Abscisic Acid Induction of Vacuolar H+-ATPase Activity in Mesembryanthemum crystallinum Is Developmentally Regulated

Abscisic acid (ABA) has been implicated as a key component in water-deficit-induced responses, including those triggered by drought, NaCl, and low- temperature stress. In this study a role for ABA in mediating the NaCl-stress-induced increases in tonoplast H⁺-translocating ATPase (V-ATPase) and Na⁺/H⁺ antiport activity in Mesembryanthemum crystallinum, leading to vacuolar Na⁺ sequestration, were investigated. NaCl or ABA treatment of adult M. crystallinum plants induced V-ATPase H⁺ transport activity, and when applied in combination, an additive effect on V-ATPase stimulation was observed. In contrast, treatment of juvenile plants with ABA did not induce V-ATPase activity, whereas NaCl treatment resulted in a similar response to that observed in adult plants. Na⁺/H⁺ antiport activity was induced in both juvenile and adult plants by NaCl, but ABA had no effect at either developmental stage. Results indicate that ABA-induced changes in V-ATPase activity are dependent on the plant reaching its adult phase, whereas NaCl-induced increases in V-ATPase and Na⁺/H⁺ antiport activity are independent of plant age. This suggests that ABA-induced V-ATPase activity may be linked to the stress-induced, developmentally programmed switch from C₃ metabolism to Crassulacean acid metabolism in adult plants, whereas, vacuolar Na⁺ sequestration, mediated by the V-ATPase and Na⁺/H⁺ antiport, is regulated through ABA-independent pathways.     

SOS2 promotes salt tolerance in part by interacting with the vacuolar H+-ATPase and upregulating its transport activity

The salt overly sensitive (SOS) pathway is critical for plant salt stress tolerance and has a key role in regulating ion transport under salt stress. To further investigate salt tolerance factors regulated by the SOS pathway, we expressed an N-terminal fusion of the improved tandem affinity purification tag to SOS2 (NTAP-SOS2) in sos2-2 mutant plants. Expression of NTAP-SOS2 rescued the salt tolerance defect of sos2-2 plants, indicating that the fusion protein was functional in vivo. Tandem affinity purification of NTAP-SOS2-containing protein complexes and subsequent liquid chromatography-tandem mass spectrometry analysis indicated that subunits A, B, C, E, and G of the peripheral cytoplasmic domain of the vacuolar H⁺-ATPase (V-ATPase) were present in a SOS2-containing protein complex. Parallel purification of samples from control and salt-stressed NTAP-SOS2/sos2-2 plants demonstrated that each of these V-ATPase subunits was more abundant in NTAP-SOS2 complexes isolated from salt-stressed plants, suggesting that the interaction may be enhanced by salt stress. Yeast two-hybrid analysis showed that SOS2 interacted directly with V-ATPase regulatory subunits B1 and B2. The importance of the SOS2 interaction with the V-ATPase was shown at the cellular level by reduced H⁺ transport activity of tonoplast vesicles isolated from sos2-2 cells relative to vesicles from wild-type cells. In addition, seedlings of the det3 mutant, which has reduced V-ATPase activity, were found to be severely salt sensitive. Our results suggest that regulation of V-ATPase activity is an additional key function of SOS2 in coordinating changes in ion transport during salt stress and in promoting salt tolerance.     

Requirement for Ergosterol in V-ATPase Function Underlies Antifungal Activity of Azole Drugs

Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H⁺-ATPase function, which is known to be essential for fungal virulence. Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma ⁻ phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca²⁺ and H⁺ surges triggered by the antimicrobial agent amiodarone, and impaired Ca²⁺ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.     

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.     

Regulation of the Fast Vacuolar Channel by Cytosolic and Vacuolar Potassium

At resting cytosolic Ca²⁺, passive K⁺ conductance of a higher plant tonoplast is likely dominated by fast vacuolar (FV) channels. This patch-clamp study describes K⁺-sensing behavior of FV channels in Beta vulgaris taproot vacuoles. Variation of K⁺ between 10 and 400 mM had little effect on the FV channel conductance, but a pronounced one on the open probability. Shift of the voltage dependence by cytosolic K⁺ could be explained by screening of the negative surface charge with a density σ = 0.25 e⁻/nm². Vacuolar K⁺ had a specific effect on the FV channel gating at negative potentials without significant effect on closed-open transitions at positive ones. Due to K⁺ effects at either membrane side, the potential at which the FV channel has minimal activity was always situated at ~50 mV below the potassium equilibrium potential, E_K⁺. At tonoplast potentials below or equal to E_K⁺, the FV channel open probability was almost independent on the cytosolic K⁺ but varied in a proportion to the vacuolar K⁺. Therefore, the release of K⁺ from the vacuole via FV channels could be controlled by the vacuolar K⁺ in a feedback manner; the more K⁺ is lost the lower will be the transport rate.     

