Adenosine Triphosphatase Complex Research Articles

Vacuolar H+-ATPase determines daughter cell fates through asymmetric segregation of the nucleosome remodeling and deacetylase complex.

Asymmetric cell divisions (ACDs) generate two daughter cells with identical genetic information but distinct cell fates through epigenetic mechanisms. However, the process of partitioning different epigenetic information into daughter cells remains unclear. Here, we demonstrate that the nucleosome remodeling and deacetylase (NuRD) complex is asymmetrically segregated into the surviving daughter cell rather than the apoptotic one during ACDs in Caenorhabditis elegans. The absence of NuRD triggers apoptosis via the EGL-1-CED-9-CED-4-CED-3 pathway, while an ectopic gain of NuRD enables apoptotic daughter cells to survive. We identify the vacuolar H+-adenosine triphosphatase (V-ATPase) complex as a crucial regulator of NuRD's asymmetric segregation. V-ATPase interacts with NuRD and is asymmetrically segregated into the surviving daughter cell. Inhibition of V-ATPase disrupts cytosolic pH asymmetry and NuRD asymmetry. We suggest that asymmetric segregation of V-ATPase may cause distinct acidification levels in the two daughter cells, enabling asymmetric epigenetic inheritance that specifies their respective life-versus-death fates.

MiR-1 sustains muscle physiology by controlling V-ATPase complex assembly.

Muscle function requires unique structural and metabolic adaptations that can render muscle cells selectively vulnerable, with mutations in some ubiquitously expressed genes causing myopathies but sparing other tissues. We uncovered a muscle cell vulnerability by studying miR-1, a deeply conserved, muscle-specific microRNA whose ablation causes various muscle defects. Using Caenorhabditis elegans, we found that miR-1 represses multiple subunits of the ubiquitous vacuolar adenosine triphosphatase (V-ATPase) complex, which is essential for internal compartment acidification and metabolic signaling. V-ATPase subunits are predicted miR-1 targets in animals ranging from C. elegans to humans, and we experimentally validated this in Drosophila. Unexpectedly, up-regulation of V-ATPase subunits upon miR-1 deletion causes reduced V-ATPase function due to defects in complex assembly. These results reveal V-ATPase assembly as a conserved muscle cell vulnerability and support a previously unknown role for microRNAs in the regulation of protein complexes.

Cohesin extrudes DNA loops

Molecular Biology DNA is folded into loops in eukaryotic cells by a process that depends on a ring-shaped adenosine triphosphatase complex called cohesin. Davidson et al. and Kim et al. now show that in the presence of the NIPBLMAU2 protein complex, the human cohesin complex can function as a molecular motor that extrudes DNA loops with high speed in vitro. In contrast to how it mediates sister chromatid cohesion, cohesin does not appear to entrap DNA topologically during loop extrusion. The results provide direct evidence for the loop extrusion model of chromatin organization and suggest that genome architecture is highly dynamic. Science , this issue p. [1338][1], p. [1345][2] [1]: /lookup/doi/10.1126/science.aaz3418 [2]: /lookup/doi/10.1126/science.aaz4475

Human cohesin compacts DNA by loop extrusion.

Cohesin is a chromosome-bound, multisubunit adenosine triphosphatase complex. After loading onto chromosomes, it generates loops to regulate chromosome functions. It has been suggested that cohesin organizes the genome through loop extrusion, but direct evidence is lacking. Here, we used single-molecule imaging to show that the recombinant human cohesin-NIPBL complex compacts both naked and nucleosome-bound DNA by extruding DNA loops. DNA compaction by cohesin requires adenosine triphosphate (ATP) hydrolysis and is force sensitive. This compaction is processive over tens of kilobases at an average rate of 0.5 kilobases per second. Compaction of double-tethered DNA suggests that a cohesin dimer extrudes DNA loops bidirectionally. Our results establish cohesin-NIPBL as an ATP-driven molecular machine capable of loop extrusion.

