Vacuolar Proton ATPase Research Articles

A search for doxycycline-dependent mutations that increase Drosophila melanogaster life span identifies the VhaSFD, Sugar baby, filamin, fwd and Cctl genes

Screening for conditional mutations that increase Drosophila life span has identified genes implicated in membrane transport, phospholipid metabolism and signaling, and actin cytoskeleton organization.

The intramacrophagic environment of Brucella suis and bacterial response

Phagocytes have developed various antimicrobial defense mechanisms to eliminate pathogens. They comprise the oxidative burst, acidification of phagosomes, or fusion of phagosomes with lysosomes. Facultative intracellular bacteria, in return, have developed strategies counteracting the host cell defense, resulting in intramacrophagic survival. Until lately, only very little was known about the phagosomal compartment containing Brucella spp., the environmental conditions the bacteria encounter, and the pathogen’s stress response. Recently, we have determined that the phagosomes acidify rapidly to a pH of 4.0–4.5 following infection, but this early acidification is crucial for intracellular replication as neutralization results in bacterial elimination. A vacuolar proton-ATPase is responsible for this phenomenon that is not linked to phagosome–lysosome fusion. On the contrary, in vitro reconstitution assays revealed association only between phagosomes containing killed B. suis and lysosomes, describing the absence of phagolysosome fusion due to specific recognition inhibition for live bacteria. Further evidence for the necessity of an intact, acidic phagosome as a predominant niche of brucellae in macrophages was obtained with a strain of B. suis secreting listeriolysin. It partially disrupts the phagosomal membranes and fails to multiply intracellularly. How does B. suis adapt to this environment? We have identified and studied a series of genes that are involved in this process of adaptation. The bacterial heat shock protein and chaperone DnaK is induced in phagocytes and it is essential for intracellular multiplication. A low-level, constitutive expression of dnaK following promoter exchange does not restore intramacrophagic survival. Another chaperone and heat shock protein, ClpB, belonging to the family of ClpATPases, is important for the resistance of B. suis to several in vitro stresses, but does not contribute to intramacrophagic survival of the pathogen. Additional bacterial genes specifically induced within the phagocyte were identified by an intramacrophagic screen of random promoter fusions to the reporter gene gfp. A large majority of these genes are encoding proteins involved in transport of nutrients (sugars, amino acids), or cofactors, such as nickel. Analysis of the intracellular gene activation reveals that low oxygen tension is encountered by B. suis. Altogether, these results suggest three major stress conditions encountered by brucellae in the phagosome: acid stress, starvation and low oxygen tension.

Abnormal Acidification of Melanoma Cells Induces Tyrosinase Retention in the Early Secretory Pathway

In tyrosinase-positive amelanotic melanoma cells, inactive tyrosinase accumulates in the endoplasmic reticulum. Based on studies described here, we propose that aberrant vacuolar proton ATPase (V-ATPase)-mediated proton transport in melanoma cells disrupts tyrosinase trafficking through the secretory pathway. Amelanotic but not melanotic melanoma cells or normal melanocytes display elevated proton export as observed by the acidification of the extracellular medium and their ability to maintain neutral intracellular pH. Tyrosinase activity and transit through the Golgi were restored by either maintaining the melanoma cells in alkaline medium (pH 7.4-7.7) or by restricting glucose uptake. The translocation of tyrosinase out of the endoplasmic reticulum and the induction of cell pigmentation in the presence of the ionophore monensin or the specific V-ATPase inhibitors concanamycin A and bafilomycin A1 supported a role for V-ATPases in this process. Because it was previously shown that V-ATPase activity is increased in solid tumors in response to an acidified environment, the appearance of hypopigmented cells in tyrosinase-positive melanoma tumors may indicate the onset of enhanced glycolysis and extracellular acidification, conditions known to favor metastatic spread and resistance to weak base chemotherapeutic drugs.

In Vivo Treatment With Granulocyte Colony-Stimulating Factor Does Not Delay Apoptosis in Human Neutrophils by Increasing the Expression of the Vacuolar Proton ATPase

BackgroundNeutrophils die by apoptosis, and in vivo administration of granulocyte colony-stimulating factor (G-CSF) delays this apoptotic cell death. G-CSF administered in vitro correlates delayed apoptosis with upregulation of the vacuolar...

