Mouse oocytes sequester aggregated proteins in degradative super-organelles.

Oocytes are among the longest-lived cells in the body and need to preserve their cytoplasm to support proper embryonic development. Protein aggregation is a major threat for intracellular homeostasis in long-lived cells. How oocytes cope with protein aggregation during their extended life is unknown. Here, we find that mouse oocytes accumulate protein aggregates in specialized compartments that we named endolysosomal vesicular assemblies (ELVAs). Combining live-cell imaging, electron microscopy, and proteomics, we found that ELVAs are non-membrane-bound compartments composed of endolysosomes, autophagosomes, and proteasomes held together by a protein matrix formed by RUFY1. Functional assays revealed that in immature oocytes, ELVAs sequester aggregated proteins, including TDP-43, and degrade them upon oocyte maturation. Inhibiting degradative activity in ELVAs leads to the accumulation of protein aggregates in the embryo and is detrimental for embryo survival. Thus, ELVAs represent a strategy to safeguard protein homeostasis in long-lived cells.

NDST3 deacetylates α-tubulin and suppresses V-ATPase assembly and lysosomal acidification.

Lysosomes are key organelles maintaining cellular homeostasis in health and disease. Here, we report the identification of N-deacetylase and N-sulfotransferase 3 (NDST3) as a potent regulator of lysosomal functions through an unbiased genetic screen. NDST3 constitutes a new member of the histone deacetylase (HDAC) family and catalyzes the deacetylation of α-tubulin. Loss of NDST3 promotes assembly of the V-ATPase holoenzyme on the lysosomal membrane and thereby increases the acidification of the organelle. NDST3 is downregulated in tissues and cells from patients carrying the C9orf72 hexanucleotide repeat expansion linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Deficiency in C9orf72 decreases the level of NDST3, and downregulation of NDST3 exacerbates the proteotoxicity of poly-dipeptides generated from the C9orf72 hexanucleotide repeats. These results demonstrate a previously unknown regulatory mechanism through which microtubule acetylation regulates lysosomal activities and suggest that NDST3 could be targeted to modulate microtubule and lysosomal functions in relevant diseases.

Inhibition of endocytic uptake of severe acute respiratory syndrome coronavirus 2 and endo-lysosomal acidification by diphenoxylate.

Cell culture-based screening of a chemical library identified diphenoxylate as an antiviral agent against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The observed 50% effective concentrations ranged between 1.4 and 4.9 µM against the original wild-type strain and its variants. Time-of-addition experiments indicated that diphenoxylate is an entry blocker targeting a host factor involved in viral infection. Fluorescence microscopic analysis visualized that diphenoxylate prevented SARS-CoV-2 particles from penetrating the cell membrane and also impaired endo-lysosomal acidification. Diphenoxylate exhibited a synergistic inhibitory effect on SARS-CoV-2 infection in human lung epithelial Calu-3 cells when combined with a transmembrane serine protease 2 (TMPRSS2) inhibitor, nafamostat. This synergy suggested that efficient antiviral activity is achieved by blocking both TMPRSS2-mediated early and endosome-mediated late SARS-CoV-2 entry pathways. The antiviral efficacy of diphenoxylate against SARS-CoV-2 was reproducible in a human tonsil organoids system. In a transgenic mouse model expressing the obligate SARS-CoV-2 receptor, human angiotensin-converting enzyme 2, intranasal administration of diphenoxylate (10 mg/kg/day) significantly reduced the viral RNA copy number in the lungs by 70% on day 3. This study underscores that diphenoxylate represents a promising core scaffold, warranting further exploration for chemical modifications aimed at developing a new class of clinically effective antiviral drugs against SARS-CoV-2.

Tubulin deacetylase NDST3 modulates lysosomal acidification: Implications in neurological diseases.