DNA loop extrusion by human cohesin.

Eukaryotic genomes are folded into loops and topologically associating domains, which contribute to chromatin structure, gene regulation, and gene recombination. These structures depend on cohesin, a ring-shaped DNA-entrapping adenosine triphosphatase (ATPase) complex that has been proposed to form loops by extrusion. Such an activity has been observed for condensin, which forms loops in mitosis, but not for cohesin. Using biochemical reconstitution, we found that single human cohesin complexes form DNA loops symmetrically at rates up to 2.1 kilo-base pairs per second. Loop formation and maintenance depend on cohesin's ATPase activity and on NIPBL-MAU2, but not on topological entrapment of DNA by cohesin. During loop formation, cohesin and NIPBL-MAU2 reside at the base of loops, which indicates that they generate loops by extrusion. Our results show that cohesin and NIPBL-MAU2 form an active holoenzyme that interacts with DNA either pseudo-topologically or non-topologically to extrude genomic interphase DNA into loops.

Doa1 is a MAD adaptor for Cdc48.

Dislocation of polypeptides from the mitochondrial outer membrane by the p97/Cdc48–Ufd1–Npl4 adenosine triphosphatase complex is essential for mitochondria-associated degradation and Parkin-mediated mitophagy. In this issue, Wu et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510098) identify Doa1 as a pivotal adaptor that recruits substrates to Cdc48 for processing.

Polarization of the vacuolar adenosine triphosphatase delineates a transition to high-grade pancreatic intraepithelial neoplasm lesions.

A functional vacuolar adenosine triphosphatase (v-ATPase) complex regulates canonical Wnt/β-catenin signaling. The goal of this study was to identify the distribution of the v-ATPase in human and murine models of pancreatic intraepithelial neoplasms (PanINs) and assess its role in Wnt/β-catenin signaling. We evaluated the immunolabeling pattern of the v-ATPase in human PanIN specimens and murine PanIN-1 and PanIN-2 lesions obtained from Ptf1a(Cre/+); LSL-Kras(G12D) mice. Wnt/β-catenin signaling was interrogated in primary PanIN cells by examining the phosphorylated levels of its surface coreceptor, low-density lipoprotein receptor-related protein-6 (LRP6), and its intracellular effector, nonphosphorylated β-catenin. The response of primary PanIN cells to epidermal growth factor (EGF) was assessed in the absence and presence of the v-ATPase inhibitor, concanamycin. In advanced (PanIN-2), but not early (PanIN-1), lesions, the v-ATPase assumed a polarized phenotype. Blocking the v-ATPase disrupted Wnt/β-catenin signaling in primary PanIN cells despite significantly higher levels of the total and activated Wnt cell surface coreceptor, LRP6. Vacuolar adenosine triphosphatase blockade significantly decreased the total and activated levels of EGF receptor, a determinant of PanIN progression. The activation of EGF receptor and its intracellular mediator, p44/42 mitogen-activated protein kinase, was also reduced by v-ATPase blockade. This led to diminished proliferation in response to EGF ligand. The v-ATPase regulates Wnt/β-catenin and EGF receptor signaling in PanINs.

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

Biochemical, Enzymatic and Molecular Properties of Renin/Prorenin Mediated by (Pro)Renin Receptor

(Pro)renin receptor, (P)RR, exhibited its crucial role in the renin angiotensin (RA) system and also in such organ development as the brain and retina. The binding of (pro)renin to (P)RR mediates the enhanced production of angiotensin-I, which stimulates the tissue-specific RA system and intracellular signal transduction by activating mitogen-activated protein kinase (ERK) 1/2 and p38 pathways. These characteristics together develop the organ damage, especially in diabetes and hypertension. (P)RR acts as an adaptor between Wnt receptors and the vacuolar H+-adenosine triphosphatase (V-ATPase) complex. (P)RR is expressed on the membrane and intracellularly in the golgi apparatus and endoplasmic reticulum. ADAM19 and Furin cleavage sites in the (P)RR molecule have been identified. A soluble form of (P)RR has been detected in the cell culture medium and plasma that can bind renin/prorenin. This review describes recent topics on (pro)renin receptor research in the context of its biochemical and molecular aspects associated with end-stage organ damage. Keywords: Decoy peptide, “Hinge non-proteolytic activation, peptide, PCP/Fz pathway, prorenin, (pro)renin receptor, renin, V-H+ATPase, Wnt/, β -catenin pathway