The Polyphosphate Bodies of Chlamydomonas reinhardtii Possess a Proton-pumping Pyrophosphatase and Are Similar to Acidocalcisomes

Acidocalcisomes are acidic calcium storage compartments described initially in trypanosomatid and apicomplexan parasites. In this work, we describe organelles with properties similar to acidocalcisomes in the green alga Chlamydomonas reinhardtii. Nigericin and NH(4)Cl released (45)Ca(2+) from preloaded permeabilized cells, suggesting the incorporation of a significant amount of this cation into an acidic compartment. X-ray microanalysis of the electron-dense vacuoles or polyphosphate bodies of C. reinhardtii showed large amounts of phosphorus, magnesium, calcium, and zinc. Immunofluorescence microscopy, using antisera raised against a peptide sequence of the vacuolar type proton pyrophosphatase (H(+)-PPase) of Arabidopsis thaliana which is conserved in the C. reinhardtii enzyme, indicated localization in the plasma membrane, in intracellular vacuoles, and the contractile vacuole where it colocalized with the vacuolar proton ATPase (V-H(+)-ATPase). Purification of the electron-dense vacuoles using iodixanol density gradients indicated a preferential localization of the H(+)-PPase and the V-H(+)-ATPase activities in addition to high concentrations of PP(i) and short and long chain polyphosphate, but lack of markers for mitochondria and chloroplasts. In isolated electron-dense vacuoles, PP(i)-driven proton translocation was stimulated by potassium ions and inhibited by the PP(i) analog aminomethylenediphosphonate. Potassium fluoride, imidodiphosphate, N,N'-dicyclohexylcarbodiimide, and N-ethylmaleimide also inhibited PP(i) hydrolysis in the isolated organelles in a dose-dependent manner. These results indicate that the electron-dense vacuoles of C. reinhardtii are very similar to acidocalcisomes with regard to their chemical composition and the presence of proton pumps. Polyphosphate was also localized to the contractile vacuole by 4',6-diamidino-2-phenylindole staining, suggesting, with the immunochemical data, a link between these organelles and the acidocalcisomes.

Immunochemical analysis of the vacuolar proton-ATPase B-subunit in the gills of a euryhaline stingray (Dasyatis sabina): effects of salinity and relation to Na(+)/K(+)-ATPase.

In the gills of freshwater teleost fishes, vacuolar proton-ATPase (V-H(+)-ATPase) is found on the apical membrane of pavement and chloride (Na(+)/K(+)-ATPase-rich) cells, and is an important transporter for energizing Na(+) uptake and H(+) excretion. In the gills of elasmobranch fishes, the V-H(+)-ATPase has not been extensively studied and its expression in freshwater individuals has not been examined. The goals of this study were to examine the effects of environmental salinity on the expression of V-H(+)-ATPase in the gills of an elasmobranch (the Atlantic stingray, Dasyatis sabina) and determine if V-H(+)-ATPase and Na(+)/K(+)-ATPase are expressed in the same cells. We found that gills from freshwater stingrays had the highest relative abundance of V-H(+)-ATPase and greatest number of V-H(+)-ATPase-rich cells, using immunoblotting and immunohistochemistry, respectively. When freshwater animals were acclimated to sea water for 1 week, V-H(+)-ATPase abundance and the number of V-H(+)-ATPase-rich cells decreased significantly. Atlantic stingrays from seawater environments were characterized by the lowest expression of V-H(+)-ATPase and least number of V-H(+)-ATPase-rich cells. In contrast to teleost fishes, localization of V-H(+)-ATPase in freshwater stingray gills was not found in pavement cells and occurred on the basolateral membrane in cells that are presumably rich in mitochondria. In freshwater stingrays acclimated to sea water and seawater stingrays, V-H(+)-ATPase localization appeared qualitatively to be stronger in the cytoplasm, which may suggest the transporter was stored in vesicles. Using a double-immunolabeling technique, we found that V-H(+)-ATPase and Na(+)/K(+)-ATPase occurred in distinct cells, which suggests there may be two types of mitochondrion-rich cells in the elasmobranch gill epithelium. Based on these findings, we propose a unique model of NaCl and acid-base regulation where the V-H(+)-ATPase-rich cells and Na(+)/K(+)-ATPase-rich cells are the sites of Cl(-) uptake/HCO(3)(-) excretion and Na(+) uptake/H(+) excretion, respectively.

