A functional chimeric membrane subunit of an ion-translocating ATPase

A chimeric transport protein was made by expression of a fusion of thearsB genes fromEscherichia coli plasmid R773 andStaphylococcus aureus plasmid pI258. The two genes were fused to encode a functional protein with first eight membrane spanning -helices of theS. aureus and the last four helices of theE. coli protein. The hybrid protein provided arsenite resistance and transport. When anarsA gene was expressed in trans with the ArsB proteins encoded by the R773, pI258 and fusion genes, arsenite efflux was dependent on chemical but not electrochemical energy. The Ars system is hypothesized to be a novel transport system that functions as a primary ATP-driven pump or a secondary carrier, depending on the subunit composition of the complex.     

Structure-function relationships in an anion-translocating ATPase

The ArsAB ATPase is an efflux pump located in the inner membrane of Escherichia coli. This transport ATPase confers resistance to arsenite and antimonite by their extrusion from the cells. The pump is composed of two subunits, the catalytic ArsA subunit and the membrane subunit ArsB. The complex is similar in many ways to ATP-binding cassette ('ABC') transporters, which typically have two groups of six transmembrane-spanning helical segments and two nucleotide-binding domains (NBDs). The 45 kDa ArsB protein has 12 transmembrane-spanning segments. ArsB contains the substrate translocation pathway and is capable of functioning as an anion uniporter. The 63 kDa ArsA protein is a substrate-activated ATPase. It has two homologous halves, A1 and A2, which are clearly the result of an ancestral gene duplication and fusion. Each half has a consensus NBD. The mechanism of allosteric activation of the ArsA ATPase has been elucidated by a combination of molecular genetics and biochemical, structural and kinetic analyses. Conformational changes produced by binding of substrates, activator and/or products could be revealed by stopped-flow fluorescence measurements with single-tryptophan derivatives of ArsA. The results demonstrate that the rate-limiting step in the overall reaction is a slow isomerization between two conformations of the enzyme. Allosteric activation increases the rate of this isomerization such that product release becomes rate-limiting, thus accelerating catalysis. ABC transporters, which exhibit similar substrate activation of ATPase activity, can undergo similar conformational changes to overcome a rate-limiting step. Thus the ArsAB pump is a useful model for elucidating mechanistic aspects of the ABC superfamily of transport ATPases.     

Role of conserved histidine residues in metalloactivation of the ArsA ATPase

The ArsA ATPase is the catalytic subunit of a pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. Arsenite or antimonite allosterically activates the ArsA ATPase activity. ArsA homologues from eubacteria, archaea and eukarya have a signature sequence (DTAPTGHT) that includes a conserved histidine. The ArsA ATPase has two such conserved motifs, one in the NH₂-terminal (A1) half and the other in the COOH-terminal (A2) half of the protein. These sequences have been proposed to be signal transduction domains that transmit the information of metal occupancy at the allosteric to the catalytic site to activate ATP hydrolysis. The role of the conserved residues His148 and His453, which reside in the A1 and A2 signal transduction domains respectively, was investigated by mutagenesis to create H148A, H453A or H148A/H453A ArsAs. Each altered protein exhibited a decrease in the V _max of metalloid-activated ATP hydrolysis, in the order wild type ArsA>H148A>H453A>H148A/H453A. These results suggest that the histidine residues play a role in transmission of the signal between the catalytic and allosteric sites.     

Mutagenesis of a nucleotide-binding site of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. The operon encodes three structural genes arsA, arsB, and arsC. The anion pump requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and was demonstrated to bind ATP by photoaffinity labeling with [alpha-32P]ATP. Analysis of the amino acid sequence deduced from the nucleotide sequence of the arsA gene suggests that the ArsA protein contains two potential nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. A combination of site-directed and bisulfite mutagenesis was used to alter the glycine-rich region of the N-terminal putative nucleotide-binding sequence G15KGGVGKTS23. Four mutant proteins (G18----D, G18----R, G20----S, and T22----I) were analyzed. Strains bearing the mutated plasmids were all arsenite sensitive and were unable to extrude arsenite. Each purified mutant protein lacked oxyanion-stimulated ATPase activity and ATP binding. These results suggest that the N-terminal sequence is part of a nucleotide-binding domain required for catalysis.     

