Involvement of plasma membrane H+-ATPase in the nitrate-nutrition uptake and utilization in indica rice.

Utilization of nitrogen by crops is essential for sustainable agriculture. The transport of nitrate (NO3-) across the plasma membrane is a critical gateway for N uptake and subsequent utilization. This process requires proton (H+) coupled cotransport, which is driven by proton motive force, provided by plasma membrane (PM) H+-ATPase. In this report, two indica rice varieties [Meixiangzhan 2 (MXZ) and Jifengyou 1002 (JFY)] in South China were selected and cultivated in hydroponic solution with 0.5 mM or 2.0 mM NO3- as the N source. The JFY exhibited stronger growth with higher biomass than MXZ under both 0.5 mM and 2.0 mM NO3-. PM H+-ATPase activity of JFY roots was significantly higher than that of MXZ. The higher PM H+-ATPase activity in JFY was consistent with a higher abundance of PM H+-ATPase protein and higher transcription levels of OSAs, such as OSA2, OSA7 and OSA8 in roots, OSA3, OSA7 and OSA8 in leaves. The expression of nitrate transporters (OsNRT1;1b, OsNRT2.1, OsNRT2.2, and OsNAR2.1) were also higher in roots or shoots of JFY than those in MXZ. Under 0.5 mM and 2.0 mM NO3-, the NO3- absorption and translocation rate, nitrate content, as well as nitrate reductase (NR) activity were all significantly higher in JFY, as compared to those in MXZ. Taken together, in JFY and MXZ, a higher level of PM H+-ATPase protein and higher activity coupled with greater efficiency in nitrate uptake, translocation and assimilation, suggesting the existence of a close correlation between PM H+-ATPase and nitrate utilization in indica rice. PM H+-ATPase may one of the elite genes that can contribute to nitrate use efficiency in rice.

Ionic regulatory strategies of crabs: the transition from water to land.

Terrestrial crabs (brachyurans and anomurans) have invaded land following a variety of pathways from marine and/or via freshwater environments. This transition from water to land requires physiological, ecological, and behavioral adaptations to allow the exploitation of these new environmental conditions. Arguably, the management of salt and water balance (e.g., osmoregulation) is integral for their survival and success in an environment where predominantly low-salinity aquatic (e.g., freshwater) water sources are found, sometimes in only minimal amounts. This requires a suite of morphological and biochemical modifications, especially at the branchial chamber of semi-terrestrial and terrestrial crabs to allow reprocessing of urine to maximize ion uptake. Using knowledge gained from electrophysiology, biochemistry, and more recent molecular biology techniques, we present summarized updated models for ion transport for all major taxonomic groups of terrestrial crabs. This is an exciting and fast-moving field of research, and we hope that this review will stimulate further study. Terrestrial crabs retain their crown as the ideal model group for studying the evolutionary pathways that facilitated terrestrial invasion.

Regulation mechanism of exogenous nitric oxide on phenanthrene uptake by ryegrass roots.

Polycyclic aromatic hydrocarbons (PAHs) constitute a category of persistent organic contaminants that possess the potential to induce carcinogenic, teratogenic, and mutagenic consequences. Our previous findings have revealed that plant roots actively take up PAHs through co-transport with protons, and auxin can promote PAHs uptake by wheat roots. It remains unclear whether nitric oxide (NO), a signaling molecule involved in numerous physiological processes in plants and downstream of auxin, can affect PAHs uptake by plant roots. In our study, 50 µmol/L sodium nitroprusside (SNP) significantly enhanced phenanthrene uptake after 4 h of exposure. After the addition of SNP (50 µmol/L), the H+ flux on root surface increased, and H+-ATPase activity was activated, indicating that exogenous NO promotes phenanthrene uptake by plant roots via activating H+-ATPase. By studying the effects of 50 µmol/L cyclic guanosine monophosphate (cGMP), 5 mmol/L Ca2+, and 50 µmol/L adenosine monophosphate (AMP) on phenanthrene uptake by ryegrass roots and measuring root calcium-dependent protein kinases (CDPK) activity, we demonstrated that exogenous NO promotes phenanthrene uptake through the signaling pathway of NO, cGMP, Ca2+, CDPK, 14-3-3 protein and H+-ATPase. The results contribute significant insights into elucidating the underlying mechanisms of NO modulating PAHs absorption by plant roots, thereby offering crucial strategies for advancing food safety measures and enhancing the phytoremediation potential of soils and waters contaminated with PAHs.

