Assembly of the yeast vacuolar H+-ATPase and ATP hydrolysis occurs in the absence of subunit c''

The V-ATPases are ubiquitous enzymes of eukaryotes. They are involved in many cellular processes via their ability to pump protons across biological membranes. They are two domain enzymes comprising an ATP hydrolysing sector and a proton translocating sector. Both sectors are functionally coupled. The proton tanslocating sector, V0, is comprised of five polypeptides in an as yet undetermined stoichiometry. In V0 three homologous proteins, subunit c, c' and c' have previously been reported to be essential for assembly of the enzyme. However, we report that subunit c' is not essential for assembly but is for functional coupling of the enzyme.     

The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations

The Na+,K+-ATPase transforms the energy of ATP to the maintenance of steep electrochemical gradients for sodium and potassium across the plasma membrane. This activity is tissue specific, in particular due to variations in the expressions of the alpha subunit isoforms one through four. Several mutations in alpha2 and 3 have been identified that link the specific function of the Na+,K+-ATPase to the pathophysiology of neurological diseases such as rapid-onset dystonia parkinsonism and familial hemiplegic migraine type 2. We show a mapping of the isoform differences and the disease-related mutations on the recently determined crystal structure of the pig renal Na+,K+-ATPase and a structural comparison to Ca2+-ATPase. Furthermore, we present new experimental data that address the role of a stretch of three conserved arginines near the C-terminus of the alpha subunit (Arg1003-Arg1005).     

Measurements of Na+-occluded intermediates during the catalytic cycle of the Na+/K+-ATPase provide novel insights into the mechanism of Na+ transport

The Na⁺/K⁺-ATPase is an integral plasma membrane glycoprotein of all animal cells that couples the exchange of intracellular Na⁺ for extracellular K⁺ to the hydrolysis of ATP. The asymmetric distribution of Na⁺ and K⁺ is essential for cellular life and constitutes the physical basis of a series of fundamental biological phenomena. The pumping mechanism is explained by the Albers–Post model. It involves the presence of gates alternatively exposing Na⁺/K⁺-ATPase transport sites to the intracellular and extracellular sides and includes occluded states in which both gates are simultaneously closed. Unlike for K⁺, information is lacking about Na⁺-occluded intermediates, as occluded Na⁺ was only detected in states incapable of performing a catalytic cycle, including two Na⁺-containing crystallographic structures. The current knowledge is that intracellular Na⁺ must bind to the transport sites and become occluded upon phosphorylation by ATP to be transported to the extracellular medium. Here, taking advantage of epigallocatechin-3-gallate to instantaneously stabilize native Na⁺-occluded intermediates, we isolated species with tightly bound Na⁺ in an enzyme able to perform a catalytic cycle, consistent with a genuine occluded state. We found that Na⁺ becomes spontaneously occluded in the E1 dephosphorylated form of the Na⁺/K⁺-ATPase, exhibiting positive interactions between binding sites. In fact, the addition of ATP does not produce an increase in Na⁺ occlusion as it would have been expected; on the contrary, occluded Na⁺ transiently decreases, whereas ATP lasts. These results reveal new properties of E1 intermediates of the Albers–Post model for explaining the Na⁺ transport pathway.     

Evolutionary appearance of the plasma membrane H+-ATPase containing a penultimate threonine in the bryophyte

The plasma membrane H⁺-ATPase provides the driving force for solute transport via an electrochemical gradient of H⁺ across the plasma membrane, and regulates pH homeostasis and membrane potential in plant cells. However, the plasma membrane H⁺-ATPase in non-vascular plant bryophyte is largely unknown. Here, we show that the moss Physcomitrella patens, which is known as a model bryophyte, expresses both the penultimate Thr-containing H⁺-ATPase (pT H⁺-ATPase) and non-pT H⁺-ATPase as in the green algae, and that pT H⁺-ATPase is regulated by phosphorylation of its penultimate Thr. A search in the P. patens genome database revealed seven H⁺-ATPase genes, designated PpHA (Physcomitrella patens H⁺-ATPase). Six isoforms are the pT H⁺-ATPase; a remaining isoform is non-pT H⁺-ATPase. An apparent 95-kD protein was recognized by anti-H⁺-ATPase antibodies against an isoform of Arabidopsis thaliana and was phosphorylated on the penultimate Thr in response to a fungal toxin fusicoccin and light in protonemata, indicating that the 95-kD protein contains pT H⁺-ATPase. Furthermore, we could not detect the pT H⁺-ATPase in the charophyte alga Chara braunii, which is the closest relative of the land plants, by immunological methods. These results strongly suggest the pT H⁺-ATPase most likely appeared for the first time in bryophyte.     

