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There is no cross-resistance between arsenic trioxide and conventional chemotherapeutics. Classical multi-drug resistant (MDR) cells remain sensitive to arsenic trioxide, which may even reverse the drug resistance. Arsenic trioxide is also effective in leukemias/tumors that persist despite conventional cytotoxic or targeted drugs. We obtained a highly arsenic-resistant MDR leukemic cell line, HL-60/RS, by exposing leukemic HL-60 cells to adriamycin selection. We compared the arsenic sensitivity, and the expression and responses to arsenic of the arsenic-related transporters, MRP1, MRP2, and ASNA1, in paired parent/arsenic-resistant HL-60/RS/HL-60 and arsenic-sensitive/parental K562/ADM/K562 cells. Expression levels of MRP1, MRP2, and ASNA1 were negatively correlated with cell sensitivities to arsenic trioxide, and ASNA1 expression notably was highest in HL-60/RS cells and lowest in K562/ADM cells. Expression levels of MRP1, MRP2, and ASNA1 were significantly enhanced in HL-60/RS cells and inhibited in K562/ADM cells by arsenic trioxide treatment, compared with their parental sensitive cells, in accord with the high-resistance of HL-60/RS cells and high-sensitivity of K562/ADM cells. In conclusion, the cross-resistance of conventional chemotherapeutics-resistant leukemic cells to arsenic trioxide is determined by both levels of MRP1, MRP2, and ASNA1, and also by the responses of these transporters to arsenic stress.

Pseudomonas xanthomarina S11 is an arsenite-oxidizing bacterium isolated from an arsenic-contaminated former gold mine in Salsigne, France. This bacterium showed high resistance to arsenite and was able to oxidize arsenite to arsenate at concentrations up to 42.72 mM As[III]. The genome of this strain was sequenced and revealed the presence of three ars clusters. One of them is located on a plasmid and is organized as an “arsenic island” harbouring an aio operon and genes involved in phosphorous metabolism, in addition to the ars genes. Neither the aioXRS genes nor a specific sigma-54-dependent promoter located upstream of aioBA genes, both involved in regulation of arsenite oxidase expression in other arsenite-oxidizing bacteria, could be identified in the genome. This observation is in accordance with the fact that no difference was observed in expression of arsenite oxidase in P. xanthomarina S11, whether or not the strain was grown in the presence of As[III].

Calpains are a family of calcium-dependent cysteine proteases that are ubiquitously expressed in mammals and play critical roles in neuronal death by catalyzing substrate proteolysis. Here, we developed two-dimensional gel electrophoresis-based protease proteomics to identify putative calpain substrates. To accomplish this, cellular lysates from neuronal cells were first separated by pI, and the immobilized sample on a gel strip was incubated with a recombinant calpain and separated by molecular weight. Among 25 altered protein spots that were differentially expressed by at least 2-fold, we confirmed that arsenical pump-driving ATPase, optineurin, and peripherin were cleaved by calpain using in vitro and in vivo cleavage assays. Furthermore, we found that all of these substrates were cleaved in MN9D cells treated with either ionomycin or 1-methyl-4-phenylpyridinium, both of which cause a calcium-mediated calpain activation. Their cleavage was blocked by calcium chelator or calpain inhibitors. In addition, calpain-mediated cleavage of these substrates and its inhibition by calpeptin were confirmed in a middle cerebral artery occlusion model of cerebral ischemia, as well as a stereotaxic brain injection model of Parkinson disease. Transient overexpression of each protein was shown to attenuate 1-methyl-4-phenylpyridinium-induced cell death, indicating that these substrates may confer protection of varying magnitudes against dopaminergic injury. Taken together, the data indicate that our protease proteomic method has the potential to be applicable for identifying proteolytic substrates affected by diverse proteases. Moreover, the results described here will help us decipher the molecular mechanisms underlying the progression of neurodegenerative disorders where protease activation is critically involved.