Chloride ATPase pumps in nature: do they exist?

Five widely documented mechanisms for chloride transport across biological membranes are known: anion-coupled antiport, Na+ and H(+)-coupled symport, Cl- channels and an electrochemical coupling process. These transport processes for chloride are either secondarily active or are driven by the electrochemical gradient for chloride. Until recently, the evidence in favour of a primary active transport mechanism for chloride has been inconclusive despite numerous reports of cellular Cl(-)-stimulated ATPases coexisting, in the same tissue, with uphill ATP-dependent chloride transport. Cl(-)-stimulated ATPase activity is a ubiquitous property of practically all cells with the major location being of mitochondrial origin. It also appears that plasma membranes are sites of Cl(-)-stimulated ATPase pump activity. Recent studies of Cl(-) -stimulated ATPase activity and ATP-dependent chloride transport in the same plasma membrane system, including liposomes, strongly suggest a mediation by the ATPase in the net movement of chloride up its electrochemical gradient across the plasma membrane structure. Contemporary evidence points to the existence of Cl(-)-ATPase pumps; however, these primary active transporters exist as either P-, F- or V-type ATPase pumps depending upon the tissue under study.     

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.     

Substrate-induced dimerization of the ArsA protein, the catalytic component of an anion-translocating ATPase

The ArsA protein, the catalytic component of the plasmid-encoded resistance system for removal of the toxic oxyanions arsenite, antimonite, and arsenate from bacterial cells, catalyzes oxyanion-stimulated ATP hydrolysis. Three lines of evidence suggest that the ArsA protein functions as a homodimer. First, the ArsA protein was modified with 5'-p-fluorosulfonyl-benzoyladenosine (FSBA). Antimonite potentiated FSBA inhibition, while ATP or ADP afforded partial protection. ATP and antimonite together provided complete protection, indicating interaction of the anion- and nucleotide-binding sites. The estimated Ki values for FSBA were 0.4 mM in the absence of antimonite and 0.1 mM in the presence of antimonite, suggesting that the binding of antimonite increased the affinity of ArsA protein for FSBA. Incorporation of [14C]FSBA was examined. Extrapolation of the amount of FSBA required to inactivate the protein indicated that 1 mol of FSBA was sufficient to inhibit the activity of 1 mol of ArsA protein in the absence of substrates, while only 0.5 mol was required in the presence of the anionic substrate antimonite. Second, chemical cross-linking of the 63-kDa ArsA protein with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline resulted in formation of a species approximately twice the size of the monomer in the presence of antimonite but not ATP. Third, determination of the average mass of the ArsA protein in solution by light scattering demonstrated that the average species was 66 kDa in the absence of substrates. In the presence of antimonite the weight average molecular mass increased to a mass in excess of 100 kDa. These results are consistent with the ArsA protein existing in an equilibrium between monomer and dimer, with the equilibrium favoring dimerization upon binding of the anionic substrate. Moreover, total loss of ATPase activity in the half-modified enzyme suggests that the catalytic sites on each monomer must interact.     

Biochemistry of arsenic detoxification

All living organisms have systems for arsenic detoxification. The common themes are (a) uptake of As(V) in the form of arsenate by phosphate transporters, (b) uptake of As(III) in the form of arsenite by aquaglyceroporins, (c) reduction of As(V) to As(III) by arsenate reductases, and (d) extrusion or sequestration of As(III). While the overall schemes for arsenic resistance are similar in prokaryotes and eukaryotes, some of the specific proteins are the products of separate evolutionary pathways.     