Transcription factor SlSTOP1 regulates Small Auxin-Up RNA Genes for tomato root elongation under aluminum stress.

Aluminum (Al) stress, a prevalent constraint in acidic soils, inhibits plant growth by inhibiting root elongation through restricted cell expansion. The molecular mechanisms of Al-induced root inhibition, however, are not fully understood. This study aimed to elucidate the role of Small Auxin-up RNAs (SlSAURs), which function downstream of the key Al stress-responsive transcription factor SENSITIVE TO PROTON RHIZOTOXICITY 1 (SlSTOP1) and its enhancer STOP1-INTERACTING ZINC-FINGER PROTEIN 1 (SlSZP1), in modulating root elongation under Al stress in tomato (Solanum lycopersicum). Our findings demonstrated that tomato lines with knocked out SlSAURs exhibited shorter root lengths when subjected to Al stress. Further investigation into the underlying mechanisms revealed that SlSAURs interact with Type 2C Protein Phosphatases (SlPP2Cs), specifically D-clade Type 2C Protein Phosphatases (SlPP2C.Ds). This interaction was pivotal as it suppresses the phosphatase activity, leading to the degradation of SlPP2C.D's inhibitory effect on plasma membrane H+-ATPase. Consequently, this promoted cell expansion and root elongation under Al stress. These findings increase our understanding of the molecular mechanisms by which Al ions modulate root elongation. The discovery of the SlSAUR-SlPP2C.D interaction and its impact on H+-ATPase activity also provides a perspective on the adaptive strategies employed by plants to cope with Al toxicity, which may lead to the development of tomato cultivars with enhanced Al stress tolerance, thereby improving crop productivity in acidic soils.

ZmCRK1 negatively regulates maize's response to drought stress by phosphorylating plasma membrane H+-ATPase ZmMHA2.

Drought severely affects crop growth and yields. Stomatal regulation plays an important role in plant response to drought stress. Light-activated plasma membrane-localized proton ATPase (PM H+-ATPase) mainly promoted the stomatal opening. Abscisic acid (ABA) plays a dominant role in the stomatal closure during drought stress. It is not clear how PM H+-ATPase is involved in the regulation of ABA-induced stomatal closure. We found that a CALCIUM-DEPENDENT PROTEIN KINASE RELATED KINASE 1 (ZmCRK1), and its mutant zmcrk1 exhibited slow water loss in detached leaves, high-survival rate after drought stress, and sensitivity to stomatal closure induced by ABA. The ZmCRK1 overexpression lines are opposite. ZmCRK1 interacted with the maize PM H+-ATPase ZmMHA2. ZmCRK1 phosphorylated ZmMHA2 at the Ser-901 and inhibited its proton pump activity. ZmCRK1 overexpression lines and zmmha2 mutants had low H+-ATPase activity, resulting in impaired ABA-induced H+ efflux. Taken together, our study indicates that ZmCRK1 negatively regulates maize drought stress response by inhibiting the activity of ZmMHA2. Reducing the expression level of ZmCRK1 has the potential to reduce yield losses under water deficiency.

The Vacuolar H+-ATPase subunit C is involved in oligogalacturonide (OG) internalization and OG-triggered immunity.

In plants, the perception of cell wall fragments initiates signal transduction cascades that activate the immune response. Previous research on early protein dynamics induced by oligogalacturonides (OGs), pectin fragments acting as damage-associated molecular patterns (DAMPs), revealed significant phosphorylation changes in several proteins. Among them, the subunit C of the vacuolar H+-ATPase, known as DE-ETIOLATED 3 (DET3), was selected to elucidate its role in the OG-triggered immune response. The Arabidopsis det3 knockdown mutant exhibited defects in H2O2 accumulation, mitogen-activated protein kinases (MAPKs) activation, and induction of defense marker genes in response to OG treatment. Interestingly, the det3 mutant showed a higher basal resistance to the fungal pathogen Botrytis cinerea that, in turn, was completely reversed by the pre-treatment with OGs. Our results suggest a compromised ability of the det3 mutant to maintain a primed state over time, leading to a weaker defense response when the plant is later exposed to the fungal pathogen. Using fluorescently labelled OGs, we demonstrated that endocytosis of OGs was less efficient in the det3 mutant, implicating DET3 in the internalization process of OGs. This impairment aligns with the observed defect in the priming response in the det3 mutant, underscoring that proper internalization and signaling of OGs are crucial for initiating and maintaining a primed state in plant defense responses.

