Browse
Submit Data
Databases
API
Help

Dataset Information

27 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Crystal structure of yeast V-ATPase subunit C reveals its stator function.

ABSTRACT: Vacuolar H(+)-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells. It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis. Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase. We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase subunits, at 1.75 A resolution. The structural features suggest that subunit C functions as a flexible stator that holds together the catalytic and membrane sectors of the enzyme. A second crystal form that was solved at 2.9 A resolution supports the flexible nature of subunit C. These structures provide a framework for exploring the unique mechanistic features of V-ATPases.

SUBMITTER: Drory O

PROVIDER: S-EPMC1299189 | biostudies-literature | 2004 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Crystal structure of yeast V-ATPase subunit C reveals its stator function.

Drory Omri O Frolow Felix F Nelson Nathan N

EMBO reports 20041201 12

Vacuolar H(+)-ATPase (V-ATPase) has a crucial role in the vacuolar system of eukaryotic cells. It provides most of the energy required for transport systems that utilize the proton-motive force that is generated by ATP hydrolysis. Some, but not all, of the V-ATPase subunits are homologous to those of F-ATPase and the nonhomologous subunits determine the unique features of V-ATPase. We determined the crystal structure of V-ATPase subunit C (Vma5p), which does not show any homology with F-ATPase s ...[more]

PMID: 15540116

Similar Datasets

Domain characterization and interaction of the yeast vacuolar ATPase subunit C with the peripheral stator stalk subunits E and G.

Project description:The proton pumping activity of the eukaryotic vacuolar ATPase (V-ATPase) is regulated by a unique mechanism that involves reversible enzyme dissociation. In yeast, under conditions of nutrient depletion, the soluble catalytic V(1) sector disengages from the membrane integral V(o), and at the same time, both functional units are silenced. Notably, during enzyme dissociation, a single V(1) subunit, C, is released into the cytosol. The affinities of the other V(1) and V(o) subunits for subunit C are therefore of particular interest. The C subunit crystal structure shows that the subunit is elongated and dumbbell-shaped with two globular domains (C(head) and C(foot)) separated by a flexible helical neck region (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1148-1152). We have recently shown that subunit C is bound in the V(1)-V(o) interface where the subunit is in contact with two of the three peripheral stators (subunit EG heterodimers): one via C(head) and one via C(foot) (Zhang, Z., Zheng, Y., Mazon, H., Milgrom, E., Kitagawa, N., Kish-Trier, E., Heck, A. J., Kane, P. M., and Wilkens, S. (2008) J. Biol. Chem. 283, 35983-35995). In vitro, however, subunit C binds only one EG heterodimer (Féthière, J., Venzke, D., Madden, D. R., and Böttcher, B. (2005) Biochemistry 44, 15906-15914), implying that EG has different affinities for the two domains of the C subunit. To determine which subunit C domain binds EG with high affinity, we have generated C(head) and C(foot) and characterized their interaction with subunit EG heterodimer. Our findings indicate that the high affinity site for EGC interaction is C(head). In addition, we provide evidence that the EGC(head) interaction greatly stabilizes EG heterodimer.

| S-EPMC2915702 | biostudies-literature

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

Project description: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.

| S-EPMC4969575 | biostudies-literature

Crystal structure of the yeast inner kinetochore subunit Cep3p.

Project description:In budding yeast, the four-protein CBF3 complex (Skp1p-Ctf13p-Cep3p-Ndc10p) initiates kinetochore assembly by binding to the CDEIII locus of centromeric DNA. A Cep3p dimer recruits a Skp1p-Ctf13p heterodimer and contacts two sites on CDEIII. We report here the crystal structure, determined at 2.8 A resolution by multiple isomorphous replacement with anomalous scattering, of a truncated Cep3p (Cep3p [47-608]), comprising all but an N-terminal, Zn(2)Cys(6)-cluster, DNA-binding module. Cep3p has a well-ordered structure throughout essentially all of its polypeptide chain, unlike most yeast transcription factors, including those with Zn(2)Cys(6) clusters, such as Gal4p. This difference may reflect an underlying functional distinction: whereas any particular transcription factor must adapt to a variety of upstream activating sites, Cep3p scaffolds kinetochore assembly on centromeres uniformly configured on all 16 yeast chromosomes. We have, using the structure of Cep3p (47-608) and the known structures of Zn(2)Cys(6)-cluster domains, modeled the interaction of Cep3p with CDEIII.

| S-EPMC2288795 | biostudies-literature

Crystal structure of the yeast vacuolar ATPase heterotrimeric EGC(head) peripheral stalk complex.

Project description:Vacuolar ATPases (V-ATPases) are multisubunit rotary motor proton pumps that function to acidify subcellular organelles in all eukaryotic organisms. V-ATPase is regulated by a unique mechanism that involves reversible dissociation into V₁-ATPase and V₀ proton channel, a process that involves breaking of protein interactions mediated by subunit C, the cytoplasmic domain of subunit "a" and three "peripheral stalks," each made of a heterodimer of E and G subunits. Here, we present crystal structures of a yeast V-ATPase heterotrimeric complex composed of EG heterodimer and the head domain of subunit C (C(head)). The structures show EG heterodimer folded in a noncanonical coiled coil that is stabilized at its N-terminal ends by binding to C(head). The coiled coil is disrupted by a bulge of partially unfolded secondary structure in subunit G and we speculate that this unique feature in the eukaryotic V-ATPase peripheral stalk may play an important role in enzyme structure and regulation by reversible dissociation.

| S-EPMC3496068 | biostudies-literature

Crystal structure of the cytoplasmic N-terminal domain of subunit I, a homolog of subunit a, of V-ATPase.