Dual effects of ouabain, digoxin and proscillaridin A on the regulation of apoptosis in human fibroblasts

In this study, we looked at the effect of ouabain, digoxin and proscillaridin A on human fibroblasts. These data show that low concentrations of ouabain, digoxin and proscillaridin A can activate proliferation of human fibroblasts, suggesting that the Na+, K+-adenosine triphosphatase complex may act as a transducing receptor. It was shown that 30 nM ouabain, digoxin and proscillaridin A stimulated an antiapoptotic action by the increase in the level of phosphorylated extracellular signal-regulated kinases (P-ERK 1/2). Ouabain, digoxin and proscillaridin A only at the relatively high concentration of 300 nM increased intracellular Ca2+ concentration, activated caspase-3 and induced apoptosis in human fibroblasts. In terms of reduction in cell viability, antiproliferative and apoptotic activity, these cardiac glycosides rank in the order proscillaridin A >digoxin >ouabain.

STRUCTURAL, TRANSCRIPTIONAL, AND PHYLOGENETIC ANALYSES OF THE atpB GENE CLUSTER FROM THE PLASTID OF CRYPTOMONASΨ (CRYPTOPHYCEAE)1

ABSTRACTGenes encoding the β and ɛ subunits of the H+‐adenosine triphosphatase complex have been identified in the large single‐copy region of the plastid genome from the marine chlorophyll c‐containing alga Cryptomonas Ψ. Sequence analysis shows that the two genes do not overlap as in most land plants but, rather, are separated by a short 11‐bp intergenic region. The genes lack introns and encode polypeptides of 475 and 131 amino acids, respectively. The two genes are co‐transcribed, producing a transcript of approximately 2000 nucleotides. Primer extension analysis of the 5′ end of the transcript indicates two termini, one more prominent than the other. Sequence motifs typical of chloroplast promoters are located at — 10 and — 35 bp from both of these termini. Upstream of the atpBE operon is a gene with similarity to ORF167 of land plant plastids, and downstream is ORF281, which is homologous to a similarly located open reading frame from a brown alga. Phylogenetic analysis of atpB sequences from a variety of eubacteria and plastids shows all plastids arising from a single origin within the cyanobacteria and a clear dichotomy between the green and non‐green lineages, in agreement with trees based on plastid small‐subunit ribosomal RNA sequences.

Cytochemical study of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase activity in guinea pig facial nerve.

The localization of ouabain-sensitive, K(+)-dependent p-nitrophenylphosphatase (K-NPPase) activity, the second dephosphorylative property of the Na-K adenosine triphosphatase complex, was cytochemically studied in the intra-temporal portion of the facial nerve in normal guinea pigs using a cerium-based method. A fine-granular reaction product of the K-NPPase activity was observed on the cytoplasmic side of the axolemma of the axon cylinder, of the incisures of Schmidt-Lanterman, and of the nodes of Ranvier. No reaction product was detected on the periaxonal and outermost plasma membrane of Schwann cells and in the myelin sheath. In control tissue samples, the enzyme activity was almost completely inhibited by 10 mM ouabain, and no reaction was noted in medium without K+.

Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH.

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.

Interaction between the oligomycin sensitivity conferring protein and the F0 sector of the mitochondrial adenosinetriphosphatase complex: cooperative effect of the F1 sector.

