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.     

Inter‐subunit interaction and quaternary rearrangement defined by the central stalk of prokaryotic V1‐ATPase

V‐type ATPases (V‐ATPases) are categorized as rotary ATP synthase/ATPase complexes. The V‐ATPases are distinct from F‐ATPases in terms of their rotation scheme, architecture and subunit composition. However, there is no detailed structural information on V‐ATPases despite the abundant biochemical and biophysical research. Here, we report a crystallographic study of V₁‐ATPase, from Thermus thermophilus, which is a soluble component consisting of A, B, D and F subunits. The structure at 4.5 Å resolution reveals inter‐subunit interactions and nucleotide binding. In particular, the structure of the central stalk composed of D and F subunits was shown to be characteristic of V₁‐ATPases. Small conformational changes of respective subunits and significant rearrangement of the quaternary structure observed in the three AB pairs were related to the interaction with the straight central stalk. The rotation mechanism is discussed based on a structural comparison between V₁‐ATPases and F₁‐ATPases.     

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

Vacuolar H⁺‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V₁‐ATPase and membrane‐integral V₀ proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V₁V₀ND). We show that V₁V₀ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation.     

Some assembly required: Contributions of Tom Stevens' lab to the V‐ATPase field

Tom Stevens' lab has explored the subunit composition and assembly of the yeast V‐ATPase for more than 30 years. Early studies helped establish yeast as the predominant model system for study of V‐ATPase proton pumps and led to the discovery of protein splicing of the V‐ATPase catalytic subunit. The Vma^? phenotype, characteristic of loss‐of‐V‐ATPase activity in yeast was key in determining the enzyme's subunit composition via yeast genetics. V‐ATPase subunit composition proved to be highly conserved among eukaryotes. Genetic screens for new vma mutants led to identification of a set of dedicated V‐ATPase assembly factors and helped unravel the complex pathways for V‐ATPase assembly. In later years, exploration of the evolutionary history of several V‐ATPase subunits provided new information about the enzyme's structure and function. This review highlights V‐ATPase work in the Stevens’ lab between 1987 and 2017.      

Crystal structure of A3B3 complex of V‐ATPase from Thermus thermophilus

Vacuolar‐type ATPases (V‐ATPases) exist in various cellular membranes of many organisms to regulate physiological processes by controlling the acidic environment. Here, we have determined the crystal structure of the A₃B₃ subcomplex of V‐ATPase at 2.8 Å resolution. The overall construction of the A₃B₃ subcomplex is significantly different from that of the α₃β₃ sub‐domain in F_oF₁‐ATP synthase, because of the presence of a protruding ‘bulge’ domain feature in the catalytic A subunits. The A₃B₃ subcomplex structure provides the first molecular insight at the catalytic and non‐catalytic interfaces, which was not possible in the structures of the separate subunits alone. Specifically, in the non‐catalytic interface, the B subunit seems to be incapable of binding ATP, which is a marked difference from the situation indicated by the structure of the F_oF₁‐ATP synthase. In the catalytic interface, our mutational analysis, on the basis of the A₃B₃ structure, has highlighted the presence of a cluster composed of key hydrophobic residues, which are essential for ATP hydrolysis by V‐ATPases.     

V‐ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C

The activity of vacuolar H⁺‐ATPase (V‐ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5‐HT). 5‐HT induces, via protein kinase A, the phosphorylation of V‐ATPase subunit C and the assembly of V‐ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V‐ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK‐506) do not prevent V‐ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg²⁺ level caused by loading secretory cells with EDTA‐AM leads to the activation of proton pumping in the absence of 5‐HT, prolongs the 5‐HT‐induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V‐ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg²⁺, namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands. © 2009 Wiley Periodicals, Inc.     

N‐linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H+‐ATPase (V‐ATPase) biosynthesis

The a subunit of the V₀ membrane‐integrated sector of human V‐ATPase has four isoforms, a1‐a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N‐glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N‐glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope‐tagged human a1‐a3 subunits, with or without mutations that convert Asn to Gln at putative N‐glycosylation sites. Expression and N‐glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N‐glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild‐type a subunits after enzymatic deglycosylation. Cycloheximide‐chase experiments showed that unglycosylated subunits were turned over at a higher rate than N‐glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co‐immunoprecipitation studies showed that they were unable to associate with the V‐ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N‐glycosylation is crucial in all four human V‐ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V‐ATPase complexes.     