Neurological diseases (NDs), featured by progressive dysfunctions of the nervous system, have become a growing burden for the aging populations. N-Deacetylase and N-sulfotransferase 3 (NDST3) is known to catalyze deacetylation and N-sulfation on disaccharide substrates. Recently, NDST3 is identified as a novel deacetylase for tubulin, and its newly recognized role in modulating microtubule acetylation and lysosomal acidification provides fresh insights into ND therapeutic approaches using NDST3 as a target. Microtubule acetylation and lysosomal acidification have been reported to be critical for activities in neurons, implying that the regulators of these two biological processes, such as the previously known microtubule deacetylases, histone deacetylase 6 (HDAC6) and sirtuin 2 (SIRT2), could play important roles in various NDs. Aberrant NDST3 expression or tubulin acetylation has been observed in an increasing number of NDs, including amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD), schizophrenia and bipolar disorder, Alzheimer's disease (AD), and Parkinson's disease (PD), suggesting that NDST3 is a key player in the pathogenesis of NDs and may serve as a target for development of new treatment of NDs.

Nucleic acid uptake occurs independent of lysosomal acidification but dependent on ATP consumption during RNautophagy/DNautophagy.

RNautophagy/DNautophagy (RDA) is an autophagic process that refers to the direct uptake of nucleic acids by lysosomes for degradation. Autophagy relies on lysosomes and lysosomal acidification is crucial for the degradation of intracellular components. However, whether lysosomal acidification interferes with nucleic acid uptake during RDA is unclear. In this study, we focused on vacuolar H+-ATPase (V-ATPase), the major proton pump responsible for maintaining an acidic pH in lysosomes. Our results show that lysosomes take up nucleic acids independently of the intralysosomal acidic pH during RDA. Isolated lysosomes treated with bafilomycin A1, a potent V-ATPase inhibitor, did not degrade, but took up RNA at similar levels as the control lysosomes. Similarly, the knockdown of Atp6v1a, the gene that encodes V-ATPase catalytic subunit A, did not affect the RNA uptake ability of isolated lysosomes. In addition, we demonstrated that nucleic acid uptake by isolated lysosomes necessitates ATP consumption, although V-ATPase is not required for the uptake process. These results broaden our understanding of the mechanisms underlying nucleic acid degradation via autophagy.

Lysosomal acidification dysfunction in microglia: an emerging pathogenic mechanism of neuroinflammation and neurodegeneration.

Microglia are the resident innate immune cells in the brain with a major role in orchestrating immune responses. They also provide a frontline of host defense in the central nervous system (CNS) through their active phagocytic capability. Being a professional phagocyte, microglia participate in phagocytic and autophagic clearance of cellular waste and debris as well as toxic protein aggregates, which relies on optimal lysosomal acidification and function. Defective microglial lysosomal acidification leads to impaired phagocytic and autophagic functions which result in the perpetuation of neuroinflammation and progression of neurodegeneration. Reacidification of impaired lysosomes in microglia has been shown to reverse neurodegenerative pathology in Alzheimer's disease. In this review, we summarize key factors and mechanisms contributing to lysosomal acidification impairment and the associated phagocytic and autophagic dysfunction in microglia, and how these defects contribute to neuroinflammation and neurodegeneration. We further discuss techniques to monitor lysosomal pH and therapeutic agents that can reacidify impaired lysosomes in microglia under disease conditions. Finally, we propose future directions to investigate the role of microglial lysosomal acidification in lysosome-mitochondria crosstalk and in neuron-glia interaction for more comprehensive understanding of its broader CNS physiological and pathological implications.

Ethyl Gallate Inhibits Bovine Viral Diarrhea Virus by Promoting IFITM3 Expression, Lysosomal Acidification and Protease Activity.