Dynamics of activity and structure of the tonoplast vacuolar-type H+-ATPase in plants with differing CAM expression and in a C3 plant under salt stress

Summary Differences in the activity and structure of the vacuolar H⁺-ATPase (V-ATPase, EC 3.6.1.3) were investigated in the C₃/CAM intermediate plantKalanchoë blossfeldiana Poellnitz cv. Tom Thumb, with lower or higher expression of CAM, andHordeum vulgare cv. Carina, grown with or without 150 mM NaCl. InK. blossfeldiana ATP-hydrolysis and H⁺-transport activity were higher with higher expression of CAM than in plants with very weak CAM. This was mainly due to a larger amount of V-ATPase. Statistical analysis of the diameter of intramembrane particles (IMPs) on freeze-fractures of tonoplast vesicles showed that IMPs were larger in tonoplast vesicle preparations ofK. blossfeldiana with strong CAM expression (9.1 nm) than in preparations ofK. blossfeldiana with low CAM expression (7.3 nm). As there is evidence that the majority of IMPs on freeze-fractures of tonoplast vesicles corresponds to the V₀ domain of V-ATPase, the higher activity of V-ATPase inK. blossfeldiana with stronger CAM could be a result of additional structural changes in its membrane-integral domain. The higher activity of V-ATPase inK. blossfeldiana with stronger CAM is discussed in relation to the requirement for a higher proton pumping capacity for nocturnal malate accumulation in the vacuole. The ATP-dependent H⁺-pumping activity inH. vulgare was higher under salt stress than in control plants, while the rates of ATP-hydrolysis and the size of IMPs were not affected by the salt treatment. The data presented here indicate that different mechanisms might increase the transport capacity of V-ATPase to meet the higher requirements of secondary active transport related to CAM expression and adaptation to salt stress.Abbrevations ATP adenosine triphosphate - CAM crassulacean acid metabolism - IMP intramembrane particles - V-ATPase vacuolar proton-translocating adenosine triphosphatase - V₀ domain membrane-integral domain of V-ATPase - V₁ domain membrane-peripheral domain of V-ATPase Dedicated to Prof. Dr. Eberhard Schnepf on the occasion of his retirement     

Vacuolar ATPase Regulates Surfactant Secretion in Rat Alveolar Type II Cells by Modulating Lamellar Body Calcium

Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase) is the enzyme responsible for pumping H⁺ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase) dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1), an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca²⁺ chelator, BAPTA-AM, the protein kinase C (PKC) inhibitor, staurosporine, and the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), KN-62. Baf A1 induced Ca²⁺ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca²⁺ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca²⁺ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.     

Enhanced V-ATPase activity contributes to the improved salt tolerance of transgenic tobacco plants overexpressing vacuolar Na(+)/H (+) antiporter AtNHX1

AtNHX1, a vacuolar Na⁺/H⁺ antiporter gene from Arabidopsis thaliana, was introduced into tobacco genome via Agrobacterium tumefaciens-mediated transformation to evaluate the role of vacuolar energy providers in plants salt stress response. Compared to the wild-type plants, over-expression of AtNHX1 increased salt tolerance in the transgenic tobacco plants, allowing higher germination rates of seeds and successful seedling establishment in the presence of toxic concentrations of NaCl. More importantly, the induced Na⁺/H⁺ exchange activity in the transgenic plants was closely correlated to the enhanced activity of vacuolar H⁺-ATPase (V-ATPase) when exposed to 200 mM NaCl. In addition, inhibition of V-ATPase activity led to the malfunction of Na⁺/H⁺ exchange activity, placing V-ATPase as the dominant energy provider for the vacuolar Na⁺/H⁺ antiporter AtNHX1. V-ATPase and vacuolar Na⁺/H⁺ antiporter thus function in an additive or synergistic way. Simultaneous overexpression of V-ATPase and vacuolar Na⁺/H⁺ antiporter might be appropriate for producing plants with a higher salt tolerance ability.     

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.     

Initial Steps in the Assembly of the Vacuole-Type H+-ATPase

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H⁺-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A⁺) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A⁺) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.     

ATP synthesis catalyzed by a V-ATPase: an alternative pathway for energy conservation operating in plant vacuoles?