Three-dimensional structure of the vacuolar ATPase proton channel by electron microscopy.

Vacuolar ATPases are ATP hydrolysis-driven proton pumps found in the endomembrane system of eucaryotic cells where they are involved in pH regulation. We have determined the three-dimensional structure of the proton channel domain of the vacuolar ATPase from bovine brain clathrin-coated vesicles by electron microscopy at 21 A resolution. The model shows an asymmetric protein ring with two small openings on the luminal side and one large opening on the cytoplasmic side. The central hole on the luminal side is covered by a globular protein, while the cytoplasmic opening is covered by two elongated proteins arranged in a collar-like fashion.

ATP6H, a subunit of vacuolar ATPase involved in metal transport: evaluation in canine copper toxicosis

Copper toxicosis (CT), resulting in liver disease, occurs commonly in Bedlington terriers. Canine CT is of particular interest because identification of the causative gene may lead to the discovery of another important gene in the copper transport pathway possibly related to human copper diseases not yet identified. Homologs of the copper transporting ATPase ATP7B, defective in Wilson disease, and the copper chaperone ATOX1 were potential candidates, but both have been excluded. The CT locus in Bedlington terriers has been mapped to canine chromosome region CFA10q26, which has a syntenic human chromosome region, HAS2p13-21. The gene ATP6H, for human vacuolar proton-ATPase subunit M9.2, is associated with copper and iron transport in yeast and has been mapped to HAS2p21 and suggested as a candidate gene for CT. We cloned canine ATP6H, which encodes a predicted protein with 99% amino acid sequence identity to the orthologous human protein. Canine ATP6H shows a conserved potential metal binding site, CSVCC, and a glycosylation site, NET. The canine ATP6H is organized into four exons, with a 246-bp open reading frame. Sequence analysis of the coding regions showed no mutations in ATP6H from genomic DNA of an affected dog. We have also identified two, apparently non-transcribed canine ATP6H pseudogenes. Mapping of the true ATP6H gene and a marker closely linked to the CT locus on a canine radiation hybrid panel indicted lack of close physical association. We have therefore excluded canine ATP6H as a candidate gene for canine copper toxicosis, indicating that some other unidentified gene is responsible for this copper storage disease.

Involvement of vacuolar proton ATPase in Junin virus multiplication.

The role of vacuolar-proton ATPase (V-H+ ATPAse) on Junin virus (JV) replication was evaluated by analyzing the effect of specific inhibitors of the enzyme activity on different steps of virus multiplication cycle. The presence of the macrolide antibiotics bafilomycin A1 and concanamycin A during the first two hours of infection caused a significant reduction of extracellular infectious virus production and viral protein expression in Vero and BHK-21 cells. The inhibitory action of the compounds was mainly exerted at an early stage of the JV multiplication cycle, without affecting virus attachment to the cell but preventing virus penetration. A correlation between the inhibitory action of the compounds on intracellular compartments acidification and the reduction of JV yield was observed. The addition of concanamycin A at different times after infection indicated that the compound also interferes with the release of infectious particles to the extracellular medium. Although, intracellular transport of JV glycoproteins to the cell membrane, seems not to be affected as revealed by immunofluorescence staining. The results confirm that JV enters into the cell through the endocytic pathway as previously suggested by using lysosomotropic compounds.

Effect of bafilomycin A1, a specific inhibitor of vacuolar (V-type) proton ATPases, on the capacitation of rabbit spermatozoa

