Dmxl1 Is an Essential Mammalian Gene that Is Required for V-ATPase Assembly and Function In Vivo.

The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.

Vacuolar H+-ATPase determines daughter cell fates through asymmetric segregation of the nucleosome remodeling and deacetylase complex.

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.

Reduced vacuolar ATPase protects mice from Friend virus infection - an unintended but instructive effect in Hif-2afl mice.

During acute viral infections, innate immune cells invade inflamed tissues and face hypoxic areas. Hypoxia-inducible factors (HIFs) adapt cellular responses towards these conditions. We wanted to investigate the effects of a loss of HIF-2α in macrophages during acute Friend murine leukemia retrovirus (FV) infection in C57BL/6 mice using a Cre/loxP system. Remarkably, mice with floxed Hif-2a (Hif-2afl; Hif-2a is also known as Epas1) did not show any signs of FV infection independent of Cre activity. This prevented a detailed analysis of the role of macrophage HIF-2α for FV infection but allowed us to study a model of unexpected FV resistance. Hif-2afl mice showed a significant decrease in the expression of the Atp6v1e2 gene encoding for the E2 subunit of the vacuolar H+-ATPase, which resulted in a decreased acidification of lysosomes and limited virus entry into the cell. These findings highlight that the insertion of loxP sites is not always without functional consequences and has established a phenotype in the floxed Hif-2a mouse, which is not only unexpected, but unwanted and is of relevance for the use of this mouse strain in (at least virus) experiments.

Human cytomegalovirus deploys molecular mimicry to recruit VPS4A to sites of virus assembly.

The AAA-type ATPase VPS4 is recruited by proteins of the endosomal sorting complex required for transport III (ESCRT-III) to catalyse membrane constriction and membrane fission. VPS4A accumulates at the cytoplasmic viral assembly complex (cVAC) of cells infected with human cytomegalovirus (HCMV), the site where nascent virus particles obtain their membrane envelope. Here we show that VPS4A is recruited to the cVAC via interaction with pUL71. Sequence analysis, deep-learning structure prediction, molecular dynamics and mutagenic analysis identify a short peptide motif in the C-terminal region of pUL71 that is necessary and sufficient for the interaction with VPS4A. This motif is predicted to bind the same groove of the N-terminal VPS4A Microtubule-Interacting and Trafficking (MIT) domain as the Type 2 MIT-Interacting Motif (MIM2) of cellular ESCRT-III components, and this viral MIM2-like motif (vMIM2) is conserved across ß-herpesvirus pUL71 homologues. However, recruitment of VPS4A by pUL71 is dispensable for HCMV morphogenesis or replication and the function of the conserved vMIM2 during infection remains enigmatic. VPS4-recruitment via a vMIM2 represents a previously unknown mechanism of molecular mimicry in viruses, extending previous observations that herpesviruses encode proteins with structural and functional homology to cellular ESCRT-III components.

Genome and RNA sequencing were essential to reveal cryptic intronic variants associated to defective ATP6AP1 mRNA processing.

The diagnosis of Mendelian disorders has notably advanced with integration of whole exome and genome sequencing (WES and WGS) in clinical practice. However, challenges in variant interpretation and uncovered variants by WES still leave a substantial percentage of patients undiagnosed. In this context, integrating RNA sequencing (RNA-seq) improves diagnostic workflows, particularly for WES inconclusive cases. Additionally, functional studies are often necessary to elucidate the impact of prioritized variants on gene expression and protein function. Our study focused on three unrelated male patients (P1-P3) with ATP6AP1-CDG (congenital disorder of glycosylation), presenting with intellectual disability and varying degrees of hepatopathy, glycosylation defects, and an initially inconclusive diagnosis through WES. Subsequent RNA-seq was pivotal in identifying the underlying genetic causes in P1 and P2, detecting ATP6AP1 underexpression and aberrant splicing. Molecular studies in fibroblasts confirmed these findings and identified the rare intronic variants c.289-233C > T and c.289-289G > A in P1 and P2, respectively. Trio-WGS also revealed the variant c.289-289G > A in P3, which was a de novo change in both patients. Functional assays expressing the mutant alleles in HAP1 cells demonstrated the pathogenic impact of these variants by reproducing the splicing alterations observed in patients. Our study underscores the role of RNA-seq and WGS in enhancing diagnostic rates for genetic diseases such as CDG, providing new insights into ATP6AP1-CDG molecular bases by identifying the first two deep intronic variants in this X-linked gene. Additionally, our study highlights the need to integrate RNA-seq and WGS, followed by functional validation, in routine diagnostics for a comprehensive evaluation of patients with an unidentified molecular etiology.

Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior.

BACKGROUND: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown. RESULTS: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments. CONCLUSIONS: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

Exploring the impact of osteoprotegerin on osteoclast and precursor fusion: Mechanisms and modulation by ATP.

Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.

ATP6V1A is required for synaptic rearrangements and plasticity in murine hippocampal neurons.

AIM: Understanding the physiological role of ATP6V1A, a component of the cytosolic V1 domain of the proton pump vacuolar ATPase, in regulating neuronal development and function. METHODS: Modeling loss of function of Atp6v1a in primary murine hippocampal neurons and studying neuronal morphology and function by immunoimaging, electrophysiological recordings and electron microscopy. RESULTS: Atp6v1a depletion affects neurite elongation, stabilization, and function of excitatory synapses and prevents synaptic rearrangement upon induction of plasticity. These phenotypes are due to an overall decreased expression of the V1 subunits, that leads to impairment of lysosomal pH-regulation and autophagy progression with accumulation of aberrant lysosomes at neuronal soma and of enlarged vacuoles at synaptic boutons. CONCLUSIONS: These data suggest a physiological role of ATP6V1A in the surveillance of synaptic integrity and plasticity and highlight the pathophysiological significance of ATP6V1A loss in the alteration of synaptic function that is associated with neurodevelopmental and neurodegenerative diseases. The data further support the pivotal involvement of lysosomal function and autophagy flux in maintaining proper synaptic connectivity and adaptive neuronal properties.

Novel Environmentally Friendly RNAi Biopesticides: Targeting V-ATPase in Holotrichia parallela Larvae Using Layered Double Hydroxide Nanocomplexes.

RNA interference (RNAi)-based biopesticides offer an attractive avenue for pest control. Previous studies revealed high RNAi sensitivity in Holotrichia parallela larvae, showcasing its potential for grub control. In this study, we aimed to develop an environmentally friendly RNAi method for H. parallela larvae. The double-stranded RNA (dsRNA) of the V-ATPase-a gene (HpVAA) was loaded onto layered double hydroxide (LDH). The dsRNA/LDH nanocomplex exhibited increased environmental stability, and we investigated the absorption rate and permeability of dsRNA-nanoparticle complexes and explored the RNAi controlling effect. Silencing the HpVAA gene was found to darken the epidermis of H. parallela larvae, with growth cessation or death or mortality, disrupting the epidermis and midgut structure. Quantitative reverse transcription-polymerase chain reaction and confocal microscopy confirmed the effective absorption of the dsRNA/LDH nanocomplex by peanut plants, with distribution in roots, stems, and leaves. Nanomaterial-mediated RNAi silenced the target genes, leading to the death of pests. Therefore, these findings indicate the successful application of the nanomaterial-mediated RNAi system for underground pests, thus establishing a theoretical foundation for developing a green, safe, and efficient pest control strategy.

ATP6AP2, a regulator of LRP6/ß-catenin protein trafficking, promotes Wnt/ß-catenin signaling and bone formation in a cell type dependent manner.