Inter‐subunit interaction of gastric H+,K+‐ATPase prevents reverse reaction of the transport cycle

The gastric H⁺,K⁺‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H⁺,K⁺‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E₂P conformation. Different from Na⁺,K⁺‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E₂P to E₁P, which is likely to be relevant for the generation of a large H⁺ gradient in vivo situation.     

Molecular architecture of the N‐type ATPase rotor ring from Burkholderia pseudomallei

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N‐type rotary ATPase, in addition to an operon for a regular F‐type rotary ATPase. The molecular architecture of N‐type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N₁N_o‐type ATPase and investigated the structure and ion specificity of its membrane‐embedded c‐ring rotor by single‐particle electron cryo‐microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low‐density, low‐CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c‐ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c₁₇ ring is H⁺ specific, demonstrating that the ATPase is proton‐coupled. The c₁₇ ring stoichiometry results in a very high ion‐to‐ATP ratio of 5.7. We propose that this N‐ATPase is a highly efficient proton pump that helps these melioidosis‐causing bacteria to survive in the hostile, acidic environment of phagosomes.     

Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V‐ATPase

Glucose is the preferred carbon source for most cell types and a major determinant of cell growth. In yeast and certain mammalian cells, glucose activates the cAMP‐dependent protein kinase A (PKA), but the mechanisms of PKA activation remain unknown. Here, we identify cytosolic pH as a second messenger for glucose that mediates activation of the PKA pathway in yeast. We find that cytosolic pH is rapidly and reversibly regulated by glucose metabolism and identify the vacuolar ATPase (V‐ATPase), a proton pump required for the acidification of vacuoles, as a sensor of cytosolic pH. V‐ATPase assembly is regulated by cytosolic pH and is required for full activation of the PKA pathway in response to glucose, suggesting that it mediates, at least in part, the pH signal to PKA. Finally, V‐ATPase is also regulated by glucose in the Min6 β‐cell line and contributes to PKA activation and insulin secretion. Thus, these data suggest a novel and potentially conserved glucose‐sensing pathway and identify a mechanism how cytosolic pH can act as a signal to promote cell growth.     

MODULATION OF P‐GLYCOPROTEIN ATPASE OF HELICOVERPA ARMIGERA BY CHOLESTEROL: EFFECTS ON ATPASE ACTIVITY AND INTERACTION OF INSECTICIDES

Purified P‐glycoprotein ATPase from Helicoverpa armigera (Ha‐Pgp), reconstituted in proteoliposomes composed of phospholipids and cholesterol, shows higher ATPase activity in the presence of cholesterol than in its absence. The Ha‐Pgp ATPase activity was increased 30–40% with cholesterol. The K_M for ATP was found to be 1 and 0.8 mM in the absence and presence of cholesterol, respectively. The insecticide‐stimulated Ha‐Pgp ATPase activity was increased by 10–20% for all the insecticides in the reconstituted proteoliposomes containing cholesterol compared to those with no cholesterol. The effects of cholesterol on K_M and V_max values of insecticide‐stimulated Ha‐Pgp ATPase activity were unrelated to the size of the insecticide. Ha‐Pgp tryptophan fluorescence displayed a red shift of 3 and 8 nm in emission spectra upon binding of insecticides. Cholesterol enhances the interaction of insecticides with Ha‐Pgp. K_d values of different insecticides for binding to Ha‐Pgp were found to be lower in the presence of cholesterol in the proteoliposomes compared to its absence. Results suggest that cholesterol plays a role in the recognition and interaction of insecticides by modulating Ha‐Pgp ATPase and may be involved in efflux of insecticides from cells by the transporter.     

