The biosynthetic and endocytic pathways of secretory cells are characterized by progressive luminal acidification, a process which is crucial for posttranslational modifications and membrane trafficking. This progressive fall in luminal pH is mainly achieved by the vacuolar-type-H⁺ ATPase (V-ATPase). V-ATPases are large, evolutionarily ancient rotary proton pumps that consist of a peripheral V1 complex, which hydrolyzes ATP, and an integral membrane V0 complex, which transports protons from the cytosol into the lumen. Upon sensing the desired luminal pH, V-ATPase activity is regulated by reversible dissociation of the complex into its V1 and V0 components. Molecular details of how intraluminal pH is sensed and transmitted to the cytosol are not fully understood. Peptidylglycine α-amidating mono-oxygenase (PAM; EC 1.14.17.3), a secretory pathway membrane enzyme which shares similar topology with two V-ATPase accessory proteins (Ac45 and prorenin receptor), has a pH-sensitive luminal linker region. Immunofluorescence and sucrose gradient analysis of peptidergic cells (AtT-20) identified distinct subcellular compartments exhibiting spatial co-occurrence of PAM and V-ATPase. In vitro binding assays demonstrated direct binding of the cytosolic domain of PAM to V1H. Blue native PAGE identified heterogeneous high-molecular weight complexes of PAM and V-ATPase. A PAM-1 mutant (PAM-1/H3A) with altered pH sensitivity had diminished ability to form high-molecular weight complexes. In addition, V-ATPase assembly status was altered in PAM-1/H3A expressing cells. Our analysis of the secretory and endocytic pathways of peptidergic cells supports the hypothesis that PAM serves as a luminal pH-sensor, regulating V-ATPase action by altering its assembly status.     

Regulation of the V-ATPase along the endocytic pathway occurs through reversible subunit association and membrane localization

The lumen of endosomal organelles becomes increasingly acidic when going from the cell surface to lysosomes. Luminal pH thereby regulates important processes such as the release of internalized ligands from their receptor or the activation of lysosomal enzymes. The main player in endosomal acidification is the vacuolar ATPase (V-ATPase), a multi-subunit transmembrane complex that pumps protons from the cytoplasm to the lumen of organelles, or to the outside of the cell. The active V-ATPase is composed of two multi-subunit domains, the transmembrane V(0) and the cytoplasmic V(1). Here we found that the ratio of membrane associated V(1)/Vo varies along the endocytic pathway, the relative abundance of V(1) being higher on late endosomes than on early endosomes, providing an explanation for the higher acidity of late endosomes. We also found that all membrane-bound V-ATPase subunits were associated with detergent resistant membranes (DRM) isolated from late endosomes, raising the possibility that association with lipid-raft like domains also plays a role in regulating the activity of the proton pump. In support of this, we found that treatment of cells with U18666A, a drug that leads to the accumulation of cholesterol in late endosomes, affected acidification of late endosome. Altogether our findings indicate that the activity of the vATPase in the endocytic pathway is regulated both by reversible association/dissociation and the interaction with specific lipid environments.     

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.     

Modulation of the actin cytoskeleton via gelsolin regulates vacuolar H+-ATPase recycling

The role of the actin cytoskeleton in regulating membrane protein trafficking is complex and depends on the cell type and protein being examined. Using the epididymis as a model system in which luminal acidification is crucial for sperm maturation and storage, we now report that modulation of the actin cytoskeleton by the calcium-activated actin-capping and -severing protein gelsolin plays a key role in regulating vacuolar H(+)-ATPase (V-ATPase) recycling. Epididymal clear cells contain abundant V-ATPase in their apical pole, and an increase in their cell-surface V-ATPase expression correlates with an increase in luminal proton secretion. We have shown that apical membrane accumulation of V-ATPase is triggered by an elevation in cAMP following activation of bicarbonate-regulated soluble adenylyl cyclase in response to alkaline luminal pH (Pastor-Soler, N., Beaulieu, V., Litvin, T. N., Da Silva, N., Chen, Y., Brown, D., Buck, J., Levin, L. R., and Breton, S. (2003) J. Biol. Chem. 278, 49523-49529). Here, we show that clear cells express high levels of gelsolin, indicating a potential role in the functional activity of these cells. When jasplakinolide was used to overcome the severing action of gelsolin by polymerizing actin, complete inhibition of the alkaline pH- and cAMP-induced apical membrane accumulation of V-ATPase was observed. Conversely, when gelsolin-mediated actin filament elongation was inhibited using a 10-residue peptide (PBP10) derived from the phosphatidylinositol 4,5-bisphosphate-binding region (phosphoinositide-binding domain 2) of gelsolin, significant V-ATPase apical membrane mobilization was induced, even at acidic luminal pH. In contrast, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) and the phospholipase C inhibitor U-73122 inhibited the alkaline pH-induced V-ATPase apical accumulation. Thus, maintenance of the actin cytoskeleton in a depolymerized state by gelsolin facilitates calcium-dependent apical accumulation of V-ATPase in response to luminal pH alkalinization. Gelsolin is present in other cell types that express the V-ATPase in their plasma membrane and recycling vesicles, including kidney intercalated cells and osteoclasts. Therefore, modulation of the actin cortex by this severing and capping protein may represent a common mechanism by which these cells regulate their rate of proton secretion.     