Roles of transmembrane segment M1 of Na+,K+-ATPase and Ca2+-ATPase, the gatekeeper and the pivot

In this review we summarize mutagenesis work on the structure–function relationship of transmembrane segment M1 in the Na⁺,K⁺-ATPase and the sarco(endo)plasmic reticulum Ca²⁺-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca²⁺ interaction/occlusion in Ca²⁺-ATPase and K⁺ interaction/occlusion in Na⁺,K⁺-ATPase. Leu⁶⁵ of the Ca²⁺-ATPase and Leu⁹⁹ of the Na⁺,K⁺-ATPase, located at homologous positions in M1, function as gate-locking residues that restrict the mobility of the side chain of the cation binding/gating residue of transmembrane segment M4, Glu³⁰⁹/Glu³²⁹. A pivot formed between a pair of a glycine and a bulky residue in M1 and M3 seems critical to the opening of the extracytoplasmic gate in both the Ca²⁺-ATPase and the Na⁺,K⁺-ATPase. All numbering of Na⁺,K⁺-ATPase amino acid residues in this article refers to the sequence of the rat α₁-isoform.     

Effect of hyperbaric oxygenation on the Na+, K+-ATPase and membrane fluidity of cerebrocortical membranes after experimental subarachnoid hemorrhage

It is reported that CNS hemorrage causes membrane dysfunction and may exacerbate this damage as a result of secondary ischemia or hypoxia. Since hyperbaric oxygenation improves oxygen metabolism, it may reduce this membrane damage. The present study was conducted to reveal whether hyperbaric oxygenation influences membrane alteration after hemorrhage. Thirty minutes after subarachnoid hemorrhage induction, rats were treated with hyperbaric oxygenation 2 ATA for 1 hour. Rats were decapitated 2 hours after subarachnoid hemorrhage induction. Na⁺, K⁺-ATPase activity measurement, and spin-label studies were performed on crude synpatosomal membranes. Subarachnoid hemorrhage decreased Na⁺, K⁺-ATPase activity. Spin label studies showed that hydrophobic portions of near the membrane surface became more rigid and the mobility of the membrane protein labeled sulfhydryl groups decreased after subarachnoid hemorrhage. Hyperbaric oxygenation significantly ameliorated most of the subarachnoid hemorrhage induced alterations. We conclude that hyperbaric oxygenation may be a beneficial treatment for acute subarachnoid hemorrhage.     

Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima

The hypothesis that the primary Na+-pump, Na+-ATPase, functions in the plasma membrane (PM) of halotolerant microalga Dunaliella maritima was tested using membrane preparations from this organism enriched with the PM vesicles. The pH profile of ATP hydrolysis catalyzed by the PM fractions exhibited a broad optimum between pH 6 and 9. Hydrolysis in the alkaline range was specifically stimulated by Na+ ions. Maximal sodium dependent ATP hydrolysis was observed at pH 7.5-8.0. On the assumption that the ATP-hydrolysis at alkaline pH values is related to a Na+-ATPase activity, we investigated two ATP-dependent processes, sodium uptake by the PM vesicles and generation of electric potential difference (Deltapsi) across the vesicle membrane. PM vesicles from D. maritima were found to be able to accumulate 22Na+ upon ATP addition, with an optimum at pH 7.5-8.0. The ATP-dependent Na+ accumulation was stimulated by the permeant NO3- anion and the protonophore CCCP, and inhibited by orthovanadate. The sodium accumulation was accompanied by pronounced Deltapsi generation across the vesicle membrane. The data obtained indicate that a primary Na+ pump, an electrogenic Na+-ATPase of the P-type, functions in the PM of marine microalga D. maritima.     