Beef heart mitchondrial oligomycin sensitivity conferring protein (OSCP) labeled with [14C]-N-ethylmaleimide ([14C]OSCP) at the only cysteine residue, Cys-118, present in the sequence [Ovchinnikov, Y. A., Modyanov, N. N., Grinkevich, V. A., Aldanova, N. A., Trubetskaya, O. E., Nazimov, I.V., Hundal, T., & Ernster, L. (1984) FEBS Lett. 166, 19-22] exhibits full biological activity in a reconstituted F0-F1 system [Dupuis, A., Issartel, J. P., Lunardi, J., Satre, M., & Vignais, P. V. (1985) Biochemistry 24, 728-733]. The binding parameters of [14C]OSCP with respect to the F0 sector of submitochondrial particles largely depleted of F1 and OSCP (AUA particles) have been explored. In the absence of added F1, a limited number of high-affinity OSCP binding sites were detected in the AUA particles (20-40 pmol/mg of particles); under these conditions, the low-affinity binding sites for OSCP were essentially not saturable. Addition of F1 to the particles promoted high-affinity binding for OSCP, with an apparent Kd of 5 nM, a value 16 times lower than the Kd relative to the binding of OSCP to F1 in the absence of particles. Saturation of the F1 and OSCP binding sites of AUA particles was attained with about 200 pmol of both F1 and OSCP added per milligram of particles. The oligomycin-dependent inhibition of F1-ATPase bound to AUA particles was assayed as a function of bound OSCP. At subsaturating concentrations of F1, the dose-effect curves were rectilinear until inhibition of ATPase activity by oligomycin was virtually complete, and maximal inhibition was obtained for an OSCP to F1 ratio of 1 (mol/mol).(ABSTRACT TRUNCATED AT 250 WORDS)

37] Generation of a protonmotive force in anaerobic bacteria by end-product efflux

Publisher Summary This chapter deals with the generation of a Protonmotive force in anaerobic bacteria by end-product efflux. The protonmotive force acts as a driving force or regulatory parameter in a large number of different metabolic processes. Until recently only three primary proton pumps have been described in bacteria: (1) membranebound electron transfer chains; (2) the proton-translocating adenosine triphosphatase (ATPase) complex; and (3) in halobacteria, the light-driven proton pump bacteriorhodopsin. A number of experimental approaches can supply information about the generation of metabolic energy by end-product efflux: (1) uptake experiments, to demonstrate carrier activity and to obtain information on the H + /end-product stoichiometry; (2) efflux experiments from preloaded membranes, to demonstrate end-product efflux driven Ap generation (and subsequent ATP synthesis); (3) simultaneous measurements of the end-product gradient and Ap in growing or actively metabolizing cells, to obtain information on the H + / end-product stoichiometry and possible contributions of energy recycling to the overall production of metabolic energy; and (4) yield experiments under various conditions.

Characterization of detergent-solubilized adenosine triphosphatase of chromaffin granule membranes

Purified bovine chromaffin granule membranes contain approximately 24 pmol/mg protein (16 copies per granule) of an F 1-like adenosine 5′-triphosphatase, and 340 pmol/mg protein (200 copies per granule) of a low-molecular weight protein which reacts covalently with dicyclohexylcarbodiimide. These co-purify on electrofocusing and exclusion chomatography and are apparently components of a proton-translocating adenosine triphosphatase complex, that is involved in maintaining the high concentration of catecholamines in the granules. The membranes contain another adenosine 5'-triphosphatase, of lower molecular weight, which is sensitive to inhibition by vanadate but relatively insensitive to dicyclohexylcarbodiimide. The function of this enzyme is unknown.