Mammalian spermatozoa maintain precisely regulated ionic gradients that must be modified during capacitation and the acrosome reaction. In other cell types, ionic gradients are mainly regulated by the presence in plasma membranes of three metabolically different types of ATPases. The modifications induced during in vitro capacitation of rabbit spermatozoa by the specific inhibition of V-type H+-ATPases with bafilomycin A were studied. We used chlortetracycline binding to rabbit spermatozoa to monitor capacitation, and the coomassie brilliant blue method to identify acrosome-reacted sperm cells. There was a significant difference between the percentage of epididymal (66 +/- 7%) and ejaculated (43 +/- 11%) spermatozoa capacitated in vitro, after a 6-h incubation period in the presence of Ca2+ without ATPase inhibitor. The presence of bafilomycin significantly reduced these numbers (25 +/- 11 and 16+/- 8%, epididymal and ejaculated spermatozoa, respectively) and eliminated the difference. Ejaculated spermatozoa capacitated in the absence of bafilomycin showed a linear increase in the percentage of acrosome reactions induced by the addition of A23187 (12 +/- 5, 23+/- 6 and 31 +/- 5 after 15, 30 and 45 min). The presence of 0.2 micromol l-1 bafilomycin during the capacitation incubation induced a significant decrease in the acrosome reaction percentages (4 +/- 2, 8 +/- 3 and 14 +/- 4 after 15, 30 and 45 min). The addition of bafilomycin after the capacitating period had no effect upon the induction of the acrosome reaction by A23187. These results indicate that vacuolar ATPases play an important role during rabbit sperm capacitation. However, once the spermatozoa have been capacitated, V-type ATPases do not have a significant participation during the acrosome reaction.

Stimulation of p38 MAP kinase reduces acidosis and Na + overload in preconditioned hepatocytes

Ischemic preconditioning has been shown to improve liver resistance to hypoxia/reperfusion damage. A signal pathway involving A 2A-adenosine receptor, G i-proteins, protein kinase C and p38 MAP kinase is responsible for the development of hypoxic preconditioning in hepatocytes. However, the coupling of this signal pathway with the mechanisms responsible for cytoprotection is still unknown. We have observed that stimulation of A 2A-adenosine receptors or of p38 MAPK by CGS21680 or anisomycin, respectively, appreciably reduced intracellular acidosis and Na + accumulation developing during hypoxia. These effects were reverted by p38 MAPK inhibitor SB203580 as well as by blocking vacuolar proton ATPase with bafilomycin A 1. SB203580 and bafilomycin A 1 also abolished the cytoprotective action exerted by both CGS21680 and anisomycin. We propose that the stimulation of p38 MAPK by preconditioning might increase hepatocyte resistance to hypoxia by activating proton extrusion through vacuolar proton ATPase, thus limiting Na + overload promoted by Na +-dependent acid buffering systems.

Entry of poliovirus into cells is blocked by valinomycin and concanamycin A.

Poliovirus contains a virus particle devoid of a lipid envelope that does not require an intact pH to enter into susceptible cells. Thus, the blockade of pH gradient generated in endosomes is not sufficient to impede the translocation of poliovirus particles to the cytoplasm, suggesting that translocation takes place at the plasma membrane. Measuring both viral protein synthesis and eIF4G-1 cleavage mediated by poliovirus protease 2A has been used to monitor productive entry of poliovirus into cells. Translation of the input poliovirus RNA produces enough 2A(pro) to cleave eIF4G-1, providing a sensitive assay to estimate poliovirus RNA delivery to the cytoplasm followed by its translation. Combination of concanamycin A, a vacuolar proton-ATPase inhibitor, and valinomycin, an ionophore that promotes K(+) efflux from cells, powerfully prevented poliovirus infection. Moreover, modifying the ionic conditions of the culture medium (increasing the concentration of K(+) and decreasing the concentration of Na(+)), together with concanamycin A, also significantly interfered with poliovirus entry. These findings suggest that poliovirus RNA requires an intact concentration of K(+) ions inside the cells to be uncoated and to gain access to the cytoplasm. In addition, the actual contribution of concanamycin A (as well as other inhibitors of endocytosis) to the total inhibition of productive poliovirus entry points to the idea that at least some percentage of polioviral subparticles translocates from the endosomes.