Involvement of Na+/H+ exchangers in induction of cyclooxygenase-2 by vacuolar-type (H+)-ATPase inhibitors in RAW 264 cells

In the mouse macrophage-like cell line RAW 264, vacuolar-type (H(+))-ATPase (V-ATPase) inhibitors, bafilomycin A(1) and concanamycin A, increased the level of cyclooxygenase (COX)-2 protein and its mRNA. The V-ATPase inhibitor-induced expression of COX-2 was suppressed by inhibitors of c-jun N-terminal kinase (JNK) and nuclear factor-kappaB, and by inhibitors of Na(+)/H(+) exchangers (NHEs). The bafilomycin A(1)-induced activation of JNK but not degradation of IkappaB-alpha was suppressed by NHE inhibitors and by an inhibitor of Na(+)/Ca(2+) exchanger SN-6. These results suggested that V-ATPase inhibitors induce the expression of COX-2 via NHE-dependent and -independent pathways.     

Ferredoxin-NADP+ reductase from Plasmodium falciparum undergoes NADP+-dependent dimerization and inactivation: functional and crystallographic analysis

The completion of the Plasmodium falciparum genome sequence has recently promoted the search for new antimalarial drugs. More specifically, metabolic pathways of the apicoplast, a key organelle for survival of the parasite, have been recognized as potential targets for the development of specific new antimalarial agents. As most apicomplexan parasites, P. falciparum displays a plant-type ferredoxin-NADP(+) reductase, yielding reduced ferredoxin for essential biosynthetic pathways in the apicoplast. Here we report a molecular, kinetic and ligand binding characterization of the recombinant ferredoxin-NADP(+) reductase from P. falciparum, in the light of current data available for plant ferredoxin-NADP(+) reductases. In parallel with the functional characterization, we describe the crystal structures of P. falciparum ferredoxin-NADP(+) reductase in free form and in complex with 2'-phospho-AMP (at 2.4 and 2.7 A resolution, respectively). The enzyme displays structural properties likely to be unique to plasmodial reductases. In particular, the two crystal structures highlight a covalent dimer, which relies on the oxidation of residue Cys99 in two opposing subunits, and a helix-coil transition that occurs in the NADP-binding domain, triggered by 2'-phospho-AMP binding. Studies in solution show that NADP(+), as well as 2'-phospho-AMP, promotes the formation of the disulfide-stabilized dimer. The isolated dimer is essentially inactive, but full activity is recovered upon disulfide reduction. The occurrence of residues unique to the plasmodial enzyme, and the discovery of specific conformational properties, highlight the NADP-binding domain of P. falciparum ferredoxin-NADP(+) reductase as particularly suited for the rational development of antimalarial compounds.     

2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters

The diuretic drug amiloride and its numerous derivatives are competitive inhibitors of mammalian Na(+)/H(+) antiporters and other eukaryotic antiporters. Most prokaryotic antiporters, including the major NhaA family of enterobacteria, are resistant to these compounds. We show that 2-aminoperimidine (AP), a guanidine-containing naphthalene derivative with some similarity to amiloride, acts as a specific inhibitor of NhaA from Escherichia coli. Similar concentrations (IC(50) of 0.9 muM) inhibit the proton motive force dependent Na(+)(Li(+))/H(+) exchange reaction in inside-out sub-bacterial vesicles (at 10 mM NaCl, pH 8) as well as the initial rate of (22)Na(+)/Na(+) exchange mediated by pure NhaA in proteoliposomes. The inhibitor is specific to NhaA type antiporters, so AP is a new tool to study the mechanism and roles of NhaA antiporters of enterobacteria as well as the molecular basis of inhibition by an amiloride-like compound.     

Interactions of phosphate groups of ATP and Aspartyl phosphate with the sarcoplasmic reticulum Ca2+-ATPase: an FTIR study

Phosphate binding to the sarcoplasmic reticulum Ca2+-ATPase was studied by time-resolved Fourier transform infrared spectroscopy with ATP and isotopically labeled ATP ([beta-18O2, betagamma-18O]ATP and [gamma-18O3]ATP). Isotopic substitution identified several bands that can be assigned to phosphate groups of bound ATP: bands at 1260, 1207, 1145, 1110, and 1085 cm(-1) are affected by labeling of the beta-phosphate, bands likely near 1154, and 1098-1089 cm(-1) are affected by gamma-phosphate labeling. The findings indicate that the strength of interactions of beta- and gamma- phosphate with the protein are similar to those in aqueous solution. Two bands, at 1175 and 1113 cm(-1), were identified for the phosphate group of the ADP-sensitive phosphoenzyme Ca2E1P. They indicate terminal and bridging P-O bond strengths that are intermediate between those of ADP-insensitive phosphoenzyme E2P and the model compound acetyl phosphate in water. The bridging bond of Ca2E1P is weaker than for acetyl phosphate, which will facilitate phosphate transfer to ADP, but is stronger than for E2P, which will make the Ca2E1P phosphate less susceptible to attack by water.     

