Human V-ATPase function is positively and negatively regulated by TLDc proteins.

Proteins that contain a highly conserved TLDc domain (Tre2/Bub2/Cdc16 LysM domain catalytic) offer protection against oxidative stress and are widely implicated in neurological health and disease. How this family of proteins exerts their function, however, is poorly understood. We have recently found that the yeast TLDc protein, Oxr1p, inhibits the proton pumping vacuolar ATPase (V-ATPase) by inducing disassembly of the pump. While loss of TLDc protein function in mammals shares disease phenotypes with V-ATPase defects, whether TLDc proteins impact human V-ATPase activity directly is unclear. Here we examine the effects of five human TLDc proteins, TLDC2, NCOA7, OXR1, TBC1D24, and mEAK7 on the activity of the human V-ATPase. We find that while TLDC2, TBC1D24, and the TLDc domains of OXR1 and NCOA7 inhibit V-ATPase by inducing enzyme disassembly, mEAK7 activates the pump. The data thus shed new light both on mammalian TLDc protein function and V-ATPase regulation.

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.

The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

Vacuolar H+-ATPases (V-ATPases) acidify several organelles in all eukaryotic cells and export protons across the plasma membrane in a subset of cell types. V-ATPases are multisubunit enzymes consisting of a peripheral subcomplex, V1, that is exposed to the cytosol and an integral membrane subcomplex, Vo, that contains the proton pore. The Vo a-subunit is the largest membrane subunit and consists of two domains. The N-terminal domain of the a-subunit (aNT) interacts with several V1 and Vo subunits and serves to bridge the V1 and Vo subcomplexes, while the C-terminal domain contains eight transmembrane helices, two of which are directly involved in proton transport. Although there can be multiple isoforms of several V-ATPase subunits, the a-subunit is encoded by the largest number of isoforms in most organisms. For example, the human genome encodes four a-subunit isoforms that exhibit a tissue- and organelle-specific distribution. In the yeast S. cerevisiae, the two a-subunit isoforms, Golgi-enriched Stv1 and vacuolar Vph1, are the only V-ATPase subunit isoforms. Current structural information indicates that a-subunit isoforms adopt a similar backbone structure but sequence variations allow for specific interactions during trafficking and in response to cellular signals. V-ATPases are subject to several types of environmental regulation that serve to tune their activity to their cellular location and environmental demands. The position of the aNT domain in the complex makes it an ideal target for modulating V1-Vo interactions and regulating enzyme activity. The yeast a-subunit isoforms have served as a paradigm for dissecting interactions of regulatory inputs with subunit isoforms. Importantly, structures of yeast V-ATPases containing each a-subunit isoform are available. Chimeric a-subunits combining elements of Stv1NT and Vph1NT have provided insights into how regulatory inputs can be integrated to allow V-ATPases to support cell growth under different stress conditions. Although the function and distribution of the four mammalian a-subunit isoforms present additional complexity, it is clear that the aNT domains of these isoforms are also subject to multiple regulatory interactions. Regulatory mechanisms that target mammalian a-subunit isoforms, and specifically the aNT domains, will be described. Altered V-ATPase function is associated with multiple diseases in humans. The possibility of regulating V-ATPase subpopulations via their isoform-specific regulatory interactions are discussed.

Tender love and disassembly: How a TLDc domain protein breaks the V-ATPase.

Vacuolar ATPases (V-ATPases, V1 Vo -ATPases) are rotary motor proton pumps that acidify intracellular compartments, and, when localized to the plasma membrane, the extracellular space. V-ATPase is regulated by a unique process referred to as reversible disassembly, wherein V1 -ATPase disengages from Vo proton channel in response to diverse environmental signals. Whereas the disassembly step of this process is ATP dependent, the (re)assembly step is not, but requires the action of a heterotrimeric chaperone referred to as the RAVE complex. Recently, an alternative pathway of holoenzyme disassembly was discovered that involves binding of Oxidation Resistance 1 (Oxr1p), a poorly characterized protein implicated in oxidative stress response. Unlike conventional reversible disassembly, which depends on enzyme activity, Oxr1p induced dissociation can occur in absence of ATP. Yeast Oxr1p belongs to the family of TLDc domain containing proteins that are conserved from yeast to mammals, and have been implicated in V-ATPase function in a variety of tissues. This brief perspective summarizes what we know about the molecular mechanisms governing both reversible (ATP dependent) and Oxr1p driven (ATP independent) V-ATPase dissociation into autoinhibited V1 and Vo subcomplexes.

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.

Functional reconstitution of vacuolar H+-ATPase from Vo proton channel and mutant V1-ATPase provides insight into the mechanism of reversible disassembly.

The vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is an ATP-dependent proton pump that acidifies subcellular compartments in all eukaryotic organisms. V-ATPase activity is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel subcomplexes, a process that is poorly understood on the molecular level. V-ATPase is a rotary motor, and recent structural analyses have revealed different rotary states for disassembled V1 and Vo, a mismatch that is likely responsible for their inability to reconstitute into holo V-ATPase in vitro Here, using the model organism Saccharomyces cerevisiae, we show that a key impediment for binding of V1 to Vo is the conformation of the inhibitory C-terminal domain of subunit H (HCT). Using biolayer interferometry and biochemical analyses of purified mutant V1-ATPase and Vo proton channel reconstituted into vacuolar lipid-containing nanodiscs, we further demonstrate that disruption of HCT's V1-binding site facilitates assembly of a functionally coupled and stable V1Vo-ATPase. Unlike WT, this mutant enzyme was resistant to MgATP hydrolysis-induced dissociation, further highlighting HCT's role in the mechanism of V-ATPase regulation. Our findings provide key insight into the molecular events underlying regulation of V-ATPase activity by reversible disassembly.

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary-motor proton pumps that acidify intracellular compartments and, in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo H is required for MgATPase activity in holo-V-ATPase but also for stabilizing the MgADP-inhibited state in membrane-detached V1 However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full-length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full-length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H or HNT with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly.

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.

The Presynaptic v-ATPase Reversibly Disassembles and Thereby Modulates Exocytosis but Is Not Part of the Fusion Machinery.

Vacuolar H+-ATPase (v-ATPase) is a multi-subunit complex comprising two domains: the cytosolic V1 domain catalyzing ATP hydrolysis and the membranous V0 sector translocating protons across membranes. In addition to proton pumping, a direct function of the V0 proteolipid ring in membrane fusion has been proposed for yeast vacuolar fusion and synaptic vesicle exocytosis in Drosophila. Here, we show in cultured hippocampal neurons that in recycling synaptic vesicles, v-ATPases are only transiently assembled in a pH-dependent fashion during the tightly coupled cycle of exo-endocytosis. Upon locking v-ATPase in an assembled state by saliphenylhalamide, we observed use- and time-dependent release depression for stimuli exceeding release of primed vesicles but no abrogation of exocytosis. Thus, the membranous V0 sector is not part of the exocytotic fusion machinery. Instead, v-ATPase modulates release upstream of docking to favor fusion of fully filled synaptic vesicles.

Responsiveness and Morphology Study of Dual Stimuli-Controlled Supramolecular Polymer.

The synthesis and characterization of a linear supramolecular polymer formed by dual host-guest recognitions is presented. The polymer linked by the orthogonal interactions of azobenzene with ß-cyclodextrin and methyl viologen with sulfonatocalix[4]arene is constructed, and the morphology change along with the formation and vanishment of host-guest interaction is investigated. The reversible disassembly-reassembly of the polymer induced by light and the redox process are monitored by UV-vis and cyclic voltammetry, respectively. The interesting morphology differences between the monomer guest (G), supramolecular polymer (P), and light dissembled product pseudorotaxane (D1) are observed and analyzed. G conducts self-assembly into a short rod with average width of 83 nm due to the molecular amphipathy and π-π interaction between naphthalene nucleuses, while P exhibits 20 nm wide line morphology. Irradiating P with 365 nm light, the corresponding aggregation D1 shows as 35 nm wide short rod.

Breaking up and making up: The secret life of the vacuolar H+ -ATPase.

The vacuolar ATPase (V-ATPase; V1 Vo -ATPase) is a large multisubunit proton pump found in the endomembrane system of all eukaryotic cells where it acidifies the lumen of subcellular organelles including lysosomes, endosomes, the Golgi apparatus, and clathrin-coated vesicles. V-ATPase function is essential for pH and ion homeostasis, protein trafficking, endocytosis, mechanistic target of rapamycin (mTOR), and Notch signaling, as well as hormone secretion and neurotransmitter release. V-ATPase can also be found in the plasma membrane of polarized animal cells where its proton pumping function is involved in bone remodeling, urine acidification, and sperm maturation. Aberrant (hypo or hyper) activity has been associated with numerous human diseases and the V-ATPase has therefore been recognized as a potential drug target. Recent progress with moderate to high-resolution structure determination by cryo electron microscopy and X-ray crystallography together with sophisticated single-molecule and biochemical experiments have provided a detailed picture of the structure and unique mode of regulation of the V-ATPase. This review summarizes the recent advances, focusing on the structural and biophysical aspects of the field.

Crystal structure of yeast V1-ATPase in the autoinhibited state.

Vacuolar ATPases (V-ATPases) are essential proton pumps that acidify the lumen of subcellular organelles in all eukaryotic cells and the extracellular space in some tissues. V-ATPase activity is regulated by a unique mechanism referred to as reversible disassembly, wherein the soluble catalytic sector, V1, is released from the membrane and its MgATPase activity silenced. The crystal structure of yeast V1 presented here shows that activity silencing involves a large conformational change of subunit H, with its C-terminal domain rotating ~150° from a position near the membrane in holo V-ATPase to a position at the bottom of V1 near an open catalytic site. Together with biochemical data, the structure supports a mechanistic model wherein subunit H inhibits ATPase activity by stabilizing an open catalytic site that results in tight binding of inhibitory ADP at another site.