Affinity Purification and Structural Features of the Yeast Vacuolar ATPase Vo Membrane Sector

The membrane sector (V_o) of the proton pumping vacuolar ATPase (V-ATPase, V₁V_o-ATPase) from Saccharomyces cerevisiae was purified to homogeneity, and its structure was characterized by EM of single molecules and two-dimensional crystals. Projection images of negatively stained V_o two-dimensional crystals showed a ring-like structure with a large asymmetric mass at the periphery of the ring. A cryo-EM reconstruction of V_o from single-particle images showed subunits a and d in close contact on the cytoplasmic side of the proton channel. A comparison of three-dimensional reconstructions of free V_o and V_o as part of holo V₁V_o revealed that the cytoplasmic N-terminal domain of subunit a (a_NT) must undergo a large conformational change upon enzyme disassembly or (re)assembly from V_o, V₁, and subunit C. Isothermal titration calorimetry using recombinant subunit d and a_NT revealed that the two proteins bind each other with a K_d of ∼5 μm. Treatment of the purified V_o sector with 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] resulted in selective release of subunit d, allowing purification of a V_oΔd complex. Passive proton translocation assays revealed that both V_o and V_oΔd are impermeable to protons. We speculate that the structural change in subunit a upon release of V₁ from V_o during reversible enzyme dissociation plays a role in blocking passive proton translocation across free V_o and that the interaction between a_NT and d seen in free V_o functions to stabilize the V_o sector for efficient reassembly of V₁V_o.     

Iejimalides A and B inhibit lysosomal vacuolar H+‐ATPase (V‐ATPase) activity and induce S‐phase arrest and apoptosis in MCF‐7 cells

Iejimalides are novel macrolides that are cytostatic or cytotoxic against a wide range of cancer cells at low nanomolar concentrations. A recent study by our laboratory characterized the expression of genes and proteins that determine the downstream effects of iejimalide B. However, little is known about the cellular target(s) of iejimalide or downstream signaling that lead to cell‐cycle arrest and/or apoptosis. Iejimalides have been shown to inhibit the activity of vacuolar H⁺‐ATPase (V‐ATPase) in osteoclasts, but how this inhibition may lead to cell‐cycle arrest and/or apoptosis in epithelial cells is not known. In this study, MCF‐7 breast cancer cells were treated with iejimalide A or B and analyzed for changes in cell‐cycle dynamics, apoptosis, lysosomal pH, cytoplasmic pH, mitochondrial membrane potential, and generation of reactive oxygen species. Both iejimalides A and B sequentially neutralize the pH of lysosomes, induce S‐phase cell‐cycle arrest, and trigger apoptosis in MCF‐7 cells. Apoptosis occurs through a mechanism that involves oxidative stress and mitochondrial depolarization but not cytoplasmic acidification. These data confirm that iejimalides inhibit V‐ATPase activity in the context of epithelial tumor cells, and that this inhibition may lead to a lysosome‐initiated cell death process. J. Cell. Biochem. 109: 634–642, 2010. © 2009 Wiley‐Liss, Inc.     

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29 , 85–90]. Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295‐amino‐acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn‐Pro‐Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti‐ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V‐ATPase (vacuolar H⁺‐ATPase) on the vesicle membrane around the CV was also detected. Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V‐ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Cellular and functional analysis of four mutations located in the mitochondrial ATPase6 gene

The smallest rotary motor of living cells, F0F1‐ATP synthase, couples proton flow—generated by the OXPHOS system—from the intermembrane space back to the matrix with the conversion of ADP to ATP. While all mutations affecting the multisubunit complexes of the OXPHOS system probably impact on the cell's output of ATP, only mutations in complex V can be considered to affect this output directly. So far, most of the F0F1‐ATP synthase variations have been detected in the mitochondrial ATPase6 gene. In this study, the four most frequent mutations in the ATPase6 gene, namely L156R, L217R, L156P, and L217P, are studied for the first time together, both in primary cells and in cybrid clones. Arginine (“R”) mutations were associated with a much more severe phenotype than Proline (“P”) mutations, in terms of both biochemical activity and growth capacity. Also, a threshold effect in both “R” mutations appeared at 50% mutation load. Different mechanisms seemed to emerge for the two “R” mutations: the F1 seemed loosely bound to the membrane in the L156R mutant, whereas the L217R mutant induced low activity of complex V, possibly the result of a reduced rate of proton flow through the A6 channel. J. Cell. Biochem. 106: 878–886, 2009. © 2009 Wiley‐Liss, Inc.     

