Caenorhabditis elegans expresses a functional ArsA Yuen-Yi Tseng, Chan-Wei Yu and Vivian Hsiu-Chuan Liao Department of Bioenvironmental Systems Engineering, National Taiwan University, Taipei, Taiwan Arsenic is a potent toxin and carcinogen. The two bio- logically relevant oxidation states of inorganic arsenic are arsenite [As(III)] and arsenate [As(V)]. In general, As(III) is more hazardous to organisms than As(V). In bacteria, high-level resistance to arsenic is conferred by the ars operon. The arsenic-resistant ars operon in Escherichia coli has both plasmid [1] and chromosomal [2] determinants. The well-characterized plasmid-borne ars operon of E. coli is composed of two regulatory (arsR and arsD ) and three structural (arsA, arsB, and arsC) genes [3,4]. The well-characterized ArsAB pump can extrude As(III) and antimonite [Sb(III)] from cells, thereby lowering the intracellular concentration of these toxic metalloids and producing resistance [5]. ArsA is the catalytic subunit of the anion pump that hydrolyzes ATP in the presence of As(III) or Sb(III) oxyanions [6]. ATP hydrolysis is coupled to the extru- sion of As(III) and Sb(III) via the ArsB transporter, which serves as both a membrane anchor for the ArsA portion and a toxic oxyanion translocating pathway [7]. Homologs of bacterial ArsA ATPase have been found in nearly every organism studied [8]. Although the function of bacterial ArsA has been identified, the ubiquity of the ArsA ATPase-dependent pathway in other organisms remains to be delineated. Here, we describe the identification and biochemical characteri- zation of the Caenorhabditis elegans homolog of the Keywords antimonite; arsenite; ASNA-1; ATPase; Caenorhabditis elegans Correspondence V. H C. Liao, Department of Bioenvironmental Systems Engineering, National Taiwan University, no. 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan Fax: +886 2 3366 3462 Tel: +886 2 3366 5239 E-mail: vivianliao@ntu.edu.tw (Received 12 January 2007, revised 12 March 2007, accepted 15 March 2007) doi:10.1111/j.1742-4658.2007.05791.x Because arsenic is the most prevalent environmental toxin, it is imperative that we understand the mechanisms of metalloid detoxification. In prokary- otes, arsenic detoxification is accomplished by chromosomal and plasmid- borne operon-encoded efflux systems. Bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Here, we describe the identifi- cation of a Caenorhabditis elegans homolog (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We evaluated the responses of wild-type and asna-1-mutant nematodes to various metal ions and found that asna-1-mutant nematodes are more sen- sitive to As(III) and Sb(III) toxicity than are wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans’ defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)–ASNA-1 fusion protein was biochemically characterized, and its properties compared with those of ArsAs. The ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 ± 0.2-fold, whereas Sb(III) stimu- lated it by 4.6 ± 0.15-fold. The results indicate that As(III)- and Sb(III)- stimulated ArsA ATPase activities are not restricted to bacteria, but extend to animals, by demonstrating that the asna-1 gene from the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism. Abbreviations MBP, maltose-binding protein; NGM, nematode growth medium. 2566 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS bacterial ArsA protein, designated ASNA-1, a puta- tive arsenite-translocating ATPase in C. elegans. Our results provide evidence that As(III)- and Sb(III)-sti- mulated ArsA ATPase activities are not restricted to bacteria, but extend to animals by demonstrating that the asna-1 gene of the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III). Results Sequence analysis of ASNA-1 Using E. coli ArsA sequence as a probe in a BLAST analysis, we identified the putative ArsA homolog in C. elegans, which we designated ASNA-1. The predic- ted version of this gene has been previously reported in the C. elegans genome database (Wormbase: http:// www.wormbase.org/) as ZK637.5 (GenBank accession