Bovine viral diarrhea virus (BVDV) is a highly contagious viral disease which causes economic losses to the cattle industry. Ethyl gallate (EG) is a phenolic acid derivative which has various potentials to modulate the host response to pathogens, such as via antioxidant activity, antibacterial activity, inhibition of the production of cell adhesion factors, and so on. This study aimed to evaluate if EG influences BVDV infection in Madin-Darby Bovine Kidney (MDBK) cells, and to understand the antiviral mechanism. Data indicated that EG effectively inhibited BVDV infection by co-treatment and post-treatment in MDBK cells with noncytotoxic doses. In addition, EG suppressed BVDV infection at an early stage of the viral life cycle by blocking entry and replication steps but not viral attachment and release. Moreover, EG strongly inhibited BVDV infection by promoting interferon-induced transmembrane protein 3 (IFITM3) expression, which localized to the cytoplasm. The protein level of cathepsin B was significantly reduced by BVDV infection, whereas with treatment with EG, it was significantly enhanced. The fluorescence intensities of acridine orange (AO) staining were significantly decreased in BVDV-infected cells but increased in EG-treated cells. Finally, Western blot and immunofluorescence analyses demonstrated that EG treatment significantly enhanced the protein levels of autophagy markers LC3 and p62. Chloroquine (CQ) significantly increased IFITM3 expression, and Rapamycin significantly decreased it. Thus, EG may regulate IFITM3 expression through autophagy. Our results showed that EG could have a solid antiviral activity on BVDV replication in MDBK cells via increased IFITM3 expression, lysosomal acidification, protease activity, and regulated autophagy. EG might have value for further development as an antiviral agent.

Lysosomal size matters.

Lysosomes are key cellular catabolic centers that also perform fundamental metabolic, signaling and quality control functions. Lysosomes are not static and they respond dynamically to intra- and extracellular stimuli triggering changes in organelle numbers, size and position. Such physical changes have a strong impact on lysosomal activity ultimately influencing cellular homeostasis. In this review, we summarize the current knowledge on lysosomal size regulation, on its physiological role(s) and association to several disease conditions.

Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes.

The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE-V1 complexes. Both purified RAVE and RAVE-V1 contain substoichiometric levels of subunit C. RAVE-V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE-V1 interacts with the N-terminal domain of Vo subunit Vph1 in vitro. RAVE-V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event.

Potential Antiviral Strategy Exploiting Dependence of SARS-CoV-2 Replication on Lysosome-Based Pathway.

The recent novel coronavirus (SARS-CoV-2) disease (COVID-19) outbreak created a severe public health burden worldwide. Unfortunately, the SARS-CoV-2 variant is still spreading at an unprecedented speed in many countries and regions. There is still a lack of effective treatment for moderate and severe COVID-19 patients, due to a lack of understanding of the SARS-CoV-2 life cycle. Lysosomes, which act as "garbage disposals" for nearly all types of eukaryotic cells, were shown in numerous studies to support SARS-CoV-2 replication. Lysosome-associated pathways are required for virus entry and exit during replication. In this review, we summarize experimental evidence demonstrating a correlation between lysosomal function and SARS-CoV-2 replication, and the development of lysosomal perturbation drugs as anti-SARS-CoV-2 agents.

AKT Ser/Thr kinase increases V-ATPase-dependent lysosomal acidification in response to amino acid starvation in mammalian cells.

