Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis.

Mitochondria and lysosomes are functionally linked, and their interdependent decline is a hallmark of aging and disease. Despite the long-standing connection between these organelles, the function(s) of lysosomes required to sustain mitochondrial health remains unclear. Here, working in yeast, we show that the lysosome-like vacuole maintains mitochondrial respiration by spatially compartmentalizing amino acids. Defects in vacuole function result in a breakdown in intracellular amino acid homeostasis, which drives age-related mitochondrial decline. Among amino acids, we find that cysteine is most toxic for mitochondria and show that elevated non-vacuolar cysteine impairs mitochondrial respiration by limiting intracellular iron availability through an oxidant-based mechanism. Cysteine depletion or iron supplementation restores mitochondrial health in vacuole-impaired cells and prevents mitochondrial decline during aging. These results demonstrate that cysteine toxicity is a major driver of age-related mitochondrial deterioration and identify vacuolar amino acid compartmentation as a cellular strategy to minimize amino acid toxicity.

A Bacterial Effector Reveals the V-ATPase-ATG16L1 Axis that Initiates Xenophagy.

Antibacterial autophagy (xenophagy) is an important host defense, but how it is initiated is unclear. Here, we performed a bacterial transposon screen and identified a T3SS effector SopF that potently blocked Salmonella autophagy. SopF was a general xenophagy inhibitor without affecting canonical autophagy. S. Typhimurium ΔsopF resembled S. flexneri ΔvirAΔicsB with the majority of intracellular bacteria targeted by autophagy, permitting a CRISPR screen that identified host V-ATPase as an essential factor. Upon bacteria-caused vacuolar damage, the V-ATPase recruited ATG16L1 onto bacteria-containing vacuole, which was blocked by SopF. Mammalian ATG16L1 bears a WD40 domain required for interacting with the V-ATPase. Inhibiting autophagy by SopF promoted S. Typhimurium proliferation in vivo. SopF targeted Gln124 of ATP6V0C in the V-ATPase for ADP-ribosylation. Mutation of Gln124 also blocked xenophagy, but not canonical autophagy. Thus, the discovery of SopF reveals the V-ATPase-ATG16L1 axis that critically mediates autophagic recognition of intracellular pathogen.

Determinants, maintenance, and function of organellar pH.

The protonation state of soluble and membrane-associated macromolecules dictates their charge, conformation, and functional activity. In addition, protons (H+ or their equivalents) partake in numerous metabolic reactions and serve as a source of electrochemical energy to drive the transmembrane transport of both organic and inorganic substrates. Stringent regulation of the intracellular pH is therefore paramount to homeostasis. Although the regulation of the cytosolic pH has been studied extensively, our understanding of the determinants of the H+ concentration ([H+]) of intracellular organelles has developed more slowly, limited by their small size and inaccessibility. Recently, however, targeting of molecular probes to the organellar lumen together with advances in genomic, proteomic, and electrophysiological techniques have led to the identification and characterization of unique pumps, channels, and transporters responsible for the establishment and maintenance of intraorganellar pH. These developments and their implications for cellular function in health and disease are the subject of this review.

Reverse-topology membrane scission by the ESCRT proteins.

The narrow membrane necks formed during viral, exosomal and intra-endosomal budding from membranes, as well as during cytokinesis and related processes, have interiors that are contiguous with the cytosol. Severing these necks involves action from the opposite face of the membrane as occurs during the well-characterized formation of coated vesicles. This 'reverse' (or 'inverse')-topology membrane scission is carried out by the endosomal sorting complex required for transport (ESCRT) proteins, which form filaments, flat spirals, tubes and conical funnels that are thought to direct membrane remodelling and scission. Their assembly, and their disassembly by the ATPase vacuolar protein sorting-associated 4 (VPS4) have been intensively studied, but the mechanism of scission has been elusive. New insights from cryo-electron microscopy and various types of spectroscopy may finally be close to rectifying this situation.

Yeast TLDc domain proteins regulate assembly state and subcellular localization of the V-ATPase.