no. NM_066564). The asna-1 locus was physically mapped to the gene cluster region of chromosome III on the cosmid, ZK637. The predicted mRNA contains an ORF of 1029 bp encoding a protein of 342 amino acids and exhibits 33% identity to E. coli ArsA ATPase. A BLAST search showed that eukaryotic ArsAs from Saccharomyces cerevisiae (Arr4p), human (hASNA-I), mouse (Asna1), and Drosophila melano- gaster exhibit 46–56% identity to C. elegans ASNA-1. Also, ASNA-1 contains a conserved Walker A motif, or P loop (GKGGVGKT), and a signal transduction domain (DTAPTGHT). Toxicity tests of metals ions To investigate whether ASNA-1 is required for As(III) and Sb(III) tolerance in the intact organism, wild-type and asna-1 deletion mutant nematodes were exposed to a range of As(III) and Sb(III) ion concentrations and the number of dead worms was scored over 24 h (Fig. 1). The proportion of worms surviving at each As(III) or Sb(III) concentration after 24 h exposure varied considerably between wild-type and asna-1- mutant worms (Fig. 1). The results showed that asna-1-mutant nematodes were more sensitive to both As(III) and Sb(III) toxicity than were the wild-type strain. The toxic effect of As(III) and Sb(III) exposure for wild-type and asna-1-mutant worms was further investigated for time dependence. Survival of wild-type and asna-1-mutant worms subjected to As(III)- and Sb(III)-induced toxicity was time dependent. As shown in Fig. 2, the mortality rate for both wild-type and asna-1-mutant worms increased as the incubation times with As(III) and Sb(III) increased. Also, the Kaplan– Meier survival curve showed that asna-1-mutant nema- todes were significantly less resistant (P<0.0001) to both As(III) and Sb(III) toxicity than the wild-type worms (Fig. 2). To further explore the protective role of ASNA-1 against other metal ions, asna-1-mutant worms were exposed to Pb(II), Cu(II), Al(III), Cr(VI), and Zn(II). The exposure concentration for these metals was based on previously reported lethal concentration (LC 50 ) val- ues for N2 worms [9]. The survival of asna-1-mutant worms treated with the aforementioned metal ions after 24 h exposure was not significantly different from that of N2 worms (data not shown). Together, the 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 20 40 60 80 100 [As(III)] m M Survival rate (%) Survival rate (%) A 0 5 10 15 20 25 0 20 40 60 80 100 [Sb(III)] m M B Fig. 1. C. elegans As(III) and Sb(III) toxicity assay. Wild-type and asna-1-mutant worms were incubated at 20 °C and the number of dead worms was scored over 24 h. The number of dead worms was determined as described in Experimental procedures. (A) Pro- portion of worms surviving a range of As(III) concentrations. (B) Proportion of worms surviving a range of Sb(III) concentrations. Values are presented as the percentage of worms still alive at a particular metal ion concentration after 24 h exposure at 20 °C; (d) wild-type worms; (j) asna-1-mutant worms. Data is represen- ted as mean ± SEM, n ¼ 6. Y Y. Tseng et al. Caenorhabditis elegans ASNA-1 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2567 toxicity results provide evidence that ASNA-1 is required for C. elegans’ defense against As(III) and Sb(III) toxicity, but not defense against other metals. Effects of As(III) and Sb(III), enzyme concentrations, and temperature on ATPase activity To biochemically characterize the C. elegans ASNA-1, we designed an expression plasmid, maltose-binding protein (MBP)–ASNA-1, to produce a plasmid MBP– ASNA-1 fusion protein in E. coli. ASNA-1 with the MBP tag was purified from E. coli cytosol using a column of amylose resin, as described in Experimental procedures. Approximately 2 mg of purified ASNA-1 protein was obtained per 500 mL of cells. Purified recombinant ASNA-1 proteins were analyzed for their ability to catalyze metalloid-stimulated ATPase activ- ity. The specific activity of the MBP–ASNA-1 fusion protein in the presence and absence of Sb(III) or As(III) was determined. MBP protein without ASNA- 1 was also expressed and purified, and its ATPase activity was analyzed. A very low