Together with its β-subunit OSTM1, ClC-7 performs 2Cl-/H+ exchange across lysosomal membranes. Pathogenic variants in either gene cause lysosome-related pathologies, including osteopetrosis and lysosomal storage. CLCN7 variants can cause recessive or dominant disease. Different variants entail different sets of symptoms. Loss of ClC-7 causes osteopetrosis and mostly neuronal lysosomal storage. A recently reported de novo CLCN7 mutation (p.Tyr715Cys) causes widespread severe lysosome pathology (hypopigmentation, organomegaly, and delayed myelination and development, \"HOD syndrome\"), but no osteopetrosis. We now describe two additional HOD individuals with the previously described p.Tyr715Cys and a novel p.Lys285Thr mutation, respectively. Both mutations decreased ClC-7 inhibition by PI(3,5)P2 and affected residues lining its binding pocket, and shifted voltage-dependent gating to less positive potentials, an effect partially conferred to WT subunits in WT/mutant heteromers. This shift predicts augmented pH gradient-driven Cl- uptake into vesicles. Overexpressing either mutant induced large lysosome-related vacuoles. This effect depended on Cl-/H+-exchange, as shown using mutants carrying uncoupling mutations. Fibroblasts from the p.Y715C patient also displayed giant vacuoles. This was not observed with p.K285T fibroblasts probably due to residual PI(3,5)P2 sensitivity. The gain of function caused by the shifted voltage-dependence of either mutant likely is the main pathogenic factor. Loss of PI(3,5)P2 inhibition will further increase current amplitudes, but may not be a general feature of HOD. Overactivity of ClC-7 induces pathologically enlarged vacuoles in many tissues, which is distinct from lysosomal storage observed with the loss of ClC-7 function. Osteopetrosis results from a loss of ClC-7, but osteoclasts remain resilient to increased ClC-7 activity.

H+-ATPases, including the phosphorylated intermediate-type (P-type) and vacuolar-type (V-type) H+-ATPases, are important ATP-driven proton pumps that generate membrane potential and provide proton motive force for secondary active transport. P- and V-type H+-ATPases have distinct structures and subcellular localizations and play various roles in growth and stress responses. A P-type H+-ATPase is mainly regulated at the posttranslational level by phosphorylation and dephosphorylation of residues in its autoinhibitory C terminus. The expression and activity of both P- and V-type H+-ATPases are highly regulated by hormones and environmental cues. In this review, we summarize the recent advances in understanding of the evolution, regulation, and physiological roles of P- and V-type H+-ATPases, which coordinate and are involved in plant growth and stress adaptation. Understanding the different roles and the regulatory mechanisms of P- and V-type H+-ATPases provides a new perspective for improving plant growth and stress tolerance by modulating the activity of H+-ATPases, which will mitigate the increasing environmental stress conditions associated with ongoing global climate change.

Vacuoles are the largest compartments in plant cells and are involved in plant development and response to abiotic and biotic stresses. Vacuolar acidification is essential for vacuoles in various physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection have never been reported. In this autophagy punctum, we discuss our recent findings about how plant viruses suppress vacuolar acidification and the degradation of autophagic bodies by directly interacting with a component of the V-ATPase to promote virus infection.

Vacuolar acidification is essential for vacuoles in diverse physiological functions. However, its role in plant defense, and whether and how pathogens affect vacuolar acidification to promote infection remain unknown. Here, we show that Barley stripe mosaic virus (BSMV) replicase γa, but not its mutant γaR569A , directly blocks acidification of vacuolar lumen and suppresses autophagic degradation to promote viral infection in plants. These were achieved via molecular interaction between γa and V-ATPase catalytic subunit B2 (VHA-B2), leading to disruption of the interaction between VHA-B2 and V-ATPase catalytic subunit E (VHA-E), which impairs the membrane localization of VHA-B2 and suppresses V-ATPase activity. Furthermore, a mutant virus BSMVR569A with the R569A point mutation possesses less viral pathogenicity. Interestingly, multiple viral infections block vacuolar acidification. These findings reveal that functional vacuolar acidification is required for plant antiviral defense and disruption of vacuolar acidification could be a general viral counter-defense strategy employed by multiple viruses.