Characteristics of membrane transport, metabolism, and target protein binding of cyclic depsipeptide destruxin E in HeLa cells.

Cyclic peptides have attracted attention as new modalities for drug development owing to their unique pharmacokinetic and pharmacodynamic properties. Destruxin E, a 19-membered cyclodepsipeptide, is a promising candidate drug for cancer therapy. The purpose of the present study was to clarify the molecular mechanisms underlying membrane transport, metabolism, and the binding for target molecules of destruxin E in human cervical carcinoma HeLa cells used as a model of cancer cells. The influx transport and the intracellular metabolism of destruxin E were non-saturable and saturable, respectively, at up to 10 µM. The intracellular amounts of destruxin E and destruxin E-diol after incubation of destruxin E with the cells significantly decreased at 4 °C compared to those at 37 °C. Destruxin E-diol, but not destruxin E, undergoes efflux transport out of cells via P-gp/MDR1/ABCB1 and BCRP/ABCG2. The epoxide hydrolase EPHX2 functions as a potent metabolizing enzyme that can convert the epoxide of destruxin E to the destruxin E-diol. Treatment with an EPHX2 inhibitor increased the intracellular destruxin E levels and enhanced the inhibitory activity of vacuolar type-H+ ATPase. These results suggest that epoxide hydrolase could be a regulatory factor for intracellular destruxin E levels and its pharmacological activity.

The Archetypal Gamma-Core Motif of Antimicrobial Cys-Rich Peptides Inhibits H+-ATPases in Target Pathogens.

Human lactoferrin (hLf) is an innate host defense protein that inhibits microbial H+-ATPases. This protein includes an ancestral structural motif (i.e., γ-core motif) intimately associated with the antimicrobial activity of many natural Cys-rich peptides. Peptides containing a complete γ-core motif from hLf or other phylogenetically diverse antimicrobial peptides (i.e., afnA, SolyC, PA1b, PvD1, thanatin) showed microbicidal activity with similar features to those previously reported for hLf and defensins. Common mechanistic characteristics included (1) cell death independent of plasma membrane (PM) lysis, (2) loss of intracellular K+ (mediated by Tok1p K+ channels in yeast), (3) inhibition of microbicidal activity by high extracellular K+, (4) influence of cellular respiration on microbicidal activity, (5) involvement of mitochondrial ATP synthase in yeast cell death processes, and (6) increment of intracellular ATP. Similar features were also observed with the BM2 peptide, a fungal PM H+-ATPase inhibitor. Collectively, these findings suggest host defense peptides containing a homologous γ-core motif inhibit PM H+-ATPases. Based on this discovery, we propose that the γ-core motif is an archetypal effector involved in the inhibition of PM H+-ATPases across kingdoms of life and contributes to the in vitro microbicidal activity of Cys-rich antimicrobial peptides.

Dual-Mediated Roles of H+-ATPase in Alleviating the Phytotoxicity of Imazethapyr to Nontarget Wheat.

The regulation solutions and mechanisms of reducing pesticide phytotoxicity to nontarget plants are not well-defined and detailed. Here, we have proposed a new detoxification strategy to control the toxic effects of herbicide imazethapyr (IM) induced in wheat seedlings from the perspective of the plasma membrane (PM) H+-ATPase. We found that the changes in PM H+-ATPase activity have a regulatory effect on the phytotoxic effects induced by IM in plants. Treatment with PM H+-ATPase activators restored the reduced auxin content and photosynthetic efficiency caused by IM, thereby promoting plant growth. Application of a PM H+-ATPase inhibitor further reduced phosphorus content and significantly increased 2,4-dihydroxy-7-methoxy-2H,1,4-benzoxazin-3(4H)one (DIMBOA) and jasmonic acid levels. These effects indicate that auxin and DIMBOA may regulate plant growth trends and detoxification effects mediated by PM H+-ATPase. This work opens a new strategy for regulating herbicide toxicity to nontarget plants from the PM H+-ATPase.

The replacement of ergosterol with alternative sterols affects the physiological function of the yeast plasma membrane, including its H+-ATPase activity and resistance to antifungal drugs.