Project description:Subunit "a" is associated with the membrane-bound (V(O)) complex of eukaryotic vacuolar H(+)-ATPase acidification machinery. It has also been shown recently to be involved in diverse membrane fusion/secretory functions independent of acidification. Here, we report the crystal structure of the N-terminal cytosolic domain from the Meiothermus ruber subunit "I" homolog of subunit a. The structure is composed of a curved long central ?-helix bundle capped on both ends by two lobes with similar ?/? architecture. Based on the structure, a reasonable model of its eukaryotic subunit a counterpart was obtained. The crystal structure and model fit well into reconstructions from electron microscopy of prokaryotic and eukaryotic vacuolar H(+)-ATPases, respectively, clarifying their orientations and interactions and revealing features that could enable subunit a to play a role in membrane fusion/secretion.

| S-EPMC3207611 | biostudies-literature

Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae.

Project description:In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.

| S-EPMC34635 | biostudies-literature

Probing subunit-subunit interactions in the yeast vacuolar ATPase by peptide arrays.

Project description:BackgroundVacuolar (H(+))-ATPase (V-ATPase; V(1)V(o)-ATPase) is a large multisubunit enzyme complex found in the endomembrane system of all eukaryotic cells where its proton pumping action serves to acidify subcellular organelles. In the plasma membrane of certain specialized tissues, V-ATPase functions to pump protons from the cytoplasm into the extracellular space. The activity of the V-ATPase is regulated by a reversible dissociation mechanism that involves breaking and re-forming of protein-protein interactions in the V(1)-ATPase - V(o)-proton channel interface. The mechanism responsible for regulated V-ATPase dissociation is poorly understood, largely due to a lack of detailed knowledge of the molecular interactions that are responsible for the structural and functional link between the soluble ATPase and membrane bound proton channel domains.Methodology/principal findingsTo gain insight into where some of the stator subunits of the V-ATPase associate with each other, we have developed peptide arrays from the primary sequences of V-ATPase subunits. By probing the peptide arrays with individually expressed V-ATPase subunits, we have identified several key interactions involving stator subunits E, G, C, H and the N-terminal domain of the membrane bound a subunit.ConclusionsThe subunit-peptide interactions identified from the peptide arrays complement low resolution structural models of the eukaryotic vacuolar ATPase obtained from transmission electron microscopy. The subunit-subunit interaction data are discussed in context of our current model of reversible enzyme dissociation.

| S-EPMC3470569 | biostudies-literature

The torque of rotary F-ATPase can unfold subunit gamma if rotor and stator are cross-linked.

Project description:During ATP hydrolysis by F(1)-ATPase subunit ? rotates in a hydrophobic bearing, formed by the N-terminal ends of the stator subunits (??)(3). If the penultimate residue at the ?-helical C-terminal end of subunit ? is artificially cross-linked (via an engineered disulfide bridge) with the bearing, the rotary function of F(1) persists. This observation has been tentatively interpreted by the unfolding of the ?-helix and swiveling rotation in some dihedral angles between lower residues. Here, we screened the domain between rotor and bearing where an artificial disulfide bridge did not impair the rotary ATPase activity. We newly engineered three mutants with double cysteines farther away from the C-terminus of subunit ?, while the results of three further mutants were published before. We found ATPase and rotary activity for mutants with cross-links in the single ?-helical, C-terminal portion of subunit ? (from ?285 to ?276 in E. coli), and virtually no activity when the cross-link was placed farther down, where the C-terminal ?-helix meets its N-terminal counterpart to form a supposedly stable coiled coil. In conclusion, only the C-terminal singular ?-helix is prone to unwinding and can form a swivel joint, whereas the coiled coil portion seems to resist the enzyme's torque.

| S-EPMC3536650 | biostudies-literature

Structure of the yeast vacuolar ATPase.

Project description:The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

| S-EPMC2602884 | biostudies-literature

Crystal structure of a central stalk subunit C and reversible association/dissociation of vacuole-type ATPase.

Project description:The vacuole-type ATPases (V-ATPases) exist in various intracellular compartments of eukaryotic cells to regulate physiological processes by controlling the acidic environment. The crystal structure of the subunit C of Thermus thermophilus V-ATPase, homologous to eukaryotic subunit d of V-ATPases, has been determined at 1.95-A resolution and located into the holoenzyme complex structure obtained by single particle analysis as suggested by the results of subunit cross-linking experiments. The result shows that V-ATPase is substantially longer than the related F-type ATPase, due to the insertion of subunit C between the V(1) (soluble) and the V(o) (membrane bound) domains. Subunit C, attached to the V(o) domain, seems to have a socket like function in attaching the central-stalk subunits of the V(1) domain. This architecture seems essential for the reversible association/dissociation of the V(1) and the V(o) domains, unique for V-ATPase activity regulation.

| S-EPMC314138 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data