The determination of flower color mainly depends on the anthocyanin biosynthesis pathway and vacuolar pH; however, unlike the former, the mechanism of vacuolar acidification in soybean remains uncharacterized at the molecular level. To investigate this mechanism, we isolated four recessive purple-blue EMS-induced flower mutants from the purple flower soybean cultivar, Pungsannamul. The petals of all the mutants had increased pH compared with those of wild Pungsannamul. One of the mutants had a single nucleotide substitution in GmPH4, a regulator gene encoding an MYB transcription factor, and the substitution resulted in a premature stop codon in its first exon. The other three mutants had nucleotide substitutions in GmPH5, a single new gene that we identified by physical mapping. It corresponds to Glyma.03G262600 in chromosome 3 and encodes a proton pump that belongs to the P3A-ATPase family. The substitutions resulted in a premature stop codon, which may be a defect in the ATP-binding capacity of GmPH5 and possibly a catalytic inefficiency of GmPH5. The result is consistent with their genetic recessiveness as well as the high pH of mutant petals, suggesting that GmPH5 is directly involved in vacuolar acidification. We also found that the expression of GmPH5 and several putative \"acidifying\" genes in the gmph4 mutant was remarkably reduced, indicating that GmPH4 may regulate the genes involved in determining the vacuolar pH of soybean petals.

ENA1 and ENA2 are P-type IID/ENA Na+/K+-ATPases required for cellular homeostasis in yeasts but remain poorly understood in filamentous fungal insect pathogens. Here, we characterized seven genes encoding five ENA1/2 homologues (ENA1a-c and ENA2a/b) and two P-type IIC/NK Na+/K+-ATPases (NK1/2) in Beauveria bassiana, an insect-pathogenic fungus serving as a main source of fungal insecticides worldwide. Most of these genes were highly responsive to alkaline pH and Na+/K+ cues at transcription level. Cellular Na+, K+ and H+ homeostasis was disturbed only in the absence of ena1a or ena2b. The disturbed homeostasis featured acceleration of vacuolar acidification, elevation of cytosolic Na+/K+ level at pH 5.0 to 9.0, and stabilization of extracellular H+ level to initial pH 7.5 during a 5-day period of submerged incubation. Despite little defect in hyphal growth and asexual development, the Δena1a and Δena2b mutants were less tolerant to metal cations (Na+, K+, Li+, Zn2+, Mn2+ and Fe3+), cell wall perturbation, oxidation, non-cation hyperosmolarity and UVB irradiation, severely compromised in insect pathogenicity via normal cuticle infection, and attenuated in virulence via hemocoel injection. The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes. Therefore, only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis, insect pathogenicity and multiple stress tolerance in B. bassiana. These findings provide a novel insight into ENA1a- and ENA2b-dependent vacuolar pH stability, cation-homeostatic process and fungal fitness to host insect and environment.

The yeast vacuolar H+-ATPase (V-ATPase) of budding yeast (Saccharomyces cerevisiae) is regulated by reversible disassembly. Disassembly inhibits V-ATPase activity under low-glucose conditions by releasing peripheral V1 subcomplexes from membrane-bound Vo subcomplexes. V-ATPase reassembly and reactivation requires intervention of the conserved regulator of H+-ATPase of vacuoles and endosomes (RAVE) complex, which binds to cytosolic V1 subcomplexes and assists reassembly with integral membrane Vo complexes. Consistent with its role, the RAVE complex itself is reversibly recruited to the vacuolar membrane by glucose, but the requirements for its recruitment are not understood. We demonstrate here that RAVE recruitment to the membrane does not require an interaction with V1 Glucose-dependent RAVE localization to the vacuolar membrane required only intact Vo complexes containing the Vph1 subunit, suggesting that the RAVE-Vo interaction is glucose-dependent. We identified a short conserved sequence in the center of the RAVE subunit Rav1 that is essential for the interaction with Vph1 in vivo and in vitro Mutations in this region resulted in the temperature- and pH-dependent growth phenotype characteristic of ravΔ mutants. However, this region did not account for glucose sensitivity of the Rav1-Vph1 interaction. We quantitated glucose-dependent localization of a GFP-tagged RAVE subunit to the vacuolar membrane in several mutants previously implicated in altering V-ATPase assembly state or glucose-induced assembly. RAVE localization did not correlate with V-ATPase assembly levels reported previously in these mutants, highlighting both the catalytic nature of RAVE\'s role in V-ATPase assembly and the likelihood of glucose signaling to RAVE independently of V1.