Sterols perform essential structural and signalling functions in living organisms. Ergosterol contributes to the fluidity, permeability, microdomain formation and functionality of proteins in the yeast membrane. In our study, desmosterol was the most successful at compensating for the lack of ergosterol in Saccharomyces cerevisiae, besides stigmasterol and sitosterol. These three sterols supported cell growth without causing severe morphological defects, unlike cholesterol, 7-dehydrocholesterol, lathosterol, cholestanol or lanosterol. Together with ergosterol, they were also able to bring the plasma membrane potential of hem1Δ cells closer to the level of the wild type. In addition, desmosterol conferred even higher thermotolerance to yeast than ergosterol. Some sterols counteracted the antifungal toxicity of polyenes, azoles and terbinafine to hem1Δ cells. Plant sterols (stigmasterol, sitosterol) and desmosterol ensured the glucose-induced activation of H+-ATPase in hem1Δ cells analogously to ergosterol, whereas cholesterol and 7-dehydrocholesterol were less effective. Exogenous ergosterol, stigmasterol, sitosterol, desmosterol and cholesterol also improved the growth of Candida glabrata and Candida albicans in the presence of inhibitory concentration of fluconazole. The proper incorporation of exogenous sterols into the membrane with minimal adverse side effects on membrane functions was mainly influenced by the structure of the sterol acyl chain, and less by their ring structures.

The fusicoccin story revisited.

Fusicoccin (FC) is one of the most studied fungal metabolites to date. The finding that the plasma membrane H+-ATPase in combination with 14-3-3 proteins acts as a high-affinity receptor for FC was a breakthrough in the field. Ever since, the binding of FC to the ATPase-14-3-3 receptor complex has taken center stage in explaining all FC-induced physiological effects. However, a more critical review shows that this is not evident for a number of FC-induced effects. This review challenges the notion that all FC-affected processes start with the binding to and activation of the plasma membrane ATPase, and raises the question of whether other proteins with a key role in the respective processes are directly targeted by FC. A second unresolved question is whether FC may be another example of a fungal molecule turning out to be a 'copy' of an as yet unknown plant molecule. In view of the evidence, albeit not conclusive, that plants indeed produce 'FC-like ligands', it is worthwhile making a renewed attempt with modern improved technology to answer this question; the answer might upgrade FC or its structural analogue(s) to the classification of plant hormone.

Indole-3-acetic acid enhances the co-transport of proton and phenanthrene mediated by TaSAUR80-5A in wheat roots.

Polycyclic aromatic hydrocarbons (PAHs) are a type of organic pollution that can accumulate in crops and hazard human health. This study used phenanthrene (PHE) as a model PAH and employed hydroponic experiments to illustrate the role of indole-3-acetic acid (IAA) in the regulation of PHE accumulation in wheat roots. At optimal concentrations, wheat roots treated with PHE + IAA showed a 46.9% increase in PHE concentration, whereas treatment with PHE + P-chlorophenoxyisobutyric acid resulted in a 38.77% reduction. Transcriptome analysis identified TaSAUR80-5A as the crucial gene for IAA-enhancing PHE uptake. IAA increases plasma membrane H+-ATPase activity, promoting active transport of PHE via the PHE/H+ cotransport mechanism. These results provide not only the theoretical basis necessary to better understand the function of IAA in PAHs uptake and transport by staple crops, but also a strategy for controlling PAHs accumulation in staple crops and enhancing phytoremediation of PAH-contaminated environments.

The Antimicrobial Activity of Human Defensins at Physiological Non-Permeabilizing Concentrations Is Caused by the Inhibition of the Plasma Membrane H+-ATPases.

