Phosphatidylinositol 3-kinase-mediated effects of glucose on vacuolar H+-ATPase assembly, translocation, and acidification of intracellular compartments in renal epithelial cells.
ABSTRACT: Vacuolar H+-ATPases (V-ATPases) are a family of ATP-driven proton pumps. They maintain pH gradients between intracellular compartments and are required for proton secretion out of the cytoplasm. Mechanisms of extrinsic control of V-ATPase are poorly understood. Previous studies showed that glucose is an important regulator of V-ATPase assembly in Saccharomyces cerevisiae. Human V-ATPase directly interacts with aldolase, providing a coupling mechanism for glucose metabolism and V-ATPase function. Here we show that glucose is a crucial regulator of V-ATPase in renal epithelial cells and that the effect of glucose is mediated by phosphatidylinositol 3-kinase (PI3K). Glucose stimulates V-ATPase-dependent acidification of the intracellular compartments in human proximal tubular cells HK-2 and porcine renal epithelial cells LLC-PK1. Glucose induces rapid ATP-independent assembly of the V1 and Vo domains of V-ATPase and extensive translocation of the V-ATPase V1 and Vo domains between different membrane pools and between membranes and the cytoplasm. In HK-2 cells, glucose stimulates polarized translocation of V-ATPase to the apical plasma membrane. The effects of glucose on V-ATPase trafficking and assembly can be abolished by pretreatment with the PI3K inhibitor LY294002 and can be reproduced in glucose-deprived cells by adenoviral expression of the constitutively active catalytic subunit p110alpha of PI3K. Taken together these data provide evidence that, in renal epithelial cells, glucose plays an important role in the control of V-ATPase-dependent acidification of intracellular compartments and V-ATPase assembly and trafficking and that the effects of glucose are mediated by PI3K-dependent signaling.
Project description: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 (V1?H). 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 V1?H with high affinity. We also found that only one molecule of HNT binds V1?H with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1?H. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H or HNT with V1?H, 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.
Project description:The vacuolar-type H<sup>+</sup>-ATPases (V-ATPase) hydrolyze ATP to pump protons across the plasma or intracellular membrane, secreting acids to the lumen or acidifying intracellular compartments. It has been implicated in tumor metastasis, renal tubular acidosis, and osteoporosis. Here, we report two cryo-EM structures of the intact V-ATPase from bovine brain with all the subunits including the subunit H, which is essential for ATPase activity. Two type-I transmembrane proteins, Ac45 and (pro)renin receptor, along with subunit c", constitute the core of the c-ring. Three different conformations of A/B heterodimers suggest a mechanism for ATP hydrolysis that triggers a rotation of subunits DF, inducing spinning of subunit d with respect to the entire c-ring. Moreover, many lipid molecules have been observed in the Vo domain to mediate the interactions between subunit c, c", (pro)renin receptor, and Ac45. These two structures reveal unique features of mammalian V-ATPase and suggest a mechanism of V1-Vo torque transmission.
Project description:Vacuolar-type H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for acidification of intracellular organelles and potential drug target. It is a multisubunit complex comprising a cytoplasmic V1 domain responsible for ATP hydrolysis and a membrane-embedded Vo domain that contributes to proton translocation across the membrane. Saccharomyces cerevisiae V-ATPase is composed of 14 subunits, deletion of any one of which results in well-defined growth defects. As the structure of V-ATPase and the function of each subunit have been well-characterized in yeast, this organism has been recognized as a preferred model for studies of V-ATPases. In this study, to assess the functional relatedness of the yeast and human V-ATPase subunits, we investigated whether human V-ATPase subunits can complement calcium- or pH-sensitive growth, acidification of the vacuolar lumen, assembly of the V-ATPase complex, and protein sorting in yeast mutants lacking the equivalent yeast genes. These assessments revealed that 9 of the 13 human V-ATPase subunits can partially or fully complement the function of the corresponding yeast subunits. Importantly, sequence similarity was not necessarily correlated with functional complementation. We also found that besides all Vo domain subunits, the V1 F subunit is required for proper assembly of the Vo domain at the endoplasmic reticulum. Furthermore, the human H subunit fully restored the level of vacuolar acidification, but only partially rescued calcium-sensitive growth, suggesting a specific role of the H subunit in V-ATPase activity. These findings provide important insights into functional homologies between yeast and human V-ATPases.
Project description: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.
Project description:The proton (H(+)) pumping vacuolar-type ATPase (V-ATPase) is a rotary enzyme that plays a pivotal role in forming intracellular acidic compartments in eukaryotic cells. In Saccharomyces cerevisiae, the membrane extrinsic catalytic V1 and the transmembrane proton-pumping Vo complexes have been shown to reversibly dissociate upon removal of glucose from the medium. However, the basis of this disassembly is largely unknown. In the earlier study, we have found that the amino-terminal ?-helical domain between Lys-33 and Lys-83 of yeast E subunit (Vma4p) in the peripheral stalk of the V1 complex has a role in glucose-dependent VoV1 assembly. Results of alanine-scanning mutagenesis within the domain revealed that the Vma4p Glu-44 is a key residue in VoV1 disassembly. Biochemical analysis on Vma4p Glu-44 to Ala, Asn, Asp, and Gln substitutions indicated that Glu-44 has a role in V-ATPase catalysis. These results suggest that Glu-44 is one of the key functional residues for subunit interaction in the V-ATPase stalk complex that allows both efficient rotation catalysis and assembly.