Tail-anchored (TA) membrane proteins destined for the secretory pathway are posttranslationally inserted into the endoplasmic reticulum (ER) membrane, but the molecular machinery for this insertion in mammalian cells remains elusive. Here we reveal a mammalian protein complex that drives the membrane insertion. We identify calcium-modulating cyclophilin ligand (CAML) as a mammal-specific receptor for TRC40, an ATPase targeting newly synthesized TA proteins, and show that CAML mediates membrane insertion of TA proteins. We show that CAML binds to WRB, an evolutionarily conserved TRC40 receptor, through the transmembrane domains and that CAML and WRB synergistically insert TA proteins into the membrane. Mutagenesis of CAML demonstrates that binding of TRC40 to CAML is required to ensure synergistic membrane insertion. Thus, identification of CAML and WRB as components of the TRC40 receptor complex represents a crucial mechanism for driving ER membrane insertion of TA proteins in mammalian cells.

Virus-infected cells are eliminated by cytotoxic T lymphocytes, which recognize viral epitopes displayed on major histocompatibility complex class I molecules at the cell surface. Herpesviruses have evolved sophisticated strategies to escape this immune surveillance. During the lytic phase of EBV infection, the viral factor BNLF2a interferes with antigen processing by preventing peptide loading of major histocompatibility complex class I molecules. Here we reveal details of the inhibition mechanism of this EBV protein. We demonstrate that BNLF2a acts as a tail-anchored protein, exploiting the mammalian Asna-1/WRB (Get3/Get1) machinery for posttranslational insertion into the endoplasmic reticulum membrane, where it subsequently blocks antigen translocation by the transporter associated with antigen processing (TAP). BNLF2a binds directly to the core TAP complex arresting the ATP-binding cassette transporter in a transport-incompetent conformation. The inhibition mechanism of EBV BNLF2a is distinct and mutually exclusive of other viral TAP inhibitors.

Calneuron-1 and -2 are neuronal EF-hand-type calcium sensor proteins that are prominently targeted to trans-Golgi network membranes and impose a calcium threshold at the Golgi for phosphatidylinositol 4-OH kinase IIIβ activation and the regulated local synthesis of phospholipids that are crucial for TGN-to-plasma membrane trafficking. In this study, we show that calneurons are nonclassical type II tail-anchored proteins that are post-translationally inserted into the endoplasmic reticulum membrane via an association of a 23-amino acid-long transmembrane domain (TMD) with the TRC40/Asna1 chaperone complex. Following trafficking to the Golgi, calneurons are probably retained in the TGN because of the length of the TMD and phosphatidylinositol 4-phosphate lipid binding. Both calneurons rapidly self-associate in vitro and in vivo via their TMD and EF-hand containing the N terminus. Although dimerization and potentially multimerization precludes TRC40/Asna1 binding and thereby membrane insertion, we found no evidence for a cytosolic pool of calneurons and could demonstrate that self-association of calneurons is restricted to membrane-inserted protein. The dimerization properties and the fact that they, unlike every other EF-hand calmodulin-like Ca(2+) sensor, are always associated with membranes of the secretory pathway, including vesicles and plasma membrane, suggests a high degree of spatial segregation for physiological target interactions.

Members of the Acr3 family of arsenite permeases confer resistance to trivalent arsenic by extrusion from cells, with members in every phylogenetic domain. In this study bacterial Acr3 homologues from Alkaliphilus metalliredigens and Corynebacterium glutamicum were cloned and expressed in Esch e richia coli. Modification of a single cysteine residue that is conserved in all analyzed Acr3 homologues resulted in loss of transport activity, indicating that it plays a role in Acr3 function. The results of treatment with thiol reagents suggested that the conserved cysteine is located in a hydrophobic region of the permease. A scanning cysteine accessibility method was used to show that Acr3 has 10 transmembrane segments, and the conserved cysteine would be predicted to be in the fourth transmembrane segment.