Wnt/ß-catenin signaling is critical for various cellular processes in multiple cell types, including osteoblast (OB) differentiation and function. Exactly how Wnt/ß-catenin signaling is regulated in OBs remain elusive. ATP6AP2, an accessory subunit of V-ATPase, plays important roles in multiple cell types/organs and multiple signaling pathways. However, little is known whether and how ATP6AP2 in OBs regulates Wnt/ß-catenin signaling and bone formation. Here we provide evidence for ATP6AP2 in the OB-lineage cells to promote OB-mediated bone formation and bone homeostasis selectively in the trabecular bone regions. Conditionally knocking out (CKO) ATP6AP2 in the OB-lineage cells (Atp6ap2Ocn-Cre) reduced trabecular, but not cortical, bone formation and bone mass. Proteomic and cellular biochemical studies revealed that LRP6 and N-cadherin were reduced in ATP6AP2-KO BMSCs and OBs, but not osteocytes. Additional in vitro and in vivo studies revealed impaired ß-catenin signaling in ATP6AP2-KO BMSCs and OBs, but not osteocytes, under both basal and Wnt stimulated conditions, although LRP5 was decreased in ATP6AP2-KO osteocytes, but not BMSCs. Further cell biological studies uncovered that osteoblastic ATP6AP2 is not required for Wnt3a suppression of ß-catenin phosphorylation, but necessary for LRP6/ß-catenin and N-cadherin/ß-catenin protein complex distribution at the cell membrane, thus preventing their degradation. Expression of active ß-catenin diminished the OB differentiation deficit in ATP6AP2-KO BMSCs. Taken together, these results support the view for ATP6AP2 as a critical regulator of both LRP6 and N-cadherin protein trafficking and stability, and thus regulating ß-catenin levels, demonstrating an un-recognized function of osteoblastic ATP6AP2 in promoting Wnt/LRP6/ß-catenin signaling and trabecular bone formation.

A high dose KRP203 induces cytoplasmic vacuoles associated with altered phosphoinositide segregation and endosome expansion.

In animal cells, vacuoles are absent, but can be induced by diseases and drugs. While phosphoinositides are critical for membrane trafficking, their role in the formation of these vacuoles remains unclear. The immunosuppressive KRP203/Mocravimod, which antagonizes sphingosine-1-phosphate receptors, has been identified as having novel multimodal activity against phosphoinositide kinases. However, the impact of this novel KRP203 activity is unknown. Here, we show that KRP203 disrupts the spatial organization of phosphoinositides and induces extensive vacuolization in tumor cells and immortalized fibroblasts. The KRP203-induced vacuoles are primarily from endosomes, and augmented by inhibition of PIKFYVE and VPS34. Conversely, overexpression of PTEN decreased KRP203-induced vacuole formation. Furthermore, V-ATPase inhibition completely blunted KRP203-induced vacuolization, pointing to a critical requirement of the endosomal maturation process. Importantly, nearly a half of KRP203-induced vacuoles are significantly decorated with PI4P, a phosphoinositide typically enriched at the plasma membrane and Golgi. These results suggest a model that noncanonical spatial reorganization of phosphoinositides by KRP203 alters the endosomal maturation process, leading to vacuolization. Taken together, this study reveals a previously unrecognized bioactivity of KRP203 as a vacuole-inducing agent and its unique mechanism of phosphoinositide modulation, providing a new insight of phosphoinositide regulation into vacuolization-associated diseases and their molecular pathologies.

Plasma exosomes impair microglial degradation of α-synuclein through V-ATPase subunit V1G1.