Characterization of Inhibitory Action of Concanamycins against Herpes Simplex Virus

Concanamycins A (Conmy A) and B (Conmy B), well-known inhibitors of the vacuolar proton-ATPase, were isolated from the culture broth of Streptomyces sp. strain FK51 as antiherpetic agents. These compounds showed potent inhibition of herpes simplex virus type 1 (HSV-1) replication in an in vitro assay system, having antiviral activities with 50% inhibitory concentrations of 0.072 and 0.51 ng/ml for Conmy A and Conmy B, respectively. While the attachment of HSV-1 to Vero cells was not inhibited, both of the compounds blocked the penetration of virus into host cells. When added to the late stages of virus replication, the concanamycins also exerted marked inhibitory effects on the production of viruses. Release of progeny viruses was found to be suppressed by the agents. SDS-PAGE analysis of isotope-labelled HSV-specific proteins revealed that the synthesis of beta proteins was moderately inhibited and some of the glycoproteins were synthesized with reduced molecular weights. Western blot analysis using antibodies against two HSV-specific glycoproteins (gC and gD) showed differences in their syntheses between untreated and Conmy A-treated cells. Syncytium formation by HSV-1 strain HF was inhibited, and small plaques with rounded cells were formed in Conmy A-treated cell cultures. When wild-type HSV-1 was serially propagated under the selective pressure of Conmy A, and the resulting progeny viruses were grown in drug-free medium, their plaque morphology of syncytium and sensitivity to Conmy A were the same as those of parent virus. From these findings, antiherpetic activities of Conmy A and B might be mainly dependent on their activities as vacuolar proton-ATPase inhibitors with intracellular translocation of glycoproteins and the inhibition of the maturation of virus glycoproteins.

Structure of the VPATPD Gene Encoding Subunit D of the Human Vacuolar Proton ATPase

The HSD11B2 and VPATPD genes encoding the human kidney isozyme of 11β-hydroxysteroid dehydrogenase (11-HSD2) and subunit D of the vacuolar proton ATPase, respectively, are located on chromosome 16q22. They are transcribed from complementary DNA strands and their 3′ ends are only 0.5 kilobase apart. Because polymorphisms in HSD11B2 have been associated with hypertension and salt sensitivity, we characterized the human VPATPD gene. It spans 19 kb and consists of 8 exons. The encoded protein is 99.5% identical to mouse subunit D at the amino acid level. An alternating purine-pyrimidine tract is located in the 3′-untranslated region of VPATPD. On genotyping 17 hypertensive subjects, no length polymorphism was found. Although VPATPD and HSD11B2 are both expressed in kidney and placenta, they are regulated differently; forskolin upregulates HSD11B2 but not VPATPD in human choriocarcinoma JEG3 cells. The functional significance of the proximity of these two genes remains to be established.

Human papillomavirus type 16 E5 protein localizes to the Golgi apparatus but does not grossly affect cellular glycosylation.

The E5 protein of papillomaviruses is a strongly hydrophobic membrane protein that can associate with the 16 kDa protein subunit of the vacuolar proton ATPase in endosomes and the Golgi apparatus resulting in raise of intraorganelle pH. We demonstrate that E5 of human papillomavirus type 16 (HPV16) when transfected into human keratinocytes localizes to the Golgi. Using FACS analysis and western blotting with a variety of lectins as well as analysing the sialylation status of a specific cell surface glycoprotein CD95 (APO-1/Fas), we show that HPV16 E5 does not grossly affect cellular glycosylation, a main Golgi function.

Vacuole acidification is not required for survival of Salmonella enterica serovar typhimurium within cultured macrophages and epithelial cells.

Phagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellular Salmonella enterica serovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential for Salmonella intracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.

Differential Involvement of Vacuolar H+-ATPase in the Refilling of Thapsigargin- and Agonist-Mobilized Ca2+ Stores

Our objective was to evaluate the role of vacuolar H+-ATPase and proton gradients in the refilling of Ca2+ stores in fura-2-loaded pancreatic acinar cells. Once depleted with a high level of ACh, the Ca2+ stores were replenished with a Ca2+-containing solution. The degree of refilling was estimated with a second release in response to either ACh (ACh-releasable store) or thapsigargin (thapsigargin-releasable store), a specific inhibitor of the endoplasmic reticulum Ca2+ pumps. Both the protonophore nigericin and folimycin, a specific inhibitor of the vacuolar H+-ATPase, reduced reuptake into the ACh-mobilized stores but not into the thapsigargin-releasable pools. These treatments effectively dissipated the subcellular pH gradients (revealed by confocal observation of the distribution of a marker for acidic compartments), and did not impair the [Ca2+]i response to ACh in control cells. Our results indicate that thapsigargin and ACh release heterogeneous Ca2+ stores which are differently operated by vacuolar proton ATPase.