Human defensins are cysteine-rich peptides (Cys-rich peptides) of the innate immune system. Defensins contain an ancestral structural motif (i.e., γ-core motif) associated with the antimicrobial activity of natural Cys-rich peptides. In this study, low concentrations of human α- and ß-defensins showed microbicidal activity that was not associated with cell membrane permeabilization. The cell death pathway was similar to that previously described for human lactoferrin, also an immunoprotein containing a γ-core motif. The common features were (1) cell death not related to plasma membrane (PM) disruption, (2) the inhibition of microbicidal activity via extracellular potassium, (3) the influence of cellular respiration on microbicidal activity, and (4) the influence of intracellular pH on bactericidal activity. In addition, in yeast, we also observed (1) partial K+-efflux mediated via Tok1p K+-channels, (2) the essential role of mitochondrial ATP synthase in cell death, (3) the increment of intracellular ATP, (4) plasma membrane depolarization, and (5) the inhibition of external acidification mediated via PM Pma1p H+-ATPase. Similar features were also observed with BM2, an antifungal peptide that inhibits Pma1p H+-ATPase, showing that the above coincident characteristics were a consequence of PM H+-ATPase inhibition. These findings suggest, for the first time, that human defensins inhibit PM H+-ATPases at physiological concentrations, and that the subsequent cytosolic acidification is responsible for the in vitro microbicidal activity. This mechanism of action is shared with human lactoferrin and probably other antimicrobial peptides containing γ-core motifs.

Exposure to Total Suspended Solids (TSS) Impacts Gill Structure and Function in Adult Zebrafish.

Total suspended solids (TSS) are a major contributor of anthropogenic impacts to aquatic systems. TSS exposure have been shown to affect the function of gills, but the mode of action is unclear. Zebrafish (Danio rerio) is emerging as an excellent model for mechanistic toxicology, and as there are no baseline studies on TSS effects in zebrafish gills, we tested the hypothesis that environmental concentrations of TSS damages gill structure and function in this species. Adult zebrafish were exposed to either 0, 10, 100, 500, 1000, or 2000 mg/L TSS for 4 days to assess the gill morphology. The minimal concentration that affected the gill structure was further tested for the distribution of key ion transporters, including Na+/K+- ATPase (NKA) and vacuolar-type H+-ATPase (VHA), using confocal microscopy. Our results reveal that TSS concentration as low as 100 mg/L alters the morphology of gills, including greater filament thickness, lamellae thickness, and epithelial lifting. This was also associated with a reduction in NKA immunoreactive (IR) cell count and intensity in the 100 mg/L TSS group, while there was neither a change in the VHA-IR cell count or expression nor the transcript abundance of atp6v1a and atp1a1a4 in the gills. Markers of stress response in these animals, including levels of cortisol, glucose, lactate, and glycogen were not altered after 4 days of TSS exposure. Overall, environmentally relevant concentrations of TSS can damage the gill structure and function in zebrafish and has the potential to enhance the toxicity of contaminants acting via the gills.

Genomic approachesidentifySTT4 as a new component in glucose-induced activation of yeast plasma membrane H+-ATPase.

Many studies have focused on identifying the signaling pathway by which addition of glucose triggers post-translational activation of the plasma membrane H+-ATPase in yeast. They have revealed that calcium signaling is involved in the regulatory pathway, supported for instance by the phenotype of mutants inARG82 that encodes an inositol kinase that phosphorylates inositol triphosphate (IP3). Strong glucose-induced calcium signaling, and high glucose-induced plasma membrane H+-ATPase activation have been observed in a specific yeast strain with the PJ genetic background. In this study, we have applied pooled-segregant whole genome sequencing, QTL analysis and a new bioinformatics methodology for determining SNP frequencies to identify the cause of this discrepancy and possibly new components of the signaling pathway. This has led to the identification of an STT4 allele with 6 missense mutations as a major causative allele, further supported by the observation that deletion of STT4 in the inferior parent caused a similar increase in glucose-induced plasma membrane H+-ATPase activation. However, the effect on calcium signaling was different indicating the presence of additional relevant genetic differences between the superior and reference strains. Our results suggest that phosphatidylinositol-4-phosphate might play a role in the glucose-induced activation of plasma membrane H+-ATPase by controlling intracellular calcium release through the modulation of the activity of phospholipase C.

Assessment of the physiological performance of the invasive oriental shrimp Palaemon macrodactylus from an atypical marine population.