Membrane topology of the ArsB protein, the membrane subunit of an anion-translocating ATPase.

The ars operon of the conjugative R-factor R773 encodes an oxyanion pump that catalyzes extrusion of arsenicals from cells of Escherichia coli. The oxyanion translocation ATPase is composed of two polypeptides, the catalytic ArsA protein and the intrinsic membrane protein, ArsB. The topology of regions of the ArsB protein in the inner membrane was determined using a variety of gene fusions. Random gene fusions with lacZ and phoA were generated using transposon mutagenesis. A series of gene fusions with blaM were constructed in vitro using a beta-lactamase fusion vector. To localize individual segments of the ArsB protein, a ternary fusion method was developed, where portions of the arsB gene were inserted in-frame between the coding regions for two heterologous proteins, in this case a portion of a newly identified arsD gene and the blaM sequence encoding the mature beta-lactamase. The location of a periplasmic loop was determined from V8 protease digestion of an ArsA-ArsB chimera. From analysis of data from 26 fusions, a topological model of the ArsB protein with 12 membrane-spanning regions is proposed.     

ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

A New Chelation Method for Determining ATPase Activity in Skeletal Muscle

Traditional methods for visualizing ATPase in sections use heavy metals that generate visible metal salfide products. These methods use unpleasant and toxic reagents. We report a safer method using a novel ferric ion chelating agent to produce highly specific, low background, and permanent staining of muscle fiber enzymes.     

Subunit composition,biosynthesis, and assembly of the yeast vacuolar proton-translocating ATPase

The yeast vacuole is acidified by a vacuolar proton-translocating ATPase (H⁺-ATPase) that closely resembles the vacuolar H⁺-ATPases of other fungi, animals, and plants. The yeast enzyme is purified as a complex of eight subunits, which include both integral and peripheral membrane proteins. The genes for seven of these subunits have been cloned, and mutant strains lacking each of the subunits (vma mutants) have been constructed. Disruption of any of the subunit genes appears to abolish the function of the vacuolar H⁺-ATPase, supporting the subunit composition derived from biochemical studies. Genetic studies of vacuolar acidification have also revealed an additional set of gene products that are required for vacuolar H⁺-ATPase activity, but may not be part of the final enzyme complex. The biosynthesis, assembly, and targeting of the enzyme is being elucidated by biochemical and cell biological studies of thevma mutants. Initial results suggest that the peripheral and integral membrane subunits may be independently assembled.     

The Structure of the Vacuolar ATPase in Neurospora crassa

The filamentous fungus Neurospora crassa contains many smallvacuoles. These organelles contain high concentrations of polyphosphates andbasic amino acids, such as arginine and ornithine. Because of their size anddensity, the vacuoles can be separated from other organelles in the cell. TheATP-driven proton pump in the vacuolar membrane is a typical V-type ATPase.We examined the size and structure of this enzyme using radiationinactivation and electron microscopy. The vacuolar ATPase is a large andcomplex enzyme, which appears to contain at least thirteen different types ofsubunits. We have characterized the genes that encode eleven of thesesubunits. In this review, we discuss the possible function and structure ofthese subunits.     

Ion channel enzyme in an oscillating electric field

To explain the electrical activation of several membrane ATPases, an electroconformational coupling (ECC) model has previously been proposed. The model explained many features of experimental data but failed to reproduce a window of the field intensity for the stimulated activity. It is shown here that if the affinities of the ion for the two conformational states of the transporter (one with binding site on the left side and the other on the right side of the membrane) are dependent on the electric field, the field-dependent transport can exhibit the observed window. The transporter may be described as a channel enzyme which opens to one side of the membrane at a time. It retains the energy-transducing ability of the earlier ECC models. Analysis of the channel enzyme in terms of the Michaelis-Menten kinetics has been done. The model reproduced the amplitude window for the electric field-induced cation pumping by (Na,K)-ATPase.     