ATPase activity of 2.4 ± 0.2 nmolÆmin )1 Æmg )1 was detected for MBP pro- tein lacking the ASNA-1 portion. In the absence of Sb(III) or As(III), a basal oxyanion-independent ATPase activity of 5.4 ± 1.3 nmolÆmin )1 Æmg )1 was measured using six different MBP–ASNA-1 fusion protein preparations. Our results showed that As(III) and Sb(III) stimulated ATPase activity of the MBP– ASNA-1 fusion protein, and that the reaction was initiated by the addition of MgCl 2 . Although the meas- ured ATPase activity varied slightly between different protein preparations, the activity increased consistently in the presence of Sb(III) or As(III). Stimulation of ATPase activity by Sb(III) and As(III) was not addit- ive. When Sb(III) and As(III) were added in a ratio of 1:10, the ATPase activity was  18% less than in the presence of Sb(III) alone, indicating that the two ani- ons bind to the same site on the ASNA-1 protein. Moreover, the effects of other metal ions, including Pb(II), Al(III), Cd(II), Cr(VI), Co(II), Cu(II), Fe(II), Hg(II), Mn(II), Ni(II), and Zn(II), on the ATPase activity of the ASNA-1 fusion protein were examined. The metal ions tested neither stimulated nor inhibited the ATPase activity of the ASNA-1 fusion protein (data not shown). The reaction rate was proportional to the amount of enzyme added, as shown in Fig. 3. Moreover, the reac- tion rate increased with increasing amounts of Sb(III) in the reaction (Fig. 3). Increasing the assay tempera- ture from 25 to 37 °C did not increase basal ATPase activity, but produced a two-fold increase in Sb(III)-stimulated ATPase activity. Based on these observations, the ATPase assay in this study was rou- tinely performed with 15 lg protein at 37 °C, and experiments were usually completed within 30 min of recovering the protein preparation. Affinity of ASNA-1 protein for substrates To examine the affinity of the ASNA-1 protein for ATP, the apparent K m for ATP was determined at pH 7.4 in the absence and presence of a saturating concentration of Sb(III) (0.5 mm) using six indepen- dently prepared MBP–ASNA-1 fusion proteins. A V max of 6.21 ± 0.24 nmolÆmin )1 Æmg )1 was observed 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 120 Hours Survival rate (%)Survival rate (%) A 0 5 10 15 20 25 30 35 40 0 20 40 60 80 100 120 Hours B Fig. 2. Time-dependent lethality of worms exposed to As(III) and Sb(III). Wild-type and asna-1-mutant worms were exposed to As(III) and Sb(III) at 0.25 m M. Worms were incubated at 20 °C and the number of dead worms was scored at different time points ranging from 1 to 36 h (± 10 min). The number of dead worms was deter- mined as described in Experimental procedures. Survival data were subjected to Kaplan–Meier survival curve analysis. (A) Proportion of wild-type (solid line) and asna-1 mutant (dash line) worms surviving with As(III) treatment. (B) Proportion of wild-type (solid line) and asna-1-mutant (dash line) worms surviving with Sb(III) treatment. Caenorhabditis elegans ASNA-1 Y Y. Tseng et al. 2568 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS for the basal ATPase activity, whereas V max for the Sb(III)-stimulated ATPase activity was 28.87 ± 3.31 nmolÆmin )1 Æmg )1 (Fig. 4A). Similar K m values of 0.21 ± 0.03 and 0.26 ± 0.09 mm were obtained in the absence and presence of Sb(III), respectively. Sb(III)- induced stimulation of ATPase activity was thus due to a 4.6-fold maximal increase in V max rather than to increased affinity of the MBP–ASNA-1 fusion protein for ATP. In addition, similar K m and V max values were observed in the presence of either 2.5 or 5.0 mm MgCl 2 (Fig. 4B). Half-maximal stimulatory concentrations of Sb(III) and As(III) on ATPase activity were determined at ATP saturation (Fig. 5). Apparent K m values for Sb(III) and As(III) were found to be 0.04 ± 0.00 and 3.54 ± 0.33 mm, respectively. The V max for Sb(III)-stimulated ATPase activity was 19.62 ± 0.16 nmolÆmin )1 Æmg )1 , whereas the As(III)-stimulated activ- ity had a V max of 11.67 ± 0.48 nmolÆmin )1 Æmg )1 . Discussion Our findings represent the first demonstration of ArsA protein-mediated