18] Cloning of the structural genes of the Escherichia coli adenosinetriphosphatase complex

Publisher Summary The structural genes (unc genes) encoding the eight proteins of the adenosine triphosphatase complex of Escherichia coli are arranged in an operon at about 83.5 min on the E. coli chromosome. Five of these genes, uncA, D, G, H, and C, code for the α, β, γ, δ and ϵ subunits of the soluble portion of the complex (F1-ATPase), respectively, and the remaining three genes, uncB, E, and F, encode the insoluble portion (F0). A number of approaches to the problem of cloning the structural genes of the unc operon have been used. A simple approach to cloning the unc structural genes uses an F′ plasmid isolation as the enriched DNA source for insertion of the operon into a multicopy plasmid vector using standard restriction and ligation procedures. A partial diploid (strain AN862) that duplicates the entire unc operon of E. coli is used as a source of the DNA to be cloned. The F′ plasmid in strain AN862 can be isolated and used as an enriched source of the unc genes. After restriction of the isolated F′ plasmid and ligation with the cloning vector, the ligation mix is used to transform an appropriate recipient strain, and selection is made for the antibiotic resistance determinant of the plasmid vector and for the ability to grow on succinate minimal medium.

Proteolipid of adenosinetriphosphatase from yeast mitochondria forms proton-selective channels in planar lipid bilayers.

Proteolipid isolated from yeast mitochondrial adenosinetriphosphatase by butanol extraction is reincorporated into lipid vesicles from which planar membranes are formed. The proteolipid permits electric conductance through the membrane. This conductance occurs through membrane channels which are highly selective for protons. Proton channels in the membrane are directly observed at high proton concentrations in the aqueous phases. Channels open and close independently from each other; their open-state conductances and lifetimes are monodisperse but influenced by the applied voltage (12 pS and 3 s, respectively, at pH 2.2 and 100 mV). Proton channels do not occur in single proteolipid molecules; the conducting structure consists of at least two polypeptide chains since channels form in a (reversible) bimolecular reaction of nonconducting forms of proteolipid. The number of proton channels at a constant proteolipid concentration changes in sharp transitions and by orders of magnitudes upon critical changes of membrane composition and pH. These transitions are caused by transitions of proteolipid organization in the membrane from a dispersed state (equilibrium between channel-forming "dimers" and a large pool of "monomers") to a state of almost complete aggregation of proteolipid which stabilizes large proton-conducting structures (probably associates of channel-forming dimers). This self-association of isolated proteolipid into structures containing proton-selective channels suggests that the six proteolipids in the adenosinetriphosphatase complex exist as a self-associating entity containing most likely three proton channels.

Proton adenosine triphosphatase complex of rat liver mitochondria. Interaction with the ATPase inhibitor peptide covalently labeled with N-hydroxysuccinimidyl-p-azidobenzoate.

Proton adenosine triphosphatase complex of rat liver mitochondria. Interaction with the ATPase inhibitor peptide covalently labeled with N-hydroxysuccinimidyl-p-azidobenzoate.

Assembly of the adenosine triphosphatase complex in Escherichia coli: assembly of F0 is dependent on the formation of specific F1 subunits.

A strain of Escherichia coli (AN1007) carrying the polar uncD436 allele which affects the operon coding for the F1-F0 adenosine triphosphatase (ATPase) complex was isolated and characterized. The uncD436 allele affected the two genes most distal to the operon promoter, i.e., uncD and uncC. Although the genes coding for the F0 portion of the ATPase complex were not affected in strains carrying this mutant allele, the lack of reconstitution of washed membranes by normal F1 ATPase suggested that a functional F0 might not be formed. This conclusion was supported by the observation that the 18,000-molecular-weight F0 subunit, coded for by the uncF gene, was absent from the membranes. Plasmid pAN36 (uncD+C+), when inserted into a strain carrying the uncD436 allele, resulted in the incorporation of the 18,000-molecular-weight F0 subunit into the membrane. A further series of experiments with Mu-induced polarity mutants, with and without plasmid pAN36, showed that the formation of both the alpha- and beta-subunits of F1 ATPase was an essential prerequisite to the incorporation into the membrane of the 18,000-molecular-weight F0 subunit and to the formation of a functional F0. Examination of the polypeptide composition of membranes from various unc mutants allowed a sequence for the normal assembly of the F1-F0 ATPase complex to be proposed.