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.     

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.     

Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p)

Vacuolar ATPases (V-ATPases) are important for many cellular processes, as they regulate pH by pumping cytosolic protons into intracellular organelles. The cytoplasm is acidified when V-ATPase is inhibited; thus we conducted a high-throughput screen of a chemical library to search for compounds that acidify the yeast cytosol in vivo using pHluorin-based flow cytometry. Two inhibitors, alexidine dihydrochloride (EC(50) = 39 μM) and thonzonium bromide (EC(50) = 69 μM), prevented ATP-dependent proton transport in purified vacuolar membranes. They acidified the yeast cytosol and caused pH-sensitive growth defects typical of V-ATPase mutants (vma phenotype). At concentrations greater than 10 μM the inhibitors were cytotoxic, even at the permissive pH (pH 5.0). Membrane fractions treated with alexidine dihydrochloride and thonzonium bromide fully retained concanamycin A-sensitive ATPase activity despite the fact that proton translocation was inhibited by 80-90%, indicating that V-ATPases were uncoupled. Mutant V-ATPase membranes lacking residues 362-407 of the tether of Vph1p subunit a of V(0) were resistant to thonzonium bromide but not to alexidine dihydrochloride, suggesting that this conserved sequence confers uncoupling potential to V(1)V(0) complexes and that alexidine dihydrochloride uncouples the enzyme by a different mechanism. The inhibitors also uncoupled the Candida albicans enzyme and prevented cell growth, showing further specificity for V-ATPases. Thus, a new class of V-ATPase inhibitors (uncouplers), which are not simply ionophores, provided new insights into the enzyme mechanism and original evidence supporting the hypothesis that V-ATPases may not be optimally coupled in vivo. The consequences of uncoupling V-ATPases in vivo as potential drug targets are discussed.     

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.     

V-ATPase, ScNhx1p and yeast vacuole fusion

Membrane fusion is the last step in trafficking pathways during which membrane vesicles fuse with target organelles to deliver cargos. It is a central cellular reaction that plays important roles in signal transduction, protein sorting and subcellular compartmentation. Recent progress in understanding the roles of ion transporters in vacuole fusion in yeast is summarized in this article. It is becoming increasingly evident that the vacuolar proton pump V-ATPase and vacuolar Na+/H+ antiporter ScNhx1p are key components of the vacuole fusion machinery in yeast. Yeast ScNhx1p regulates vacuole fusion by controlling the luminal pH. V-ATPases serve a dual role in vacuolar integrity in which they regulate both vacuole fusion and fission reactions in yeast. Fission defects are epistatic to fusion defects. Vacuole fission depends on the proton translocation activity of the V-ATPase; by contrast, the fusion reaction does not need the transport activity but requires the physical presence of the proton pump. V0, the membrane-integral sector of the V-ATPase, forms trans-complexes between the opposing vacuoles in the terminal phase of vacuole fusion where the V0trans-complexes build a continuous proteolipid channel at the fusion site to mediate the bilayer fusion.     

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.     

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.     

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.     

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.     

Binding assays of inhibitors towards selected V-ATPase domains

The macrolide antibiotic bafilomycin and the related synthetic compound SB 242784 are potent inhibitors of the vacuolar H⁺-ATPases (V-ATPase). It is currently believed that the site of action of these inhibitors is located on the membrane bound c-subunits of V-ATPases. To address the identification of the critical inhibitors binding domain, their specific binding to a synthetic peptide corresponding to the putative 4th transmembrane segment of the c-subunit was investigated using fluorescence resonance energy transfer (FRET), and for this purpose a specific formalism was derived. Another peptide of the corresponding domain of the c′ isoform, was checked for binding of bafilomycin, since it is not clear if V-ATPase inhibition can also be achieved by interaction of the inhibitor with the c′-subunit. It was concluded that bafilomycin binds to the selected peptides, whereas SB 242784 was unable to interact, and in addition for bafilomycin, its interaction with the peptides either corresponding to the c- or the c′-subunit isoforms is identical. Since the observed interactions are however much weaker as compared to the very efficient binding of both bafilomycin and SB 242784 to the whole protein, it can be concluded that assembly of all V-ATPase transmembrane segments is required for an efficient interaction.     

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.     