SummaryHundreds of proteins are anchored in intracellular membranes by a single transmembrane domain (TMD) close to the C terminus. Although these tail-anchored (TA) proteins serve numerous essential roles in cells, components of their targeting and insertion pathways have long remained elusive. Here we reveal a cytosolic TMD recognition complex (TRC) that targets TA proteins for insertion into the ER membrane. The highly conserved, 40 kDa ATPase subunit of TRC (which we termed TRC40) was identified as Asna-1. TRC40/Asna-1 interacts posttranslationally with TA proteins in a TMD-dependent manner for delivery to a proteinaceous receptor at the ER membrane. Subsequent release from TRC40/Asna-1 and insertion into the membrane depends on ATP hydrolysis. Consequently, an ATPase-deficient mutant of TRC40/Asna-1 dominantly inhibited TA protein insertion selectively without influencing other translocation pathways. Thus, TRC40/Asna-1 represents an integral component of a posttranslational pathway of membrane protein insertion whose targeting is mediated by TRC.

Desulfosporosinus sp. strain Y5 is a spore-forming bacterium capable of dissimilatory arsenate reduction coupled to the oxidation of aromatic compounds. In arsenate respiration, the arsenate respiratory reductase (ARR) catalyzes the reduction of arsenate to arsenite. Our objective is to characterize the arrA gene, encoding the ARR, for Desulfosporosinus sp. strain Y5. Oligonucleotide primers were designed based on the few arrA gene sequences available at the time and validated against positive and negative controls. The resulting arrA-amplicon of approximately 2.0kb was cloned and sequenced. The arrA from Desulfosporosinus sp. Y5 is closely related to Desulfitobacterium hafniense (similarity of 77% and 81% at the nucleotide and amino acid levels, respectively). Phylogenetic topology based on the arrA gene was partially congruent with that of 16S rRNA-based analysis. This arrA sequence will support the development of specific tracking probes for Desulfosporosinus sp. Y5 and the molecular characterization and monitoring of dissimilatory arsenate reducing bacteria.

Biosensing systems such as reporter-gene-based whole-cell assays are increasingly finding applications in biological and environmental screening. A whole-cell approach to such analyses can provide valuable information about the bioavailable level of a compound of interest. These biosensing systems rely on the molecular recognition of a specific analyte by a regulatory protein and, therefore, can detect low levels of the target analyte. In this study, Escherichia coli cells containing plasmid pSD10 were engineered to sense the model target analytes arsenite and antimonite, the target analytes in this study. The biosensing system takes advantage of the recognition of the regulatory protein, ArsR, for arsenite and antimonite to produce the reporter protein, which in this case is GFPuv. The fluorescence emitted by the GFPuv in the cells can be directly related to the concentration of the analyte in the cell, making this biosensing system useful in the detection of arsenite and/or antimonite in a variety of samples. Miniaturization of biosensing systems can further enhance their utility by decreasing reagent consumption and analysis time and by allowing for the high-throughput screening of samples. To that end, we employed a microcentrifugal microfluidics platform that has low power, space, and reagent requirements, increased speed of detection, and the potential for portability. Herein, we demonstrate for the first time the adaptation of a whole-cell sensing system to a microcentrifugal microfluidics platform. Moreover, we were able to detect our target analytes in a rapid and sensitive manner compared to conventional sensing methods.

Arsenate reductase (ArsC) from Staphylococcus aureus plasmid pI258 catalyzes the reduction of arsenate to arsenite and plays a role in bacterial heavy metal resistance. The high resolution x-ray structure of ArsC reveals the atomic details of the K+ binding site situated next to the catalytic P-loop structural motif of this redox enzyme. A full thermodynamic study of the binding characteristics of a series of monovalent cations (Li+, Na+, K+, Rb+, and Cs+) and their influence on the thermal stability of ArsC was performed with isothermal titration calorimetry, circular dichroism spectroscopy, and differential scanning calorimetry. Potassium has the largest affinity with a Ka of 3.8 x 10(3) m(-1), and the effectiveness of stabilization of ArsC by monovalent cations follows the binding affinity order: K+ > Rb+ > Cs+ > Na+ > Li+. A mutagenesis study on the K+ binding side chains showed that Asn-13 and Asp-65 are essential for potassium binding, but the impact on the stability of ArsC was the most extreme when mutating Ser-36. Additionally, the thermal stabilization by K+ is significantly reduced in the case of the ArsC E21A mutant, showing the importance of a Glu-21-coordinated water molecule in its contact with K+. Although potassium is not essential for catalysis, in its presence the kcat/KM increases with a factor of 5. Altogether, the interaction of K+ with specific residues in ArsC is an enthalpydriven process that stabilizes ArsC and increases the specific activity of this redox enzyme.