Yeast vacuoles perform crucial cellular functions as acidic degradative organelles, storage compartments, and signaling hubs. These functions are mediated by important protein complexes, including the vacuolar-type H+-ATPase (V-ATPase), responsible for organelle acidification. To gain a more detailed understanding of vacuole function, we performed cross-linking mass spectrometry on isolated vacuoles, detecting many known as well as novel protein-protein interactions. Among these, we identified the uncharacterized TLDc-domain-containing protein Rtc5 as a novel interactor of the V-ATPase. We further analyzed the influence of Rtc5 and of Oxr1, the only other yeast TLDc-domain-containing protein, on V-ATPase function. We find that both Rtc5 and Oxr1 promote the disassembly of the vacuolar V-ATPase in vivo, counteracting the role of the RAVE complex, a V-ATPase assembly chaperone. Furthermore, Oxr1 is necessary for the retention of a Golgi-specific subunit of the V-ATPase in this compartment. Collectively, our results shed light on the in vivo roles of yeast TLDc-domain proteins as regulators of the V-ATPase, highlighting the multifaceted regulation of this crucial protein complex.

Regulation of the mammalian-brain V-ATPase through ultraslow mode-switching.

Vacuolar-type adenosine triphosphatases (V-ATPases)1-3 are electrogenic rotary mechanoenzymes structurally related to F-type ATP synthases4,5. They hydrolyse ATP to establish electrochemical proton gradients for a plethora of cellular processes1,3. In neurons, the loading of all neurotransmitters into synaptic vesicles is energized by about one V-ATPase molecule per synaptic vesicle6,7. To shed light on this bona fide single-molecule biological process, we investigated electrogenic proton-pumping by single mammalian-brain V-ATPases in single synaptic vesicles. Here we show that V-ATPases do not pump continuously in time, as suggested by observing the rotation of bacterial homologues8 and assuming strict ATP-proton coupling. Instead, they stochastically switch between three ultralong-lived modes: proton-pumping, inactive and proton-leaky. Notably, direct observation of pumping revealed that physiologically relevant concentrations of ATP do not regulate the intrinsic pumping rate. ATP regulates V-ATPase activity through the switching probability of the proton-pumping mode. By contrast, electrochemical proton gradients regulate the pumping rate and the switching of the pumping and inactive modes. A direct consequence of mode-switching is all-or-none stochastic fluctuations in the electrochemical gradient of synaptic vesicles that would be expected to introduce stochasticity in proton-driven secondary active loading of neurotransmitters and may thus have important implications for neurotransmission. This work reveals and emphasizes the mechanistic and biological importance of ultraslow mode-switching.

Low-dose metformin targets the lysosomal AMPK pathway through PEN2.

Metformin, the most prescribed antidiabetic medicine, has shown other benefits such as anti-ageing and anticancer effects1-4. For clinical doses of metformin, AMP-activated protein kinase (AMPK) has a major role in its mechanism of action4,5; however, the direct molecular target of metformin remains unknown. Here we show that clinically relevant concentrations of metformin inhibit the lysosomal proton pump v-ATPase, which is a central node for AMPK activation following glucose starvation6. We synthesize a photoactive metformin probe and identify PEN2, a subunit of γ-secretase7, as a binding partner of metformin with a dissociation constant at micromolar levels. Metformin-bound PEN2 forms a complex with ATP6AP1, a subunit of the v-ATPase8, which leads to the inhibition of v-ATPase and the activation of AMPK without effects on cellular AMP levels. Knockout of PEN2 or re-introduction of a PEN2 mutant that does not bind ATP6AP1 blunts AMPK activation. In vivo, liver-specific knockout of Pen2 abolishes metformin-mediated reduction of hepatic fat content, whereas intestine-specific knockout of Pen2 impairs its glucose-lowering effects. Furthermore, knockdown of pen-2 in Caenorhabditis elegans abrogates metformin-induced extension of lifespan. Together, these findings reveal that metformin binds PEN2 and initiates a signalling route that intersects, through ATP6AP1, the lysosomal glucose-sensing pathway for AMPK activation. This ensures that metformin exerts its therapeutic benefits in patients without substantial adverse effects.