detoxification of the metalloids, As(III) and Sb(III), in an animal. Moreover, we show that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria, but extend to animals. This was shown by demonstrating that the asna-1 gene of C. elegans encodes a functional ArsA whose activity is stimulated by As(III) and Sb(III), and which is critical for As(III) and Sb(III) tolerance in the intact organism. Although the function of bac- terial ArsA has been identified, the ubiquity of the ArsA ATPase-dependent pathway in other animals remains to be delineated. E. coli ArsA hydrolyzes ATP in the presence of As(III) or Sb(III), and is the cata- lytic component of an oxyanion pump that provides resistance to As(III) or Sb(III). Several eukaryotic ArsA homologs have been identified and studied. However, they showed no biochemical functions sim- ilar to that of bacterial ArsA. Mammalian ArsA homologs have been identified in humans [10] and A 0.0 0.2 0.4 0.6 0.8 1.0 0 5 10 15 20 25 30 [ATP] m M 0.0 0.2 0.4 0.6 0.8 1.0 [ATP] m M ATPase Activity (nmol/min/mg)ATPase Activity (nmol/min/mg) 0 5 10 15 20 25 30 B Fig. 4. Affinity of the ASNA-1 protein for ATP. The ATPase activity of purified ASNA-1 protein (15 lg) was measured over a range of ATP concentrations in the absence (s) and presence (h) of 0.5 m M Sb(III) containing 5.0 mM MgCl 2 (A) and in the presence of 0.5 mM Sb(III) containing either 2.5 mM MgCl 2 (n) or 5.0 mM MgCl 2 (j) (B). The reaction was initiated by the addition of MgCl 2 after 10 min incubation of ASNA-1 protein at 37 °C. Solid and dashed lines indi- cate fitting of data to the Michaelis–Menten equation by a nonlinear regression using PRISM 4.0 software. 0 5 10 15 20 0.0 0.1 0.2 0.3 0.4 ASNA-1 (µg) ATPase Activity (nmol/min) Fig. 3. Effects of enzyme concentrations on ATPase activity. ATPase activity was measured in the presence of the indicated amounts of the ASNA-1 protein as described in Experimental proce- dures. The reaction was initiated by the addition of 5 m M MgCl 2 after 10 min incubation of the ASNA-1 protein at 37 °C with or without Sb(III) in assay buffer. At each protein concentration, the activity was measured with no oxyanion (s), or 0.1 m M (n)or 0.5 m M (h) Sb(III). Y Y. Tseng et al. Caenorhabditis elegans ASNA-1 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2569 mouse [8]. Biochemical analysis of the hASNA-I human homolog showed that it has a low level of ATPase activity, which was simulated 1.6-fold in the presence of As(III), but not in the presence of Sb(III) [11]. The mouse homolog (Asna1) has been shown to be an unlikely component of an As(III) pump in mam- mals [12]. The ArsA homolog (Arr4p) in S. cerevisiae exhibited a low level of ATPase activity but was not stimulated by As(III), Sb(III), or any other metals [13]. Therefore, it is noteworthy that, although human hASNA-I, mouse Asna1, and yeast Arr4p cDNA were isolated using homology to the bacterial ArsA, the protein they encodes is not an As(III)- and Sb(III)- stimulated ATPase, indicating that these eukaryotic ArsA homologs are biochemically distinct from that of bacterial ArsA. Moreover, the protective roles of these eukaryotic ArsA homologs against As(III) and Sb(III) remain to be studied. In both mouse [12] and C. elegans, deletion of the gene encoding the ATPase is lethal, suggesting involve- ment of the asna-1 gene in embryonic or larval devel- opment. After submission of this article, Kao et al. reported that worms lacking asna-1 gene activity arrest at the L 1 stage, even in the presence of abundant food [14]. ASNA-1 functions nonautonomously to regulate growth [14]. They further showed that ASNA-1 posi- tively regulates insulin secretion in C. elegans and mammalian cells [14]. However, the precise mechanism by which ASNA-1 protects against As(III) and Sb(III) toxicity remains to be further elucidated. Several observations, however, show the unique features of ASNA-1. First, ATPase activity of the ASNA-1 pro- tein was stimulated only by As(III) and Sb(III). Other metal ions neither stimulated nor inhibited the ATPase activity of ASNA-1. Second, although a