Effect of magnesium and ATP on ATPase of sugarcane vacuoles

Kinetic analysis of the Mg²⁺-dependence of tonoplast ATPase from suspension-cultured cells of sugarcane showed that the enzyme activity increased with increasing magnesium concentrations till 1–3 mM and then decreased consideably for higher concentrations. This kinetic could be explained by the assumption that MgATP^2- is the substrate of ATPase: MgATP^2- concentration increases with increasing concentration of magnesium till, at high concentrations of magnesium, Mg₂ATP is formed. No evidence for a direct role of Mg²⁺ as activator or inhibitor was found. These data corroborate previous findings that MgATP^2- is the sole substrate of the vacuolar ATPase of sugarcane (Thom and Komor 1984). High concentrations of ATP seemed to inhibit the ATPase. This result, however, could be traced back to interference of ATP with the Fiske-Subbarow method of phosphate determination. After adjustment of the test conditions, inhibition by ATP was no longer found. Reported data for ATPases of other plant materials, showing inhibition of enzyme activity with high magnesium or ATP concentrations, might be explicable in a similar way.Abbreviation Mes 2-(N-morpholino)ethane+Sulfonic acid     

Mechanisms of Rose Bengal inhibition on SecA ATPase and ion channel activities

SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG–SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg²⁺, mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.     

Origins and functional diversification of salinity‐responsive Na+, K+ ATPase α1 paralogs in salmonids

The Salmoniform whole‐genome duplication is hypothesized to have facilitated the evolution of anadromy, but little is known about the contribution of paralogs from this event to the physiological performance traits required for anadromy, such as salinity tolerance. Here, we determined when two candidate, salinity‐responsive paralogs of the Na⁺, K⁺ ATPase α subunit (α1a and α1b) evolved and studied their evolutionary trajectories and tissue‐specific expression patterns. We found that these paralogs arose during a small‐scale duplication event prior to the Salmoniform, but after the teleost, whole‐genome duplication. The ‘freshwater paralog’ (α1a) is primarily expressed in the gills of Salmoniformes and an unduplicated freshwater sister species (Esox lucius) and experienced positive selection in the freshwater ancestor of Salmoniformes and Esociformes. Contrary to our predictions, the ‘saltwater paralog’ (α1b), which is more widely expressed than α1a, did not experience positive selection during the evolution of anadromy in the Coregoninae and Salmonine. To determine whether parallel mutations in Na⁺, K⁺ ATPase α1 may contribute to salinity tolerance in other fishes, we studied independently evolved salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in Anabas testudineus and Oreochromis mossambicus. We found that a quarter of the mutations occurring between salmonid α1a and α1b in functionally important sites also evolved in parallel in at least one of these species. Together, these data argue that paralogs contributing to salinity tolerance evolved prior to the Salmoniform whole‐genome duplication and that strong selection and/or functional constraints have led to parallel evolution in salinity‐responsive Na⁺, K⁺ ATPase α1 paralogs in fishes.     

The vacuolar ATPase ofNeurospora crassa

The filamentous fungusNeurospora crassa has many small vacuoles which, like mammalian lysosomes, contain hydrolytic enzymes. They also store large amounts of phosphate and basic amino acids. To generate an acidic interior and to drive the transport of small molecules, the vacuolar membranes are densely studded with a proton-pumping ATPase. The vacuolar ATPase is a large enzyme, composed of 8–10 subunits. These subunits are arranged into two sectors, a complex of peripheral subunits called V₁ and an integral membrane complex called V₀. Genes encoding three of the subunits have been isolated.vma-1 andvma-2 encode polypeptides homologous to the and subunits of F-type ATPases. These subunits appear to contain the sites of ATP binding and hydrolysis.vma-3 encodes a highly hydrophobic polypeptide homologous to the proteolipid subunit of vacuolar ATPases from other organisms. This subunit may form part of the proton-containing pathway through the membrane. We have examined the structures of the genes and attempted to inactivate them.