A "Sugar-Coated" Proton Pump Comes into Focus: High-Resolution Structure of a Human V-ATPase.

In this issue of Molecular Cell, Wang et al. (2020a) present near-atomic resolution cryoEM structures of a proton-pumping vacuolar ATPase from human cells, illuminating the glycosylation of integral subunits and identifying a novel and conserved glycolipid ligand.

Structures of a Complete Human V-ATPase Reveal Mechanisms of Its Assembly.

Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are ATP-driven proton pumps comprised of a cytoplasmic V1 complex for ATP hydrolysis and a membrane-embedded Vo complex for proton transfer. They play important roles in acidification of intracellular vesicles, organelles, and the extracellular milieu in eukaryotes. Here, we report cryoelectron microscopy structures of human V-ATPase in three rotational states at up to 2.9-Å resolution. Aided by mass spectrometry, we build all known protein subunits with associated N-linked glycans and identify glycolipids and phospholipids in the Vo complex. We define ATP6AP1 as a structural hub for Vo complex assembly because it connects to multiple Vo subunits and phospholipids in the c-ring. The glycolipids and the glycosylated Vo subunits form a luminal glycan coat critical for V-ATPase folding, localization, and stability. This study identifies mechanisms of V-ATPase assembly and biogenesis that rely on the integrated roles of ATP6AP1, glycans, and lipids.

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.

Oxidative stress protein Oxr1 promotes V-ATPase holoenzyme disassembly in catalytic activity-independent manner.

The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V1 -ATPase and Vo proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted Vo and mutant V1 . Our analysis identified holoenzymes in three active rotary states, indicating that binding of V1 to Vo provides sufficient free energy to overcome Vo autoinhibition. Moreover, the structures suggest that the unequal spacing of Vo 's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V1 and Vo motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V1 -ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.

A viral protein disrupts vacuolar acidification to facilitate virus infection in plants.

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.

Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP.

Endosomal-lysosomal trafficking is accompanied by the acidification of endosomal compartments by the H+-V-ATPase to reach low lysosomal pH. Disruption of the correct pH impairs lysosomal function and the balance of protein synthesis and degradation (proteostasis). Here, we treated mammalian cells with the small dipeptide LLOMe, which is known to permeabilize lysosomal membranes, and find that LLOMe also impacts late endosomes (LEs) by neutralizing their pH without causing membrane permeabilization. We show that LLOMe leads to hyperactivation of Rab7 (herein referring to Rab7a), and disruption of tubulation and mannose-6-phosphate receptor (CI-M6PR; also known as IGF2R) recycling on pH-neutralized LEs. pH neutralization (NH4Cl) and expression of Rab7 hyperactive mutants alone can both phenocopy the alterations in tubulation and CI-M6PR trafficking. Mechanistically, pH neutralization increases the assembly of the V1G1 subunit (encoded by ATP6V1G1) of the V-ATPase on endosomal membranes, which stabilizes GTP-bound Rab7 via RILP, a known interactor of Rab7 and V1G1. We propose a novel pathway by which V-ATPase and RILP modulate LE pH and Rab7 activation in concert. This pathway might broadly contribute to pH control during physiologic endosomal maturation or starvation and during pathologic pH neutralization, which occurs via lysosomotropic compounds and in disease states.

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.

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.

No Longer Hidden Secrets of Proton Pumping: The Resolution Revolution Enlightens V-ATPases.

In this issue of Molecular Cell, Roh et al. (2018) present a high-resolution cryo-EM structure of the nanodisc-reconstituted yeast Vo proton channel that provides important new insights into subunit arrangements and the proton translocation pathway in V-type ATPases.

The 3.5-Å CryoEM Structure of Nanodisc-Reconstituted Yeast Vacuolar ATPase Vo Proton Channel.