Citric acid homeostasis patterns and its content are diversified among the fruits of citrus cultivars, but the cause remained unclear. In this study we showed that changes of citric acid content were highly associated with the expression profiles of a P-type proton pump gene (CsPH8) in the fruits of six citrus cultivars; moreover, analysis of 21 different fruit samples indicated that the correlation coefficient between titratable acid content and CsPH8 transcript level was 0.5837 with a significant level (P < 0.05). Overexpression of CsPH8 in acidless pumelo juice sacs, strawberry fruit, and tomato fruit significantly increased the titratable acid or citric acid content besides the gene transcript level. On another hand, RNA interference of CsPH8 in acidic pumelo juice sacs significantly decreased the CsPH8 transcript level and the titratable acid or citric acid content as well. In addition, severe drought significantly increased the CsPH8 transcript level besides the titratable acid content. Taken together, these findings address the function of CsPH8 in citrus vacuolar acidification, confirm that CsPH8 plays a key role in the variation of citric acid content, and supported that the acid fluctuation influenced by drought, is at least partly due to the change of CsPH8 transcript level.

The accumulation of secondary metabolites and the regulation of tissue acidity contribute to the important traits of grape berry and influence plant performance in response to abiotic and biotic factors. In several plant species a highly conserved MYB-bHLH-WD (MBW) transcriptional regulatory complex controls flavonoid pigment synthesis and transport, and vacuolar acidification in epidermal cells. An additional component, represented by a WRKY-type transcription factor, physically interacts with the complex increasing the expression of some target genes and adding specificity for other targets. Here we investigated the function of MBW(W) complexes involving two MYBs (VvMYB5a and VvMYB5b) and the WRKY factor VvWRKY26 in grapevine (Vitis vinifera L.). Using transgenic grapevine plants we showed that these complexes affected different aspects of morphology, plant development, pH regulation, and pigment accumulation. Transcriptomic analysis identified a core set of putative target genes controlled by VvMYB5a, VvMYB5b, and VvWRKY26 in different tissues. Our data indicated that VvWRKY26 enhances the expression of selected target genes induced by VvMYB5a/b. Among these targets are genes involved in vacuolar hyper-acidification, such as the P-type ATPases VvPH5 and VvPH1, and trafficking, and genes involved in the biosynthesis of flavonoids. In addition, VvWRKY26 is recruited specifically by VvMYB5a, reflecting the functional diversification of VvMYB5a and VvMYB5b. The expression of MBWW complexes in vegetative organs, such as leaves, indicates a possible function of vacuolar hyper-acidification in the repulsion of herbivores and/or in developmental processes, as shown by defects in transgenic grape plants where the complex is inactivated.

To prevent or ameliorate Alzheimer\'s disease (AD) we must understand its molecular basis. AD develops over decades but detailed molecular analysis of AD brains is limited to postmortem tissue where the stresses initiating the disease may be obscured by compensatory responses and neurodegenerative processes. Rare, dominant mutations in a small number of genes, but particularly the gene PRESENILIN 1 (PSEN1), drive early onset of familial AD (EOfAD). Numerous transgenic models of AD have been constructed in mouse and other organisms, but transcriptomic analysis of these models has raised serious doubts regarding their representation of the disease state. Since we lack clarity regarding the molecular mechanism(s) underlying AD, we posit that the most valid approach is to model the human EOfAD genetic state as closely as possible. Therefore, we sought to analyse brains from zebrafish heterozygous for a single, EOfAD-like mutation in their PSEN1-orthologous gene, psen1. We previously introduced an EOfAD-like mutation (Q96_K97del) into the endogenous psen1 gene of zebrafish. Here, we analysed transcriptomes of young adult (6-month-old) entire brains from a family of heterozygous mutant and wild type sibling fish. Gene ontology (GO) analysis implies effects on mitochondria, particularly ATP synthesis, and on ATP-dependent processes including vacuolar acidification.