(Pro)renin receptor (ATP6AP2) depletion arrests As4.1 cells in the G0/G1 phase thereby increasing formation of primary cilia

The (pro)renin receptor [(P)RR, ATP6AP2] is a multifunctional transmembrane protein that activates local renin–angiotensin systems, but also interacts with Wnt pathways and vacuolar H⁺‐ATPase (V‐ATPase) during organogenesis. The aim of this study was to characterize the role of ATP6AP2 in the cell cycle in more detail. ATP6AP2 down‐regulation by siRNA in renal As4.1 cells resulted in a reduction in the rate of proliferation and a G0/G1 phase cell cycle arrest. We identified a number of novel target genes downstream of ATP6AP2 knock‐down that were related to the primary cilium (Bbs‐1, Bbs‐3, Bbs‐7, Rabl5, Ttc26, Mks‐11, Mks‐5, Mks‐2, Tctn2, Nme7) and the cell cycle (Pierce1, Clock, Ppif). Accordingly, the number of cells expressing the primary cilium was markedly increased. We found no indication that these effects were dependent of V‐ATPase activity, as ATP6AP2 knock‐down did not affect lysosomal pH and bafilomycin A neither influenced the ciliary expression pattern nor the percentage of ciliated cells. Furthermore, ATP6AP2 appears to be essential for mitosis. ATP6AP2 translocated from the endoplasmatic reticulum to mitotic spindle poles (pro‐, meta‐ and anaphase) and the central spindle bundle (telophase) and ATP6AP2 knock‐down results in markedly deformed spindles. We conclude that ATP6AP2 is necessary for cell division, cell cycle progression and mitosis. ATP6AP2 also inhibits ciliogenesis, thus promoting proliferation and preventing differentiation.     

Conserved V‐ATPase c subunit plays a role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking

In plant cells, the vacuolar‐type H⁺‐ATPases (V‐ATPase) are localized in the tonoplast, Golgi, trans‐Golgi network and endosome. However, little is known about how V‐ATPase influences plant growth, particularly with regard to the V‐ATPase c subunit (VHA‐c). Here, we characterized the function of a VHA‐c gene from Puccinellia tenuiflora (PutVHA‐c) in plant growth. Compared to the wild‐type, transgenic plants overexpressing PutVHA‐c in Arabidopsis thaliana exhibit better growth phenotypes in root length, fresh weight, plant height and silique number under the normal and salt stress conditions due to noticeably higher V‐ATPase activity. Consistently, the Arabidopsis atvha‐c5 mutant shows reduced V‐ATPase activity and retarded plant growth. Furthermore, confocal and immunogold electron microscopy assays demonstrate that PutVHA‐c is mainly localized to endosomal compartments. The treatment of concanamycin A (ConcA), a specific inhibitor of V‐ATPases, leads to obvious aggregation of the endosomal compartments labelled with PutVHA‐c‐GFP. Moreover, ConcA treatment results in the abnormal localization of two plasma membrane (PM) marker proteins Pinformed 1 (AtPIN1) and regulator of G protein signalling‐1 (AtRGS1). These findings suggest that the decrease in V‐ATPase activity blocks endosomal trafficking. Taken together, our results strongly suggest that the PutVHA‐c plays an important role in plant growth by influencing V‐ATPase‐dependent endosomal trafficking.     

Assembly and Regulation of the Yeast Vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells. Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V₁) sector, which contains the ATP-binding sites, from the membrane (V₀) sector, which contains the proton pore. A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes. We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure. In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V₁ and V₀, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V₁ sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly.     

Structure of V-type ATPase from Clostridium fervidus by electron microscopy

F-type and V-type ATPases couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes and have similarities in structure and mechanism. In both types of enzymes three main parts can be distinguished: headpiece, membrane-bound piece and stalk region. We report on structural details of the membrane sector and stalk region, including the stator, of V-type ATPase from Clostridium fervidus, as determined by electron microscopy. Besides visualization of the stator structure, one of the main findings is that in certain projections the central stalk connecting V1 and V₀ makes an angle of about 70° with the membrane. Implications for the subunit arrangement in V-type and F-type ATPase are discussed.