The electrochemical H⁺ gradient generated in tonoplast vesicles isolated from maize seeds was found to be able to drive the reversal of the catalytic cycle of both vacuolar H⁺-pumps (Façanha and de Meis, 1998). Here we describe the reversibility of the vacuolar V-type H⁺-ATPase (V-ATPase) even in the absence of the H⁺ gradient in a water-Me₂SO co-solvent mixture, resulting in net synthesis of [γ-³²P]ATP from [³²P]P_i and ADP. The water-Me₂SO (5 to 20 %) media promoted inhibition of both PP_i hydrolysis and synthesis reactions whereas it slightly affected the ATP hydrolysis and clearly stimulated the ATP synthesis, which was unaffected by uncoupling agents (FCCP, Triton X-100 or NH₄⁺). This effect of Me₂SO on the ATP⇔³²P exchange reaction seems to be related to a decrease of the apparent K_m of the V-ATPase for P_i. The results are in accordance to the concept that the energetics of ATP synthesis catalysis depends on the solvation energies interacting in the enzyme microenvironment. A possible physiological significance of this phenomenon for the metabolism of desiccation-tolerant plant cells is discussed.Key words: bind energy, proton pumps, proton gradient, DMSO, corn seeds, V₁V₀-ATPase, membrane bound H⁺-pyrophosphatase     

Altered Na+ and Li+ Homeostasis in Saccharomyces cerevisiae Cells Expressing the Bacterial Cation Antiporter NhaA

The bacterial Na⁺(Li⁺)/H⁺ antiporter NhaA has been expressed in the yeast Saccharomyces cerevisiae. NhaA was present in both the plasma membrane and internal membranes, and it conferred lithium but not sodium tolerance. In cells containing the yeast Ena1-4 (Na⁺, Li⁺) extrusion ATPase, the extra lithium tolerance conferred by NhaA was dependent on a functional vacuolar H⁺ ATPase and correlated with an increase of lithium in an intracellular pool which exhibited slow efflux of cations. In yeast mutants without (Na⁺, Li⁺) ATPase, lithium tolerance conferred by NhaA was not dependent on a functional vacuolar H⁺ ATPase and correlated with a decrease of intracellular lithium. NhaA was able to confer sodium tolerance and to decrease intracellular sodium accumulation in a double mutant devoid of both plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase. These results indicate that the bacterial antiporter NhaA expressed in yeast is functional at both the plasma membrane and the vacuolar membrane. The phenotypes conferred by its expression depend on the functionality of plasma membrane (Na⁺, Li⁺) ATPase and vacuolar H⁺ ATPase.     

The glycolytic enzyme aldolase mediates assembly, expression, and activity of vacuolar H+-ATPase

Vacuolar H(+)-ATPases (V-ATPases) are a family of highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. How ATP is supplied for V-ATPase-mediated hydrolysis and for coupling of proton transport is poorly understood. We have reported that the glycolytic enzyme aldolase physically associates with V-ATPase. Here we show that aldolase interacts with three different subunits of V-ATPase (subunits a, B, and E). The binding sites for the V-ATPase subunits on aldolase appear to be on distinct interfaces of the glycolytic enzyme. Aldolase deletion mutant cells were able to grow in medium buffered at pH 5.5 but not at pH 7.5, displaying a growth phenotype similar to that observed in V-ATPase subunit deletion mutants. Abnormalities in V-ATPase assembly and protein expression observed in aldolase deletion mutant cells could be fully rescued by aldolase complementation. The interaction between aldolase and V-ATPase increased dramatically in the presence of glucose, suggesting that aldolase may act as a glucose sensor for V-ATPase regulation. Taken together, these findings provide functional evidence that the ATP-generating glycolytic pathway is directly coupled to the ATP-hydrolyzing proton pump through physical interaction between aldolase and V-ATPase.     

Organ-Specific Responses of Vacuolar H~+-ATPase in the Shoots and Roots of C_3 Halophyte Suaeda salsa to NaCl

Suaeda salsa L. is a halophytic species that is well adapted to high salinity. In order to understand its salt tolerance mechanism, we examined the growth and vacuolar H⁺-ATPase (V-ATPase) response to NaCl within the shoots and roots. The growth of shoots, but not roots, was dramatically stimulated by NaCl. Cl⁻ and Na⁺ were mainly accumulated in shoots. V-ATPase activity was significantly increased by NaCl in roots and especially in shoots. Interestingly, antisera ATP95 and ATP88b detected three V₁ subunits (66, 55 and 36 KDa) of V-ATPase only in shoots, while an 18 kDa V₀ subunit of V-ATPase was detected by both antisera in shoots and roots. It suggested that the tissue-specific characteristics of V-ATPase were related to the different patterns of growth and ion accumulation in shoots and roots of S. salsa.     

A Lithium-Sensitive and Sodium-Tolerant 3′-Phosphoadenosine-5′-Phosphatase Encoded by halA from the Cyanobacterium Arthrospira platensis Is Closely Related to Its Counterparts from Yeasts and Plants

3′-Phosphoadenosine-5′-phosphatase (PAPase) is required for the removal of toxic 3′-phosphoadenosine-5′-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg²⁺ for its activity and could use PAP or 3′-phosphoadenosine-5′-phosphosulfate as a substrate. HalA is sensitive to Li⁺ (50% inhibitory concentration [IC₅₀] = 3.6 mM) but only slightly sensitive to Na⁺ (IC₅₀ = 600 mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li⁺ and Na⁺, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li⁺ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.