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump that is essential for cellular homeostasis. V-ATPase activity is controlled by the regulated assembly of the enzyme from its component V1 and V0 domains. We previously reported that amino acid starvation rapidly increases V-ATPase assembly and activity in mammalian lysosomes, but the signaling pathways controlling this effect are unknown. In testing inhibitors of pathways important for controlling cellular metabolism, we found here that the cAMP-dependent protein kinase (PKA) inhibitor H89 increases lysosomal V-ATPase activity and blocks any further change upon starvation. The AMP-activated protein kinase (AMPK) inhibitor dorsomorphin decreased lysosomal V-ATPase activity and also blocked any increase upon starvation. However, CRISPR-mediated gene editing revealed that PKA and AMPK are not required for the starvation-dependent increase in lysosomal V-ATPase activity, indicating that H89 and dorsomorphin modify V-ATPase activity through other cellular targets. We next found that the AKT Ser/Thr kinase (AKT) inhibitor MK2206 blocks the starvation-dependent increase in lysosomal V-ATPase activity without altering basal activity. Expression of AKT1 or AKT3, but not AKT2, was required for increased lysosomal V-ATPase activity in response to amino acid starvation in mouse fibroblasts. Finally, HEK293T cells expressing only AKT1 responded normally to starvation, whereas cells expressing only AKT2 displayed a significantly reduced increase in V-ATPase activity and assembly upon starvation. These results show that AKT is required for controlling the rapid response of lysosomal V-ATPase activity to changes in amino acid availability and that this response depends on specific AKT isoforms.

Manganese co-localizes with calcium and phosphorus in Chlamydomonas acidocalcisomes and is mobilized in manganese-deficient conditions.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Cytosolic glucosylceramide regulates endolysosomal function in Niemann-Pick type C disease.

Niemann-Pick type C disease (NPCD) is a neurodegenerative disease associated with increases in cellular cholesterol and glycolipids and most commonly caused by defective NPC1, a late endosomal protein. Using ratiometric probes we find that NPCD cells show increased endolysosomal pH. In addition U18666A, an inhibitor of NPC1, was found to increase endolysosomal pH, and the number, size and heterogeneity of endolysosomal vesicles. NPCD fibroblasts and cells treated with U18666A also show disrupted targeting of fluorescent lipid BODIPY-LacCer to high pH vesicles. Inhibiting non-lysosomal glucocerebrosidase (GBA2) reversed increases in endolysosomal pH and restored disrupted BODIPY-LacCer trafficking in NPCD fibroblasts. GBA2 KO cells also show decreased endolysosomal pH. NPCD fibroblasts also show increased expression of a key subunit of the lysosomal proton pump vATPase on GBA2 inhibition. The results are consistent with a model where both endolysosomal pH and Golgi targeting of BODIPY-LacCer are dependent on adequate levels of cytosolic-facing GlcCer, which are reduced in NPC disease.

Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus.

Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG-immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus.

Mycobacterium tuberculosis WhiB3 Responds to Vacuolar pH-induced Changes in Mycothiol Redox Potential to Modulate Phagosomal Maturation and Virulence.

The ability of Mycobacterium tuberculosis to resist intraphagosomal stresses, such as oxygen radicals and low pH, is critical for its persistence. Here, we show that a cytoplasmic redox sensor, WhiB3, and the major M. tuberculosis thiol, mycothiol (MSH), are required to resist acidic stress during infection. WhiB3 regulates the expression of genes involved in lipid anabolism, secretion, and redox metabolism, in response to acidic pH. Furthermore, inactivation of the MSH pathway subverted the expression of whiB3 along with other pH-specific genes in M. tuberculosis. Using a genetic biosensor of mycothiol redox potential (EMSH), we demonstrated that a modest decrease in phagosomal pH is sufficient to generate redox heterogeneity in EMSH of the M. tuberculosis population in a WhiB3-dependent manner. Data indicate that M. tuberculosis needs low pH as a signal to alter cytoplasmic EMSH, which activates WhiB3-mediated gene expression and acid resistance. Importantly, WhiB3 regulates intraphagosomal pH by down-regulating the expression of innate immune genes and blocking phagosomal maturation. We show that this block in phagosomal maturation is in part due to WhiB3-dependent production of polyketide lipids. Consistent with these observations, MtbΔwhiB3 displayed intramacrophage survival defect, which can be rescued bypharmacological inhibition of phagosomal acidification. Last, MtbΔwhiB3 displayed marked attenuation in the lungs of guinea pigs. Altogether, our study revealed an intimate link between vacuolar acidification, redox physiology, and virulence in M. tuberculosis and discovered WhiB3 as crucial mediator of phagosomal maturation arrest and acid resistance in M. tuberculosis.