Accessory subunit Ac45 controls the V-ATPase in the regulated secretory pathway

The vacuolar (H(+))-ATPase (V-ATPase) is crucial for multiple processes within the eukaryotic cell, including membrane transport and neurotransmitter secretion. How the V-ATPase is regulated, e.g. by an accessory subunit, remains elusive. Here we explored the role of the neuroendocrine V-ATPase accessory subunit Ac45 via its transgenic expression specifically in the Xenopus intermediate pituitary melanotrope cell model. The Ac45-transgene product did not affect the levels of the prohormone proopiomelanocortin nor of V-ATPase subunits, but rather caused an accumulation of the V-ATPase at the plasma membrane. Furthermore, a higher abundance of secretory granules, protrusions of the plasma membrane and an increased Ca(2+)-dependent secretion efficiency were observed in the Ac45-transgenic cells. We conclude that in neuroendocrine cells Ac45 guides the V-ATPase through the secretory pathway, thereby regulating the V-ATPase-mediated process of Ca(2+)-dependent peptide secretion.     

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.     

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.     

Reversible Association between the V1 and V0 Domains of Yeast Vacuolar H+-ATPase Is an Unconventional Glucose-Induced Effect

The yeast vacuolar H⁺-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V₁), containing the ATP binding sites, and an integral membrane domain (V₀), forming the proton pore. Dissociation of the V₁ and V₀ domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V₁ and V₀ domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.     

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.     

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.     

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.     

The molecular chaperone calnexin associates with the vacuolar H(+)-ATPase from oat seedlings.

Acidification of endomembrane compartments by the vacuolar-type H(+)-ATPase (V-ATPase) is central to many cellular processes in eukaryotes, including osmoregulation and protein sorting. The V-ATPase complex consists of a peripheral sector (V1) and a membrane integral sector (V0); however, it is unclear how the multimeric enzyme is assembled. A 64-kD polypeptide that had copurified with oat V-ATPase subunits has been identified as calnexin, an integral protein on the endoplasmic reticulum. To determine whether calnexin interacted physically with the V-ATPase, microsomal membranes were Triton X-100 solubilized, and the protein-protein interaction was analyzed by coimmunoprecipitation. Monoclonal antibodies against calnexin precipitated both calnexin and V-ATPase subunits, including A and B and those of 44, 42, 36, 16, and 13 kD. A monoclonal antibody against subunit A precipitated the entire V-ATPase complex as well as calnexin and BiP, an endoplasmic reticulum lumen chaperone. The results support our hypothesis that both calnexin and BiP act as molecular chaperones in the folding and assembly of newly synthesized V1V0-ATPases at the endoplasmic reticulum.     

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.     

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.     

Molecular cloning and characterization of a novel V-ATPase associated protein, DVA9.2, from human dendritic cells

The vacuolar proton-ATPase (V-ATPase) is a ubiquitous ATP-driven H(+) transporter that functions in numerous cell processes. Accumulating evidence shows important roles of V-ATPase in tumor metastasis and antigen presentation of dendritic cells (DC). A novel V-ATPase associated protein, designated as DVA9.2 (dendritic cell-derived V-ATPase associated protein of 9.2 kDa), has been identified from a human DC cDNA library by large-scale random sequencing. Full length cDNA of DVA9.2 encodes an 81-residue protein that shares 70-80% homology with human V-ATPase subunit M9.2. Distant relationship is also found with Vma21p, a yeast protein required for V-ATPase assembly. DVA9.2 contains a conserved domain, ATP synthase subunit H (pafm05493), and two membrane-spanning helices. DVA9.2 mRNA is detectable in several human tumor cell lines as well as some human normal cells and tissues. Moreover, the inducible expression of DVA9.2 mRNA in DC during maturation is observed. DVA9.2 displays integration with membrane and main localization in lysosome, endoplasmic reticulum and Golgi-associated organelles, only less at the plasma membrane. In addition, DVA9.2 is co-localized with V(0)-sector subunit a. Silencing of DVA9.2 by small interfering RNA (siRNA) does not affect the V-ATPase activity in cell membrane fractions or attenuate the migration and invasion in breast cancer MDA-MB-231 cells. These results indicate that DVA9.2, as a novel V-ATPase-associated protein, is not essential for the activity of V-ATPase complex and may be involved in functions of DC.     