The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg(2+) and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg(2+) and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis).

Inorganic arsenic is an important environmental toxicant of both natural and anthropogenic sources. It is a human carcinogen for which appropriate animal models of most arsenic-induced cancers are missing. Although methylation of inorganic arsenic has been considered its primary mechanism for detoxification, the results of recent investigations disagree. We have investigated 17 species of non-human primates, including great apes, New and Old World monkeys and prosimians, and have found that thirteen of them lacked hepatic arsenite methyltransferase activity in vitro. Four primate species, three from the Old World genus Macaca, and one of three animals from the New World genus Saimiri, had arsenite methyltransferase activity. That all the tissues examined were viable was demonstrated by their all having arsenate reductase activity. These data suggest that methylation of inorganic arsenic is not a detoxification mechanism for many non-human primates. Thus, alternative methods of detoxifying inorganic arsenic in mammals need to be considered and investigated. In addition, there appears to be a phylogenetic component to having arsenite methyltransferase activity, as evidenced by the result of our study of the Macaca species.

ArsA is the catalytic subunit of the arsenical pump, coupling ATP hydrolysis to the efflux of arsenicals through the ArsB membrane protein. It is a paradigm for understanding the structure-function of the nucleotide binding domains (NBD) of medically important efflux pumps, such as P-glycoprotein, because it has two sequence-related, interacting NBD, for which the structure is known. On the basis of a rigorous analysis of the pre-steady-state kinetics of nucleotide binding and hydrolysis, we propose a model in which ArsA alternates between two mutually exclusive conformations as follows: the ArsA(1) conformation in which the A1 site is closed but the A2 site open; and the ArsA(2) conformation, in which the A1 and A2 sites are open and closed, respectively. Antimonite elicits its effects by sequestering ArsA in the ArsA(1) conformation, which catalyzes rapid ATP hydrolysis at the A2 site to drive ArsA between conformations that have high (nucleotide-bound ArsA) and low affinity (nucleotide-free ArsA) for Sb(III). ArsA potentially utilizes this process to sequester Sb(III) from the medium and eject it into the channel of ArsB.

Pseudomonas fluorescens MSP3 isolated from sea water was resistant to arsenate. This bacterium harbored no plasmids, indicating that arsenic resistance was chromosomally encoded. The chromosomal DNA from MSP3 when transformed onto Escherichia coli DH5alpha using pBluescript exhibited resistance to sodium arsenate and sodium arsenite. Three clones MSA1, MSA2, and MSI3 containing the ars genes were obtained and further subcloning resulted in three fragments of size 2.2, 2.6, and 2.1 kb for pMSA11, pMSA12, and pMSI13, respectively, which contained the genes arsRBC of the arsenic operon. An efflux mechanism of detoxification was observed which was ATP dependent. The resistance mechanism was encoded from a single operon which consisted of an arsenite inducible repressor that regulates the expression of arsenate reductase (ars C) and inner membrane associated arsenite export system encoded by ars B. The chromosomal operon was cloned, sequenced, and found to consist of three cistrons, named as ars R, ars B, and ars C. Southern hybridization and mating experiments confirmed the functioning of the ars genes in the operon, thereby conferring increased resistance to sodium arsenate and sodium arsenite.

Arsenate reductase (ArsC) encoded by Staphylococcus aureus arsenic-resistance plasmid pI258 reduces intracellular As(V) (arsenate) to the more toxic As(III) (arsenite). In order to study the structure of ArsC and to unravel biochemical and physical properties of this redox enzyme, wild type enzyme and a number of cysteine mutants were overproduced soluble in Escherichia coli. In this paper we describe a novel purification method to obtain high production levels of highly pure enzyme. A reversed-phase method was developed to separate and analyze the many different forms of ArsC. The oxidation state and the methionine oxidized forms were determined by mass spectroscopy.

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.