Isolated epsilon subunit of Bacillus subtilis F1-ATPase binds ATP

Previously, we demonstrated ATP binding to the isolated epsilon subunit of F1-ATPase from thermophilic Bacillus PS3 [Kato-Yamada Y., Yoshida M. (2003) J. Biol. Chem. 278, 36013]. However, whether it is a general feature of the epsilon subunit from other sources is yet unclear. Here, using a sensitive method to detect weak interactions between fluorescently labeled epsilon subunit and nucleotide, it was shown that the epsilon subunit of F1-ATPase from Bacillus subtilis also bound ATP. The dissociation constant for ATP binding at room temperature was calculated to be 2 mM, which may be suitable for sensing cellular ATP concentration in vivo.     

Position 170 of Rabbit Na+/glucose cotransporter (rSGLT1) lies in the Na+ pathway; modulation of polarity/charge at this site regulates charge transfer and carrier turnover

Positions 163, 166, and 173, within the putative external loop joining transmembrane segments IV and V of rabbit Na(+)/glucose cotransporter, form part of its Na(+) interaction and voltage-sensing domain. Since a Q170C mutation within this region exhibits anomalous behavior, its function was further investigated. We used Xenopus oocytes coinjected with mouse T-antigen to enhance Q170C expression, and the two-microelectrode voltage-clamp technique. For Q170C, alpha-methyl D-glucopyranoside, phloridzin, and Na(+) affinity values are equivalent to those of wild-type; but turnover is reduced approximately 50%. Decreased [Na(+)] reduces Q170C, but not wild-type, charge transfer. Q170C presteady-state currents exhibit three time constants, tau, identical to wild-type. MTSES decreases maximal alpha-methyl D-glucopyranoside-induced currents by approximately 64% and Na(+) leak by approximately 55%; phloridzin and Na(+) affinity are unchanged. MTSES also reduces charge transfer (dithiothreitol-reversible) and Q170C turnover by approximately 60-70%. MTSEA and MTSET protect against MTSES, but neither affect Q170C function. MTSES has no obvious effect on the tau-values. Q170A behaves the same as Q170C. The mutation Q170E affects voltage sensitivity and reduces turnover, but also appears to influence Na(+) interaction. We conclude that 1), glutamine 170 lies in the Na(+) pathway in rabbit Na(+)/glucose cotransporter and 2), altered polarity and charge at position 170 affect a cotransporter conformational state and transition, which is rate-limiting, but probably not associated with empty carrier reorientation.     

Subcellular distribution of carbonic anhydrase and Na+,K+-ATPase in the brain of the hyt/hyt hypothyroid mice

Activities of carbonic anhydrase and Na⁺,K⁺-ATPase in tissue homogenates and in subcellular fractions from different brain regions were studied in inherited primary hypothyroid (hyt/hyt) mice. The body weight, the weight of different brain regions, and the plasma thyroxine and triiodothyronine levels of hyt/hyt mice were significantly lower than those of the age-matched hyt/+ controls. In tissue homogenates of cerebral cortex, brain stem and cerebellum of hypothyroid mice, the activity of carbonic anhydrase (units/mg protein) was 59.2, 57.6, and 43.2%, and the activity of Na⁺,K⁺-ATPase (nmol Pi/mg protein/min) was 73.7, 74.4 and 68.7%, respectively, of that in corresponding regions of euthyroid littermates. The decrease in enzyme activity in tissue homogenates was also reflected in different subcellular fractions. In cerebral cortex and brain stem, carbonic anhydrase activity in cytosol, myelin and mitochondrial fractions of hypothyroid mice was about 45–50% of that in euthyroid mice, while in cerebellum the carbonic anhydrase activity in these subcellular fractions of hyt/hyt mice was only 33–38% of that in hyt/+ controls. Na⁺,K⁺-ATPase activity in myelin fraction of different brain regions of hyt/hyt mice was about 34–42% of that in hyt/+ mice, while in mitochondria, synaptosome and microsome fractions were about 44–52, 46–53, and 66–68%, respectively of controls. These data indicate that the activity of both carbonic anhydrase and Na⁺,K⁺-ATPase was affected more in the myelin than other subcellular fractions and more in the cerebellum than cerebral cortex and brain stem by deficiency of thyroid hormones. A reduction in the activity of transport enzymes in brain tissues as a result of thyroid hormone deficiency during the critical period of development may underlie permanent nervous disorders in primary hypothyroidism.     