Intestinal expression of H+ V-ATPase in the mosquito Aedes albopictus is tightly associated with gregarine infection

Vacuolar ATPase (V-ATPase) is a family of ATP-dependent proton pumps expressed on the plasma membrane and endomembranes of eukaryotic cells. Acidification of intracellular compartments, such as lysosomes, endosomes, and parasitophorous vacuoles, mediated by V-ATPase is essential for the entry by many enveloped viruses and invasion into or escape from host cells by intracellular parasites. In mosquito larvae, V-ATPase plays a role in regulating alkalization of the anterior midgut. We extracted RNA from larval tissues of Aedes albopictus, cloned the full-length sequence of mRNA of V-ATPase subunit A, which contains a poly-A tail and 2,971 nucleotides, and expressed the protein. The fusion protein was then used to produce rabbit polyclonal antibodies, which were used as a tool to detect V-ATPase in the midgut and Malpighian tubules of mosquito larvae. A parasitophorous vacuole was formed in the midgut in response to invasion by Ascogregarina taiwanensis, confining the trophozoite(s). Acidification was demonstrated within the vacuole using acridine orange staining. It is concluded that gregarine sporozoites are released by ingested oocysts in the V-ATPase-energized high-pH environment. The released sporozoites then invade and develop in epithelial cells of the posterior midgut. Acidification of the parasitophorous vacuoles may be mediated by V-ATPase and may facilitate exocytosis of the vacuole confining the trophozoites from the infected epithelial cells for further extracellular development.     

The RAVE complex is essential for stable assembly of the yeast V-ATPase

Vacuolar proton-translocating ATPases are composed of a peripheral complex, V(1), attached to an integral membrane complex, V(o). Association of the two complexes is essential for ATP-driven proton transport and is regulated post-translationally in response to glucose concentration. A new complex, RAVE, was recently isolated and implicated in glucose-dependent reassembly of V-ATPase complexes that had disassembled in response to glucose deprivation (Seol, J. H., Shevchenko, A., and Deshaies, R. J. (2001) Nat. Cell Biol. 3, 384-391). Here, we provide evidence supporting a role for RAVE in reassembly of the V-ATPase but also demonstrate an essential role in V-ATPase assembly under other conditions. The RAVE complex associates reversibly with V(1) complexes released from the membrane by glucose deprivation but binds constitutively to cytosolic V(1) sectors in a mutant lacking V(o) sectors. V-ATPase complexes from cells lacking RAVE subunits show serious structural and functional defects even in glucose-grown cells or in combination with a mutation that blocks disassembly of the V-ATPase. RAVE small middle dotV(1) interactions are specifically disrupted in cells lacking V(1) subunits E or G, suggesting a direct involvement for these subunits in interaction of the two complexes. Skp1p, a RAVE subunit involved in many different signal transduction pathways, binds stably to other RAVE subunits under conditions that alter RAVE small middle dotV(1) binding; thus, Skp1p recruitment to the RAVE complex does not appear to provide a signal for V-ATPase assembly.     

PKR1 encodes an assembly factor for the yeast V-type ATPase

Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Delta cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector.     

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.     

Vma9p (subunit e) is an integral membrane V0 subunit of the yeast V-ATPase

The Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of 14 subunits distributed between a peripheral V1 subcomplex and an integral membrane V0 subcomplex. Genome-wide screens have led to the identification of the newest yeast V-ATPase subunit, Vma9p. Vma9p (subunit e) is a small hydrophobic protein that is conserved from fungi to animals. We demonstrate that disruption of yeast VMA9 results in the failure of V1 and V0 V-ATPase subunits to assemble onto the vacuole and in decreased levels of the subunit a isoforms Vph1p and Stv1p. We also show that Vma9p is an integral membrane protein, synthesized and inserted into the endoplasmic reticulum (ER), which then localizes to the limiting membrane of the vacuole. All V0 subunits and V-ATPase assembly factors are required for Vma9p to efficiently exit the ER. In the ER, Vma9p and the V0 subunits interact with the V-ATPase assembly factor Vma21p. Interestingly, the association of Vma9p with the V0-Vma21p assembly complex is disrupted with the loss of any single V0 subunit. Similarly, Vma9p is required for V0 subunits Vph1p and Vma6p to associate with the V0-Vma21p complex. In contrast, the proteolipids associate with Vma21p even in the absence of Vma9p. These results demonstrate that Vma9p is an integral membrane subunit of the yeast V-ATPase V0 subcomplex and suggest a model for the arrangement of polypeptides within the V0 subcomplex.