The ars operon of plasmid R773 encodes an ATP-dependent extrusion pump for arsenite and antimonite in Escherichia coli. The ArsA ATPase is the catalytic subunit of the pump protein, with two nucleotide binding consensus sequences, one in the NH2-terminal half and one in the COOH-terminal half of the protein. A 12-residue consensus sequence (DTAPTGHTIRLL) has been identified in ArsA homologs from eubacteria, archebacteria, fungi, plants, and animals. ArsA enzymes were constructed containing single tryptophan residues at either end of this conserved sequence. The emission spectrum of the fluorescence of the tryptophan on the COOH-terminal end (Trp-159) indicated a relatively hydrophilic environment for this residue. An increase in intrinsic tryptophan fluorescence and a blue shift of the maximum emission wavelength were observed upon addition of MgATP, indicating movement of Trp-159 into a relatively less polar environment. No fluorescence response was observed with MgADP, with nonhydrolyzable ATP analogs, or with MgATP by catalytically inactive enyzmes. This suggests that the location Trp-159 is shifted only during hydrolysis of ATP. In contrast, the emission spectrum of Trp-141, located on the NH2-terminal side of the consensus sequence, indicated a relatively nonpolar environment. The maximum emission wavelength red shifted upon addition of MgADP. MgATP slowly produced a response that correlated with product formation, suggesting that the environment of Trp-141 is sensitive only to MgADP binding. Thus, during ATP hydrolysis the COOH-terminal end of the conserved domain moves into a less polar environment, whereas the NH2-terminal end moves into a more hydrophilic environment as product is formed. A hypothesis is presented in which the conserved domain of ArsA and homologs is an energy transduction domain involved in transmission of the energy of ATP hydrolysis to biological functions such as transport.

The arsenical resistance (ars) operon of the conjugative R-factor R773 confers resistance to arsenical and antimonial compounds in Escherichia coli, where resistance results from active extrusion of arsenite catalyzed by the products of the arsA and arsB genes. Previous in vivo studies on the energetics of arsenite extrusion showed that expression of both genes produced an ATP-coupled arsenite extrusion system that was independent of the electrochemical proton gradient. In contrast, in cells expressing only the arsB gene, arsenite extrusion was coupled to electrochemical energy and independent of ATP, suggesting that the Ars transport system exhibits a dual mode of energy coupling depending on the subunit composition. In vitro the ArsA-ArsB complex has been shown to catalyze ATP-coupled uptake of 73AsO2−1 in everted membrane vesicles. However, transport catalyzed by ArsB alone has not previously been observed in vitro. In this study we demonstrate everted membrane vesicles prepared from cells expressing only arsB exhibit uptake of 73AsO2−1 coupled to electrochemical energy.

The 141-residue ArsC arsenate reductase of plasmid R773 has an essential cysteine residue, Cys-12. The pKa of Cys-12 was determined to be 6.4, compared with a pKa of 8.3 for free cysteine. The possibility of the formation of an ion pair between Cys-12 and a basic residue was investigated. Enzymatic activity was rapidly inactivated by the histidine-modifying reagent diethylpyrocarbonate. The codons for the two histidine residues in ArsC, His-8 and His-88, were changed by site-directed mutagenesis. Cells expressing arsCH88R, arsCH88S, arsCH88W, or arsCH88V genes retained arsenate resistance, and the purified proteins had wild type level of reductase activity. Cells expressing arsCH8P, arsCH8S, arsCH8G, or arsCH8R genes were each sensitive to arsenate, and the purified H8P, H8G, and H8R proteins each lacked enzymatic activity. Using the single histidine proteins it was shown that both histidines react with diethylpyrocarbonate but that only reaction with His-8 resulted in inactivation. The pKa value of Cys-12 was determined to be 6.3 in the H8R enzyme and 8.3 in the H8G enzyme. These results indicate that His-8 is essential for catalytic activity and that a positively charged residue is required at position 8 to lower the pKa of the cysteine thiolate at position 12.