Regulation of Phagolysosomal Digestion by Caveolin-1 of the Retinal Pigment Epithelium Is Essential for Vision.

Caveolin-1 associates with the endo/lysosomal machinery of cells in culture, suggesting that it functions at these organelles independently of its contribution to cell surface caveolae. Here we explored mice lacking caveolin-1 specifically in the retinal pigment epithelium (RPE). The RPE supports neighboring photoreceptors via diurnal phagocytosis of spent photoreceptor outer segment fragments. Like mice lacking caveolin-1 globally, (RPE)CAV1(-/-) mice developed a normal RPE and neural retina but showed reduced rod photoreceptor light responses, indicating that lack of caveolin-1 affects photoreceptor function in a non-cell-autonomous manner. (RPE)CAV1(-/-) RPE in situ showed normal particle engulfment but delayed phagosome clearance and reversed diurnal profiles of levels and activities of lysosomal enzymes. Therefore, eliminating caveolin-1 specifically impairs phagolysosomal degradation by the RPE in vivo. Endogenous caveolin-1 was recruited to maturing phagolysosomes in RPE cells in culture. Consistent with these in vivo data, a moderate increase (to ∼ 2.5-fold) or decrease (by half) of caveolin-1 protein levels in RPE cells in culture was sufficient to accelerate or impair phagolysosomal digestion, respectively. A mutant form of caveolin-1 that fails to reach the cell surface augmented degradation like wild-type caveolin-1. Acidic lysosomal pH and increased protease activity are essential for digestion. We show that halving caveolin-1 protein levels significantly alkalinized lysosomal pH and decreased lysosomal enzyme activities. Taken together, our results reveal a novel role for intracellular caveolin-1 in modulating phagolysosomal function. Moreover, they show, for the first time, that organellar caveolin-1 significantly affects tissue functionality in vivo.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes.

Vacuolar ATPase in phagosome-lysosome fusion.

The vacuolar H(+)-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V0 sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V0 a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V0 sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V0 mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly.

The vacuolar H(+)-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly.

TAZ deficiency impairs the autophagy-lysosomal pathway through NRF2 dysregulation and lysosomal dysfunction.

Transcriptional coactivator with a PDZ-binding motif (TAZ) plays a key role in normal tissue homeostasis and tumorigenesis through interaction with several transcription factors. In particular, TAZ deficiency causes abnormal alveolarization and emphysema, and persistent TAZ overexpression contributes to lung cancer and pulmonary fibrosis, suggesting the possibility of a complex mechanism of TAZ function. Recent studies suggest that nuclear factor erythroid 2-related factor 2 (NRF2), an antioxidant defense system, induces TAZ expression during tumorigenesis and that TAZ also activates the NRF2-mediated antioxidant pathway. We thus thought to elucidate the cross-regulation of TAZ and NRF2 and the underlying molecular mechanisms and functions. TAZ directly interacted with NRF2 through the N-terminal domain and suppressed the transcriptional activity of NRF2 by preventing NRF2 from binding to DNA. In addition, the return of NRF2 to basal levels after signaling was inhibited in TAZ deficiency, resulting in sustained nuclear NRF2 levels and aberrantly increased expression of NRF2 targets. TAZ deficiency failed to modulate optimal NRF2 signaling and concomitantly impaired lysosomal acidification and lysosomal enzyme function, accumulating the abnormal autophagy vesicles and reactive oxygen species and causing protein oxidation and cellular damage in the lungs. TAZ restoration to TAZ deficiency normalized dysregulated NRF2 signaling and aberrant lysosomal function and triggered the normal autophagy-lysosomal pathway. Therefore, TAZ is indispensable for the optimal regulation of NRF2-mediated autophagy-lysosomal pathways and for preventing pulmonary damage caused by oxidative stress and oxidized proteins.