INTRODUCTION: Microglia are the main phagocytes in the brain and can induce neuroinflammation. Moreover, they are critical to alpha-synuclein (α-syn) aggregation and propagation. Plasma exosomes derived from patients diagnosed with Parkinson's disease (PD-exo) reportedly evoked α-syn aggregation and inflammation in microglia. In turn, microglia internalized and released exosomal α-syn, enhancing α-syn propagation. However, the specific mechanism through which PD-exo influences α-syn degradation remains unknown. METHODS: Exosomes were extracted from the plasma of patients with PD by differential ultracentrifugation, analyzed using electron microscopy (EM) and nanoparticle flow cytometry, and stereotaxically injected into the unilateral striatum of the mice. Transmission EM was employed to visualize lysosomes and autophagosomes in BV2 cells, and lysosome pH was measured with LysoSensor Yellow/Blue DND-160. Cathepsin B and D, lysosomal-associated membrane protein 1 (LAMP1), ATP6V1G1, tumor susceptibility gene 101 protein, calnexin, α-syn, ionized calcium binding adaptor molecule 1, and NLR family pyrin domain containing 3 were evaluated using quantitative polymerase chain reaction or western blotting, and α-syn, LAMP1, and ATP6V1G1 were also observed by immunofluorescence. Small interfering ribonucleic acid against V1G1 was transfected into BV2 cells and primary microglia using Lipofectamine® 3000. A PD mouse model was established via injection with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into mice. A lentiviral-mediated strategy to overexpress ATP6V1G1 in the brain of MPTP-treated mice was employed. Motor coordination was assessed using rotarod and pole tests, and neurodegeneration in the mouse substantia nigra and striatum tissues was determined using immunofluorescence histochemical and western blotting of tyrosine hydroxylase. RESULTS: PD-exo decreased the expression of V1G1, responsible for the acidification of intra- and extracellular milieu. This impairment of lysosomal acidification resulted in the accumulation of abnormally swollen lysosomes and decreased lysosomal enzyme activities, impairing lysosomal protein degradation and causing α-syn accumulation. Additionally, V1G1 overexpression conferred the mice neuroprotection during MPTP exposure. CONCLUSION: Pathogenic protein accumulation is a key feature of PD, and compromised V-type ATPase dysfunction might participate in PD pathogenesis. Moreover, V1G1 overexpression protects against neuronal toxicity in an MPTP-based PD mouse model, which may provide opportunities to develop novel therapeutic interventions for PD treatment.

Alkaliptosis induction counteracts paclitaxel-resistant ovarian cancer cells via ATP6V0D1-mediated ABCB1 inhibition.

Paclitaxel serves as the cornerstone chemotherapy for ovarian cancer, yet its prolonged administration frequently culminates in drug resistance, presenting a substantial challenge. Here we reported that inducing alkaliptosis, rather than apoptosis or ferroptosis, effectively overcomes paclitaxel resistance. Mechanistically, ATPase H+ transporting V0 subunit D1 (ATP6V0D1), a key regulator of alkaliptosis, plays a pivotal role by mediating the downregulation of ATP-binding cassette subfamily B member 1 (ABCB1), a multidrug resistance protein. Both ATP6V0D1 overexpression through gene transfection and pharmacological enhancement of ATP6V0D1 protein stability using JTC801 effectively inhibit ABCB1 upregulation, resulting in growth inhibition in drug-resistant cells. Additionally, increasing intracellular pH to alkaline (pH 8.5) via sodium hydroxide application suppresses ABCB1 expression, whereas reducing the pH to acidic conditions (pH 6.5) with hydrochloric acid amplifies ABCB1 expression in drug-resistant cells. Collectively, these results indicate a potentially effective therapeutic strategy for targeting paclitaxel-resistant ovarian cancer by inducing ATP6V0D1-dependent alkaliptosis.

LC3B labeling of the parasitophorous vacuole membrane of Plasmodium berghei liver stage parasites depends on the V-ATPase and ATG16L1.

The protozoan parasite Plasmodium, the causative agent of malaria, undergoes an obligatory stage of intra-hepatic development before initiating a blood-stage infection. Productive invasion of hepatocytes involves the formation of a parasitophorous vacuole (PV) generated by the invagination of the host cell plasma membrane. Surrounded by the PV membrane (PVM), the parasite undergoes extensive replication. During intracellular development in the hepatocyte, the parasites provoke the Plasmodium-associated autophagy-related (PAAR) response. This is characterized by a long-lasting association of the autophagy marker protein, and ATG8 family member, LC3B with the PVM. LC3B localization at the PVM does not follow the canonical autophagy pathway since upstream events specific to canonical autophagy are dispensable. Here, we describe that LC3B localization at the PVM of Plasmodium parasites requires the V-ATPase and its interaction with ATG16L1. The WD40 domain of ATG16L1 is crucial for its recruitment to the PVM. Thus, we provide new mechanistic insight into the previously described PAAR response targeting Plasmodium liver stage parasites.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway as a critical route affecting the toxic effects of lead on HK-2 cells.