The gene encoding the mouse homologue of the human osteoclast-specific 116-kDa V-ATPase subunit bears a deletion in osteosclerotic ( oc/oc) mutants

Osteosclerosis ( oc) is an autosomal recessive lethal mutation that impairs bone resorption by osteoclasts, and induces a general increase of bone density in affected mice. Genetic mapping of the oc mutation was used as a backbone in a positional cloning approach in the pericentromeric region of mouse chromosome 19. Perfect cosegregation of the osteopetrotic phenotype with polymorphic markers enabled the construction of a sequence-ready bacterial artificial chromosome (BAC) contig of this region. Genomic sequencing of a 200-kb area revealed the presence of the mouse homologue to the human gene encoding the osteoclast-specific 116-kDa subunit of the vacuolar proton pump. This gene was located recently on human 11q13, a genomic region conserved with proximal mouse chromosome 19. Sequencing of the 5′ end of the gene in oc/oc mice showed a 1.6-kb deletion, including the translation start site, which impairs genuine transcription of this subunit. The inactivation of this osteoclast-specific vacuolar proton ATPase subunit could be responsible for the lack of this enzyme in the apical membranes of osteoclast cells in oc/oc mice, thereby preventing the resorption function of these cells, which leads to the osteopetrotic phenotype.

Ischemic preconditioning reduces Na(+) accumulation and cell killing in isolated rat hepatocytes exposed to hypoxia.

Short periods of ischemia followed up by reperfusion are known to protect the heart against injury caused by a subsequent sustained ischemia. This phenomenon, known as ischemic preconditioning, has also been recently shown to reduce ischemic liver damage, but the mechanisms involved are still unknown. By using isolated hepatocytes as an in vitro model of liver preconditioning, we have investigated the possible effect of preconditioning on intracellular pH and Na(+) homeostasis. Freshly isolated rat hepatocytes were preconditioned by 10 minutes of incubation under hypoxic conditions followed up by 10 minutes of reoxygenation and subsequently exposed to 90 minutes of hypoxia. Although preconditioning did not ameliorate adenosine triphosphate (ATP) depletion, preconditioned hepatocytes exhibited an increased resistance to cell killing during hypoxic incubation. Intracellular acidosis and Na(+) accumulation developing during hypoxia were appreciably reduced in preconditioned cells. The effects of preconditioning on intracellular pH, Na(+) homeostasis, and cytotoxicity were mimicked by stimulating protein kinase C (PKC) with 4beta-phorbol-12-myristate-13-acetate (PMA) or 1,2 dioctanoyl-glycerol (1,2 DOG). Conversely, inhibiting PKC with chelerythrine or blocking vacuolar proton ATPase (V-ATPase) with bafilomycin A(1) abolished the protection given by preconditioning or by PMA treatment on hypoxic acidosis, Na(+) overload, and hepatocyte killing. Similarly, the addition of Na(+) ionophore monensin also reverted the cytoprotection exerted by preconditioning. This indicated that ischemic preconditioning of isolated hepatocytes decreased cell killing during hypoxia by preventing intracellular Na(+) accumulation. We propose that, after preconditioning, the stimulation of PKC might activate proton extrusion through V-ATPase, thus, limiting intracellular acidosis and Na(+) overload promoted by Na(+)-dependent acid buffering systems.

Vacuole acidification is required for trans-SNARE pairing, LMA1 release, and homotypic fusion.

Vacuole fusion occurs in three stages: priming, in which Sec18p mediates Sec17p release, LMA1 (low M(r) activity 1) relocation, and cis-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex disassembly; docking, mediated by Ypt7p and trans-SNARE association; and fusion of docked vacuoles. Ca(2+) and calmodulin regulate late stages of the reaction. We now show that the vacuole proton gradient, generated by the vacuolar proton ATPase, is needed for trans-SNARE complex formation during docking and hence for the subsequent LMA1 release. Though neither the vacuolar Pmc1p Ca(2+)-ATPase nor the Vcx1p Ca(2+)/H(+) exchanger are needed for the fusion reaction, they participate in Ca(2+) and Delta mu(H)(+) homeostasis. Fusion itself does not require the maintenance of trans-SNARE pairs.