Since 2000, a well-established population of the invasive oriental shrimp Palaemon macrodactylus has been present in fully marine conditions in the southwestern Atlantic Ocean (~38° S). To assess the physiological performance of this atypical population restricted to fully marine conditions, we conducted a laboratory experiment in which individuals were transferred from 35 ‰S (local seawater) to 2 ‰S; 5 ‰S; 10 ‰S; 20 ‰S; 50 ‰S and 60‰ for short (6 h), medium (48 h), and long (>504 h) acclimation periods. We measured the time course response of relevant parameters in the shrimp's hemolymph; activity of Na+, K+-ATPase (NKA), and V-H+-ATPase (VHA); and muscle water content. Shrimp showed great osmoregulatory plasticity, being able to survive for long periods between 5 ‰S and 50 ‰S, whereas no individual survived after transfer to either 2 ‰S or 60 ‰S. Shrimp hyper-regulated hemolymph osmolality at 5 ‰S and 10 ‰S, hypo-regulated at 35 ‰S and 50 ‰S, and isosmoticity was close to 20 ‰S. Compared to 35 ‰S, prolonged acclimation to 5 ‰S caused a decrease in hemolymph osmolality (~34%) along with sodium and chloride concentrations (~24%); the NKA and VHA activities decreased by ~52% and ~88%, respectively, while muscle water content was tightly regulated. Our results showed that the atypical population of P. macrodactylus studied here lives in a chronic hypo-osmo-ion regulatory state and suggest that fully marine conditions contribute to its poor performance at the lower limit of salinity tolerance (<5 ‰S).

Carbonic Anhydrase 2 Deletion Delays the Growth of Kidney Cysts Whereas Foxi1 Deletion Completely Abrogates Cystogenesis in TSC.

Tuberous sclerosis complex (TSC) presents with renal cysts and benign tumors, which eventually lead to kidney failure. The factors promoting kidney cyst formation in TSC are poorly understood. Inactivation of carbonic anhydrase 2 (Car2) significantly reduced, whereas, deletion of Foxi1 completely abrogated the cyst burden in Tsc1 KO mice. In these studies, we contrasted the ontogeny of cyst burden in Tsc1/Car2 dKO mice vs. Tsc1/Foxi1 dKO mice. Compared to Tsc1 KO, the Tsc1/Car2 dKO mice showed few small cysts at 47 days of age. However, by 110 days, the kidneys showed frequent and large cysts with overwhelming numbers of A-intercalated cells in their linings. The magnitude of cyst burden in Tsc1/Car2 dKO mice correlated with the expression levels of Foxi1 and was proportional to mTORC1 activation. This is in stark contrast to Tsc1/Foxi1 dKO mice, which showed a remarkable absence of kidney cysts at both 47 and 110 days of age. RNA-seq data pointed to profound upregulation of Foxi1 and kidney-collecting duct-specific H+-ATPase subunits in 110-day-old Tsc1/Car2 dKO mice. We conclude that Car2 inactivation temporarily decreases the kidney cyst burden in Tsc1 KO mice but the cysts increase with advancing age, along with enhanced Foxi1 expression.

Plasma membrane H+-ATPases in mineral nutrition and crop improvement.

Plasma membrane H+-ATPases (PMAs) pump H+ out of the cytoplasm by consuming ATP to generate a membrane potential and proton motive force for the transmembrane transport of nutrients into and out of plant cells. PMAs are involved in nutrient acquisition by regulating root growth, nutrient uptake, and translocation, as well as the establishment of symbiosis with arbuscular mycorrhizas. Under nutrient stresses, PMAs are activated to pump more H+ and promote organic anion excretion, thus improving nutrient availability in the rhizosphere. Herein we review recent progress in the physiological functions and the underlying molecular mechanisms of PMAs in the efficient acquisition and utilization of various nutrients in plants. We also discuss perspectives for the application of PMAs in improving crop production and quality.

Molecular mechanism of Oxr1p mediated disassembly of yeast V-ATPase.

The eukaryotic vacuolar H+-ATPase (V-ATPase) is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes. We recently reported that the TLDc protein Oxr1p induces V-ATPase disassembly in vitro. Whether and how Oxr1p is involved in enzyme disassembly in vivo, however, is not known. Here, using yeast genetics and fluorescence microscopy, we show that Oxr1p is essential for efficient V-ATPase disassembly in the cell. Supporting biochemical and biophysical in vitro experiments show that whereas Oxr1p-driven holoenzyme disassembly can occur in the absence of nucleotides, the presence of ATP greatly accelerates the process. ATP hydrolysis is needed, however, for subsequent release of Oxr1p so that the free V1 can adopt the autoinhibited conformation. Overall, our study unravels the molecular mechanism of Oxr1p-induced disassembly that occurs in vivo as part of the canonical V-ATPase regulation by reversible disassembly.