The molecular mechanism of transmembrane proton translocation in rotary motor ATPases is not fully understood. Here, we report the 3.5-Å resolution cryoEM structure of the lipid nanodisc-reconstituted Vo proton channel of the yeast vacuolar H+-ATPase, captured in a physiologically relevant, autoinhibited state. The resulting atomic model provides structural detail for the amino acids that constitute the proton pathway at the interface of the proteolipid ring and subunit a. Based on the structure and previous mutagenesis studies, we propose the chemical basis of transmembrane proton transport. Moreover, we discovered that the C terminus of the assembly factor Voa1 is an integral component of mature Vo. Voa1's C-terminal transmembrane α helix is bound inside the proteolipid ring, where it contributes to the stability of the complex. Our structure rationalizes possible mechanisms by which mutations in human Vo can result in disease phenotypes and may thus provide new avenues for therapeutic interventions.

Structural basis of V-ATPase VO region assembly by Vma12p, 21p, and 22p.

Vacuolar-type adenosine triphosphatases (V-ATPases) are rotary proton pumps that acidify specific intracellular compartments in almost all eukaryotic cells. These multi-subunit enzymes consist of a soluble catalytic V1 region and a membrane-embedded proton-translocating VO region. VO is assembled in the endoplasmic reticulum (ER) membrane, and V1 is assembled in the cytosol. However, V1 binds VO only after VO is transported to the Golgi membrane, thereby preventing acidification of the ER. We isolated VO complexes and subcomplexes from Saccharomyces cerevisiae bound to V-ATPase assembly factors Vma12p, Vma21p, and Vma22p. Electron cryomicroscopy shows how the Vma12-22p complex recruits subunits a, e, and f to the rotor ring of VO while blocking premature binding of V1. Vma21p, which contains an ER-retrieval motif, binds the VO:Vma12-22p complex, "mature" VO, and a complex that appears to contain a ring of loosely packed rotor subunits and the proteins YAR027W and YAR028W. The structures suggest that Vma21p binds assembly intermediates that contain a rotor ring and that activation of proton pumping following assembly of V1 with VO removes Vma21p, allowing V-ATPase to remain in the Golgi. Together, these structures show how Vma12-22p and Vma21p function in V-ATPase assembly and quality control, ensuring the enzyme acidifies only its intended cellular targets.

The V-type H+-ATPase is targeted in antidiuretic hormone control of the Malpighian "renal" tubules.

Like other insects, secretion by mosquito Malpighian tubules (MTs) is driven by the V-type H+-ATPase (VA) localized in the apical membrane of principal cells. In Aedes aegypti, the antidiuretic neurohormone CAPA inhibits secretion by MTs stimulated by select diuretic hormones; however, the cellular effectors of this inhibitory signaling cascade remain unclear. Herein, we demonstrate that the VA inhibitor bafilomycin selectively inhibits serotonin (5HT)- and calcitonin-related diuretic hormone (DH31)-stimulated secretion. VA activity increases in DH31-treated MTs, whereas CAPA abolishes this increase through a NOS/cGMP/PKG signaling pathway. A critical feature of VA activation involves the reversible association of the cytosolic (V1) and membrane (Vo) complexes. Indeed, higher V1 protein abundance was found in membrane fractions of DH31-treated MTs, whereas CAPA significantly decreased V1 abundance in membrane fractions while increasing it in cytosolic fractions. V1 immunolocalization was observed strictly in the apical membrane of DH31-treated MTs, whereas immunoreactivity was dispersed following CAPA treatment. VA complexes colocalized apically in female MTs shortly after a blood meal consistent with the peak and postpeak phases of diuresis. Comparatively, V1 immunoreactivity in MTs was more dispersed and did not colocalize with the Vo complex in the apical membrane at 3 h post blood meal, representing a time point after the late phase of diuresis has concluded. Therefore, CAPA inhibition of MTs involves reducing VA activity and promotes complex dissociation hindering secretion. Collectively, these findings reveal a key target in hormone-mediated inhibition of MTs countering diuresis that provides a deeper understanding of this critical physiological process necessary for hydromineral balance.