Lead is a widespread environmental pollutant with serious adverse effects on human health, but the mechanism underlying its toxicity remains elusive. This study aimed to investigate the role of miR-584-5p / Ykt6 axis in the toxic effect of lead on HK-2 cells and the related mechanism. Our data suggested that lead exposure caused significant cytotoxicity, DNA and chromosome damage to HK-2 cells. Mechanistically, lead exposure down-regulated miR-584-5p and up-regulated Ykt6 expression, consequently, autophagosomal number and autophagic flux increased, lysosomal number and activity decreased, exosomal secretion increased. Interestingly, when miR-584-5p level was enhanced with mimic, autophagosomal number and autophagic flux decreased, lysosomal number and activity increased, ultimately, exosomal secretion was down-regulated, which resulted in significant aggravated toxic effects of lead. Further, directly blocking exosomal secretion with inhibitor GW4869 also resulted in exacerbated toxic effects of lead. Herein, we conclude that miR-584-5p / Ykt6 - mediated autophagy - lysosome - exosome pathway may be a critical route affecting the toxic effects of lead on HK-2 cells. We provide a novel insight into the mechanism underlying the toxicity of lead on human cells.

The human AAA-ATPase VPS4A isoform and its co-factor VTA1 have a unique function in regulating mammalian cytokinesis abscission.

Mutations in the human AAA-ATPase VPS4 isoform, VPS4A, cause severe neurodevelopmental defects and congenital dyserythropoietic anemia (CDA). VPS4 is a crucial component of the endosomal sorting complex required for transport (ESCRT) system, which drives membrane remodeling in numerous cellular processes, including receptor degradation, cell division, and neural pruning. Notably, while most organisms encode for a single VPS4 gene, human cells have 2 VPS4 paralogs, namely VPS4A and VPS4B, but the functional differences between these paralogs is mostly unknown. Here, we set out to investigate the role of the human VPS4 paralogs in cytokinetic abscission using a series of knockout cell lines. We found that VPS4A and VPS4B hold both overlapping and distinct roles in abscission. VPS4A depletion resulted in a more severe abscission delay than VPS4B and was found to be involved in earlier stages of abscission. Moreover, VPS4A and a monomeric-locked VPS4A mutant bound the abscission checkpoint proteins CHMP4C and ANCHR, while VPS4B did not, indicating a regulatory role for the VPS4A isoform in abscission. Depletion of VTA1, a co-factor of VPS4, disrupted VPS4A-ANCHR interactions and accelerated abscission, suggesting that VTA1 is also involved in the abscission regulation. Our findings reveal a dual role for VPS4A in abscission, one that is canonical and can be compensated by VPS4B, and another that is regulatory and may be delivered by its monomeric form. These observations provide a potential mechanistic explanation for the neurodevelopmental defects and other related disorders reported in VPS4A-mutated patients with a fully functional VPS4B paralog.

Defective Lamtor5 Leads to Autoimmunity by Deregulating v-ATPase and Lysosomal Acidification.

Despite accumulating evidence linking defective lysosome function with autoimmune diseases, how the catabolic machinery is regulated to maintain immune homeostasis remains unknown. Late endosomal/lysosomal adaptor, MAPK and mTOR activator 5 (Lamtor5) is a subunit of the Ragulator mediating mechanistic target of rapamycin complex 1 (mTORC1) activation in response to amino acids, but its action mode and physiological role are still unclear. Here it is demonstrated that Lamtor5 level is markedly decreased in peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). In parallel, the mice with myeloid Lamtor5 ablation developed SLE-like manifestation. Impaired lysosomal function and aberrant activation of mTORC1 are evidenced in Lamtor5 deficient macrophages and PBMCs of SLE patients, accompanied by blunted autolysosomal pathway and undesirable inflammatory responses. Mechanistically, it is shown that Lamtor5 is physically associated with ATP6V1A, an essential subunit of vacuolar H+-ATPase (v-ATPase), and promoted the V0/V1 holoenzyme assembly to facilitate lysosome acidification. The binding of Lamtor5 to v-ATPase affected the lysosomal tethering of Rag GTPase and weakened its interaction with mTORC1 for activation. Overall, Lamtor5 is identified as a critical factor for immune homeostasis by intergrading v-ATPase activity, lysosome function, and mTOR pathway. The findings provide a potential therapeutic target for SLE and/or other autoimmune diseases.

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.