Identification of a new chondropsin class of antitumor compound that selectively inhibits V-ATPases

We identify a new naturally occurring class of inhibitor of vacuolar H+-ATPases (V-ATPases) isolated from vacuolar membranes of Neurospora crassa and from chromaffin granule membranes of Bos taurus. To date, the new class includes six chondropsins and poecillastrin A, large polyketide-derived macrolide lactams with 33-37 membered rings. In the National Cancer Institute's 60-cell screen the chondropsin class showed a tumor cell growth inhibitory fingerprint essentially indistinguishable from that of the bafilomycin/concanamycin and the salicylihalamide/lobatamide classes of well-established V-ATPase inhibitors. Half-maximal inhibition of V-ATPase activity in vitro occurred at 0.04-0.7 microM for the fungal vacuolar V-ATPase and at 0.4 to >10 microM for the chromaffin granule V-ATPase. Thus, the new inhibitors are somewhat less potent than the other two classes, which typically have Ki values of <10 nM for V-ATPases, and the new inhibitors differ from the other two classes in their specificity. The bafilomycin class inhibits all eucaryotic V-ATPases, the salicylihalamide class inhibits mammalian V-ATPases but not fungal V-ATPases, and the new chondropsin class inhibits the N. crassa V-ATPase better than the chromaffin granule V-ATPase. Two mutations in the N. crassa V-ATPase that affect the binding of bafilomycin had small but reproducible effects on the affinity of chondropsins for the V-ATPase, suggesting the possibility of a similar mechanism of inhibition.     

Vacuolar H+-ATPase in human breast cancer cells with distinct metastatic potential: distribution and functional activity

Tumor cells thrive in a hypoxic microenvironment with an acidic extracellular pH. To survive in this harsh environment, tumor cells must exhibit a dynamic cytosolic pH regulatory system. We hypothesize that vacuolar H⁺-ATPases (V-ATPases) that normally reside in acidic organelles are also located at the cell surface, thus regulating cytosolic pH and exacerbating the migratory ability of metastatic cells. Immunocytochemical data revealed for the first time that V-ATPase is located at the plasma membrane of human breast cancer cells: prominent in the highly metastatic and inconspicuous in the lowly metastatic cells. The V-ATPase activities in isolated plasma membranes were greater in highly than in lowly metastatic cells. The proton fluxes via V-ATPase evaluated by fluorescence spectroscopy in living cells were greater in highly than in lowly metastatic cells. Interestingly, lowly metastatic cells preferentially used the ubiquitous Na⁺/H⁺ exchanger and HCO₃^–-based H⁺-transporting mechanisms, whereas highly metastatic cells used plasma membrane V-ATPases. The highly metastatic cells were more invasive and migratory than the lowly metastatic cells. V-ATPase inhibitors decreased the invasion and migration in the highly metastatic cells. Altogether, these data indicate that V-ATPases located at the plasma membrane are involved in the acquisition of a more metastatic phenotype. metastasis; intracellular pH; migration; sodium ion/hydrogen ion exchanger; bicarbonate transport     

Defective assembly of a hybrid vacuolar H-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

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.     

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.     

Mutations in subunit C of the vacuolar ATPase confer resistance to bafilomycin and identify a conserved antibiotic binding site.

Bafilomycin A1, a potent inhibitor of vacuolar H(+)-ATPases (V-ATPase), inhibited growth of Neurospora crassa in medium adjusted to alkaline pH. Ninety-eight mutant strains were selected for growth on medium (pH 7.2) containing 0.3 or 1.0 microm bafilomycin. Three criteria suggested that 11 mutant strains were altered in the V-ATPase: 1) these strains accumulated high amounts of arginine when grown at pH 5.8 in the presence of bafilomycin, 2) the mutation mapped to the locus of vma-3, which encodes the proteolipid subunit c of the V-ATPase, and 3) V-ATPase activity in purified vacuolar membranes was resistant to bafilomycin. Sequencing of the genomic DNA encoding vma-3 identified the following mutations: T32I (two strains), F136L (two strains), Y143H (two strains), and Y143N (five strains). Characterization of V-ATPase activity in the four kinds of mutant strains showed that the enzyme was resistant to bafilomycin in vitro, with half-maximal inhibition obtained at 80-400 nm compared with 6.3 nm for the wild-type enzyme. Surprisingly, the mutant enzymes showed only weak resistance to concanamycin. Interestingly, the positions of two mutations corresponded to positions of oligomycin-resistant mutations in the c subunit of F(1)F(0)-ATP synthases (F-ATPases), suggesting that bafilomycin and oligomycin utilize a similar binding site and mechanism of inhibition in the related F- and V-ATPases.     

Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis

In yeast cells, subunit a of the vacuolar proton pump (V-ATPase) is encoded by two organelle-specific isoforms, VPH1 and STV1. V-ATPases containing Vph1 and Stv1 localize predominantly to the vacuole and the Golgi apparatus/endosomes, respectively. Ratiometric measurements of vacuolar pH confirm that loss of STV1 has little effect on vacuolar pH. Loss of VPH1 results in vacuolar alkalinization that is even more rapid and pronounced than in vma mutants, which lack all V-ATPase activity. Cytosolic pH responses to glucose addition in the vph1Δ mutant are similar to those in vma mutants. The extended cytosolic acidification in these mutants arises from reduced activity of the plasma membrane proton pump, Pma1p. Pma1p is mislocalized in vma mutants but remains at the plasma membrane in both vph1Δ and stv1Δ mutants, suggesting multiple mechanisms for limiting Pma1 activity when organelle acidification is compromised. pH measurements in early prevacuolar compartments via a pHluorin fusion to the Golgi protein Gef1 demonstrate that pH responses of these compartments parallel cytosolic pH changes. Surprisingly, these compartments remain acidic even in the absence of V-ATPase function, possibly as a result of cytosolic acidification. These results emphasize that loss of a single subunit isoform may have effects far beyond the organelle where it resides.     

Functional complementation of yeast vma1 delta cells by a plant subunit A homolog rescues the mutant phenotype and partially restores vacuolar H(+)-ATPase activity

The ability of a vacuolar H(+)-ATPase (V-ATPase) subunit homolog (subunit A) from plants to rescue the vma mutant phenotype of yeast was investigated as a first step towards investigating the structure and function of plant subunits in molecular detail. Heterologous expression of cotton cDNAs encoding near-identical isoforms of subunit A in mutant vma1 delta yeast cells successfully rescued the mutant vma phenotype, indicating that subunit A of plants and yeast have retained elements essential to V-ATPases during the course of evolution. Although vacuoles become acidified, the plant-yeast hybrid holoenzyme only partially restored V-ATPase activity (approximately 60%) in mutant yeast cells. Domain substitution of divergent N- or C-termini only slightly enhanced V-ATPase activity, whereas swapping both domains acted synergistically, increasing coupled ATP hydrolysis and proton translocation by approximately 22% relative to the native plant subunit. Immunoblot analysis indicated that similar amounts of yeast, plant or plant-yeast chimeric subunits are membrane-bound. These results suggest that subunit A terminal domains contain structural information that impact V-ATPase structure and function.     

A plant proton-pumping inorganic pyrophosphatase functionally complements the vacuolar ATPase transport activity and confers bafilomycin resistance in yeast

V-ATPases (vacuolar H+-ATPases) are a specific class of multi-subunit pumps that play an essential role in the generation of proton gradients across eukaryotic endomembranes. Another simpler proton pump that co-localizes with the V-ATPase occurs in plants and many protists: the single-subunit H+-PPase [H+-translocating PPase (inorganic pyrophosphatase)]. Little is known about the relative contribution of these two proteins to the acidification of intracellular compartments. In the present study, we show that the expression of a chimaeric derivative of the Arabidopsis thaliana H+-PPase AVP1, which is preferentially targeted to internal membranes of yeast, alleviates the phenotypes associated with V-ATPase deficiency. Phenotypic complementation was achieved both with a yeast strain with its V-ATPase specifically inhibited by bafilomycin A1 and with a vma1-null mutant lacking a catalytic V-ATPase subunit. Cell staining with vital fluorescent dyes showed that AVP1 recovered vacuole acidification and normalized the endocytic pathway of the vma mutant. Biochemical and immunochemical studies further demonstrated that a significant fraction of heterologous H+-PPase is located at the vacuolar membrane. These results raise the question of the occurrence of distinct proton pumps in certain single-membrane organelles, such as plant vacuoles, by proving yeast V-ATPase activity dispensability and the capability of H+-PPase to generate, by itself, physiologically suitable internal pH gradients. Also, they suggest new ways of engineering macrolide drug tolerance and outline an experimental system for testing alternative roles for fungal and animal V-ATPases, other than the mere acidification of subcellular organelles.