Project description:Proton-translocating vacuolar-type ATPases (V-ATPases) are necessary for numerous processes in eukaryotic cells, including receptor-mediated endocytosis, protein maturation, and lysosomal acidification. In mammals, V-ATPase subunit isoforms are differentially targeted to various intracellular compartments or tissues, but how these subunit isoforms influence enzyme activity is not clear. In the yeast Saccharomyces cerevisiae, isoform diversity is limited to two different versions of the proton-translocating subunit a: Vph1p, which is targeted to the vacuole, and Stv1p, which is targeted to the Golgi apparatus and endosomes. We show that purified V-ATPase complexes containing Vph1p have higher ATPase activity than complexes containing Stv1p and that the relative difference in activity depends on the presence of lipids. We also show that VO complexes containing Stv1p could be readily purified without attached V1 regions. We used this effect to determine structures of the membrane-embedded VO region with Stv1p at 3.1-Å resolution, which we compare with a structure of the VO region with Vph1p that we determine to 3.2-Å resolution. These maps reveal differences in the surface charge near the cytoplasmic proton half-channel. Both maps also show the presence of bound lipids, as well as regularly spaced densities that may correspond to ergosterol or bound detergent, around the c-ring.
Project description:Eukaryotic V1VO-ATPases hydrolyze ATP in the V1 domain coupled to ion pumping in VO. A unique mode of regulation of V-ATPases is the reversible disassembly of V1 and VO, which reduces ATPase activity and causes silencing of ion conduction. The subunits D and F are proposed to be key in these enzymatic processes. Here, we describe the structures of two conformations of the subunit DF assembly of Saccharomyces cerevisiae (ScDF) V-ATPase at 3.1 Å resolution. Subunit D (ScD) consists of a long pair of ?-helices connected by a short helix ((79)IGYQVQE(85)) as well as a ?-hairpin region, which is flanked by two flexible loops. The long pair of helices is composed of the N-terminal ?-helix and the C-terminal helix, showing structural alterations in the two ScDF structures. The entire subunit F (ScF) consists of an N-terminal domain of four ?-strands (?1-?4) connected by four ?-helices (?1-?4). ?1 and ?2 are connected via the loop (26)GQITPETQEK(35), which is unique in eukaryotic V-ATPases. Adjacent to the N-terminal domain is a flexible loop, followed by a C-terminal ?-helix (?5). A perpendicular and extended conformation of helix ?5 was observed in the two crystal structures and in solution x-ray scattering experiments, respectively. Fitted into the nucleotide-bound A3B3 structure of the related A-ATP synthase from Enterococcus hirae, the arrangements of the ScDF molecules reflect their central function in ATPase-coupled ion conduction. Furthermore, the flexibility of the terminal helices of both subunits as well as the loop (26)GQITPETQEK(35) provides information about the regulatory step of reversible V1VO disassembly.
Project description:Progressive luminal acidification of intracellular compartments is important for their functions. Proton transport into the organelle's lumen is mediated by vacuolar ATPases (V-ATPases) large multi-subunit proton pumps organized into 2 domains, V0 and V1, working together as a rotary machine. The interaction of each subunit with specific partners plays a crucial role in controlling V-ATPase activity. Recently, we have shown that RILP, a Rab7 effector regulating late endocytic traffic and biogenesis of multivesicular bodies (MVBs), is a specific interactor of the V-ATPase subunit V1G1, a fundamental component of the peripheral stalk for correct V-ATPase assembly. RILP controls V1G1 stability and localization affecting V-ATPase assembly and function at the level of endosomes and lysosomes. The discovery of this new regulatory mechanism for V-ATPase opens new scenario to the comprehension of organelle's pH regulation and reveals a key role of RILP in controlling different aspects of endosome to lysosome transport.
Project description:Vacuolar-type ATPases (V-ATPases) are ATP-powered proton pumps involved in processes such as endocytosis, lysosomal degradation, secondary transport, TOR signalling, and osteoclast and kidney function. ATP hydrolysis in the soluble catalytic V1 region drives proton translocation through the membrane-embedded VO region via rotation of a rotor subcomplex. Variability in the structure of the intact enzyme has prevented construction of an atomic model for the membrane-embedded motor of any rotary ATPase. We induced dissociation and auto-inhibition of the V1 and VO regions of the V-ATPase by starving the yeast Saccharomyces cerevisiae, allowing us to obtain a ~3.9-Å resolution electron cryomicroscopy map of the VO complex and build atomic models for the majority of its subunits. The analysis reveals the structures of subunits ac8c'c?de and a protein that we identify and propose to be a new subunit (subunit f). A large cavity between subunit a and the c-ring creates a cytoplasmic half-channel for protons. The c-ring has an asymmetric distribution of proton-carrying Glu residues, with the Glu residue of subunit c? interacting with Arg735 of subunit a. The structure suggests sequential protonation and deprotonation of the c-ring, with ATP-hydrolysis-driven rotation causing protonation of a Glu residue at the cytoplasmic half-channel and subsequent deprotonation of a Glu residue at a luminal half-channel.
Project description:The V-ATPase is a membrane-bound protein complex which pumps protons across the membrane to generate a large proton motive force through the coupling of an ATP-driven 3-stroke rotary motor (V1) to a multistroke proton pump (Vo). This is done with near 100% efficiency, which is achieved in part by flexibility within the central rotor axle and stator connections, allowing the system to flex to minimise the free energy loss of conformational changes during catalysis. We have used electron microscopy to reveal distinctive bending along the V-ATPase complex, leading to angular displacement of the V1 domain relative to the Vo domain to a maximum of ~30°. This has been complemented by elastic network normal mode analysis that shows both flexing and twisting with the compliance being located in the rotor axle, stator filaments, or both. This study provides direct evidence of flexibility within the V-ATPase and by implication in related rotary ATPases, a feature predicted to be important for regulation and their high energetic efficiencies.