ArsA ATPase activity is allosterically activated by salts of the semimetal arsenic or antimony. Activation is associated with the presence of three cysteine residues in ArsA: Cys113, Cys172, and Cys422. To determine the distance between cysteine residues, wild type ArsA and ArsA proteins with cysteine to serine substitutions were treated with the bifunctional alkylating agent dibromobimane, which reacts with thiol pairs within 3-6 A of each other to form a fluorescent adduct. ArsA proteins in which single cysteine residues were altered by site-directed mutagenesis still formed fluorescent adducts. Proteins in which two of the three cysteine residues were substituted did not form fluorescent adducts. These results demonstrate that Cys113, Cys172, and Cys422 are in close proximity of each other. We propose a model in which As(III) or Sb(III) interacts with these three cysteines in a trigonal pyramidal geometry, forming a novel soft metal-thiol cage.

Arsenite resistance in bacteria is mediated by an efflux pump composed of the arsA and arsB gene products. We have isolated the human homolog of the bacterial arsA (hARSA-I), a member of the ATPase superfamily with no transmembrane domain. Southern and Northern analyses indicated the presence of two cross-hybridizing genes in the human genome and expression of hARSA-I in many tissues. A rabbit antiserum raised against a glutathione-S-transferase (GST)/hARSA-I fusion protein identified two cross-reacting proteins of 37 and 42 kDa by Western analysis in two different human cell lines. Overexpression of hARSA-I in the embryonal human kidney 293 cell line was accompanied by overproduction of the 37-kDa protein Biochemical analysis using the GST/hARSA-I fusion protein indicated that hARSA-I is an ATPase analogous to the bacterial ArsA. Thus, hARSA-I is a new eukaryotic member of a highly conserved ATP-binding superfamily of proteins.

Resistance to toxic oxyanions of arsenic and antimony in Escherichia coli results from active efflux of these anions out of the cell. Extrusion is an active process mediated by an ATP-dependent pump composed of two types of subunits, the integral membrane ArsB protein and the catalytic ArsA subunit. An in vitro assay for transport in everted membrane vesicles of E. coli was developed. Uptake of 73AsO2- by everted vesicles was time- and temperature-dependent and required both pump subunits. Transport required ATP; no other nucleotide, including GTP, CTP, UTP, or the nonhydrolyzable analog adenosine 5'-O-(thiotriphosphate), could substitute for ATP. Protonophores, ionophores, or inhibitors of other types of ion-motive ATPases did not inhibit arsenite uptake. The sulfhydryl reagent N-ethylmaleimide was a potent inhibitor of ATP-dependent arsenite accumulation in vesicles. The apparent Km values for ATP and arsenite were approximately 2 and 0.1 mM, respectively. Antimonite, the most potent activator of the ArsA ATPase, inhibited arsenite uptake with an apparent Ki of 10 microM.

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. Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with [alpha-32P]ATP. From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. Purified ArsA protein bound a fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine- 5'-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA. Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein.

Many membrane proteins are inserted into cellular membranes via a carboxy-terminal tail-anchor segment, but the mechanism of insertion is poorly understood. In this issue of Cell, Stefanovic and Hegde (2007) report the identification and initial characterization of a soluble ATP-dependent receptor for the insertion of newly synthesized tail-anchored membrane proteins.

The products of the arsenical resistance operon of resistance plasmid R733 form an efflux system for arsenicals. Detoxification results from active efflux of the oxyanions, preventing their concentration from reaching toxic levels. The largest polypeptide encoded by the ars operon was purified. From N-terminal sequencing the purified protein, termed the ArsA protein, was shown to correspond to the product of the arsA gene. The purified protein was demonstrated to bind ATP by two methods. First, a photoadduct of the protein with [alpha-32P]ATP was formed by irradiation at 254 nm. Second, the purified protein bound a fluorescent ATP analogue, 2',3'-o-(2,4,6)trinitrophenyl ATP, with a half-maximal affinity of 2 microM. By both assays competition was observed with ATP or ADP, but not with AMP, GTP, CTP, or UTP. In both nucleotide binding assays, Mg2+ was required, but neither arsenite nor antimonate had any affect. In contrast, the ArsA protein exhibited an ATPase activity which was dependent on the presence of arsenite or antimonate. The results suggest that the ArsA protein is the catalytic subunit of an oxyanion-translocating ATPase.