Kinetics and ion specificity of Na/Ca exchange mediated by the reconstituted beef heart mitochondrial Na/Ca antiporter

The Na⁺/Ca²⁺ antiporter was purified from beef heart mitochondria and reconstituted into liposomes containing fluorescent probes selective for Na⁺ or Ca²⁺. Na⁺/Ca²⁺ exchange was strongly inhibited at alkaline pH, a property that is relevant to rapid Ca²⁺ oscillations in mitochondria. The effect of pH was mediated entirely via an effect on the K_m for Ca²⁺. When present on the same side as Ca²⁺, K⁺ activated exchange by lowering the K_m for Ca²⁺ from 2  to 0.9 μM. The K_m for Na⁺ was 8 mM. In the absence of Ca²⁺, the exchanger catalyzed high rates of Na⁺/Li⁺ and Na⁺/K⁺ exchange. Diltiazem and tetraphenylphosphonium cation inhibited both Na⁺/Ca²⁺ and Na⁺/K⁺ exchange with IC₅₀ values of 10 and 0.6 μM, respectively. The V_max for Na⁺/Ca²⁺ exchange was increased about fourfold by bovine serum albumin, an effect that may reflect unmasking of an autoregulatory domain in the carrier protein.     

Membrane topology of loop 13-14 of the Na/glucose cotransporter (SGLT1): A SCAM and fluorescent labelling study

The accessibility of the hydrophilic loop between putative transmembrane segments XIII and XIV of the Na⁺/glucose cotransporter (SGLT1) was studied in Xenopus oocytes, using the substituted cysteine accessibility method (SCAM) and fluorescent labelling. Fifteen cysteine mutants between positions 565 and 664 yielded cotransport currents of similar amplitude than the wild-type SGLT1 (wtSGLT1). Extracellular, membrane-impermeant MTSES⁽⁻⁾ and MTSET⁽⁺⁾ had no effect on either cotransport or Na⁺ leak currents of wtSGLT1 but 9 mutants were affected by MTSES and/or MTSET. We also performed fluorescent labelling on SGLT1 mutants, using tetramethylrhodamine-5-maleimide and showed that positions 586, 588 and 624 were accessible. As amino acids 604 to 610 in SGLT1 have been proposed to form part of a phlorizin (Pz) binding site, we measured the K_i^Pz and K_m^αMG for wtSGLT1 and for cysteine mutants at positions 588, 605-608 and 625. Although mutants A605C, Y606C and D607C had slightly higher K_i^Pz values than wtSGLT1 with minimal changes in K_m^αMG, the effects were modest and do not support the original hypothesis. We conclude that the large, hydrophilic loop near the carboxyl terminus of SGLT1 is thus accessible to the external solution but does not appear to play a major part in the binding of phlorizin.     

Disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor and its implications for redox control and enzyme structure

Redox control of disulfide-bond formation in the H+-pyrophosphatase of Streptomyces coelicolor was investigated using cysteine mutants expressed in Escherichia coli. The wild-type enzyme, but not a cysteine-less mutant, was reversibly inactivated by oxidation. To determine the residues involved in oxidative inactivation, different cysteine residues were replaced. Analysis with a cysteine-modifying reagent revealed that the formation of a disulfide bond between cysteines 253 and 621 was responsible for enzyme inactivation. This result suggests that residues in different cytoplasmic loops are close to each other in the tertiary structure. Both cysteine residues are conserved in K+-independent (type II) H+-pyrophosphatases.     

Myosin regulatory domain orientation in skeletal muscle fibers: application of novel electron paramagnetic resonance spectral decomposition and molecular modeling methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (+/-15 degrees ) with the spin-label z axis parallel to actin, and a second population with a large distribution (+/-60 degrees ). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10 degrees of the electron microscopy model.