few ABC transporters, namely one member of the multidrug resistance-associated protein (MRP-1) and two mem- bers of the P-glycoprotein subfamily (PGP-1 and PGP- 3), have been shown to contribute to heavy metal tol- erance, including As(III) tolerance in C. elegans,no increased sensitivity to Sb(III) was found in the dele- tion mutants compared with wild-type worms [15]. Therefore, it is unlikely that the ASNA-1 protein is a component of a eukaryotic ABC transporter. Possibly the most interesting characteristic of the ASNA-1 ATPase is the fact that it was activated by the binding of As(III) or Sb(III), the same ions that are transported by the ArsAB pump in E. coli. At this point, there is no conclusive biochemical evidence that the ions which activate the hydrolytic activity of ASNA-1 are the same ions that are transported across the membrane in C. elegans. In bacteria, the ArsAB pump confers an evolutionary advantage to organisms exposed to high levels of metalloid salts, reflecting the fact that ATP-driven pumps are capable of forming higher concentration gradients than carrier proteins. Thus, the ArsAB system reduces the intracellular con- centration of metalloid ions to lower levels than can be realized by ArsB alone, providing evolutionary pres- sure to acquire an arsA gene. However, it is interesting to note that no eukaryotic ArsB orthologs have been identified to date. Therefore, C. elegans may contain a novel transporter that is possibly unrelated to ArsB but with a similar function. Isolation of the metalloid transporter is necessary before the role of ASNA-1 as a component of a nematode efflux pump for As(III) 0.0 0.5 1.0 1.5 2.0 2.5 0 5 10 15 20 25 [Sb(III)] mM A 0 2 4 6 8 10 0 2 4 6 8 10 [As(III)] mM ATPase Activity (nmol/min/mg)ATPase Activity (nmol/min/mg) B Fig. 5. Affinity of the ASNA-1 protein for Sb(III) and As(III). The half-maximal stimulatory concentrations for Sb(III) (A) and As(III) (B) were determined over a range of concentrations using 15 lgof purified ASNA-1 protein in the presence of 1 m M ATP. The reaction was initiated by the addition of 5 m M MgCl 2 after 10 min incubation of the ASNA-1 protein at 37 °C in assay buffer. The solid line indi- cates fitting of the data to the Michaelis–Menten equation by non- linear regression using PRISM 4.0 software. Caenorhabditis elegans ASNA-1 Y Y. Tseng et al. 2570 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS and Sb(III) detoxification can be examined, and this is the subject of a future study. Experimental procedures General methods All C. elegans, bacterial strains, and plasmids were supplied by the Caenorhabditis Genetics Center (University of Min- nesota, MN), which is funded by the NIH National Center for Research Resources. The exception was the cDNA clone yk747c10, which was kindly provided by Y. Kohara (National Institute of Genetics, Mishima, Japan). The following strains were used: wild-type C. elegans N2 (var. Bristol); asna-1 mutant: [tag-205(ok938)III ⁄ hT2 [bli-4(e937)let-?(q782)qIs48](I;III)], which is a homozygous lethal deletion chromosome balanced by bli-4- and GFP- marked translocation. C. elegans was grown in Petri dishes on nematode growth medium (NGM) at 20 °C using the E. coli OP50 strain as the food source. Metal ions toxicity analyses One hundred and twenty L 3 -stage hermaphrodites of wild- type (N2) or asna-1 mutant (non-GFP ok938 homozygotes) were transferred from NGM plates to Costar 24-well tissue culture plates containing 1 mL of K medium (53 mm NaCl, 32 mm KCl) [9] with appropriate concentrations of metal ions per well. Wild-type and asna-1-mutant worms were exposed to 0, 0.25, 0.5, 1.5, and 3.0 mm nominal concentra- tions of As(III) or 0, 0.25, 5, 10, and 20 mm of Sb(III). For other metal ions, wild-type and asna-1-mutant worms were exposed to 0.39 mm of Pb(II), 0.16 mm of Cu(II), 0.37 mm of Al(III), 0.54 mm of Cr(VI), and 1.48 mm Zn(II). Worms were incubated at 20 ° C and the number of dead worms was scored at different time points ranging from 1 to 36 h (± 10 min). The number of dead worms was determined by the absence of touch-provoked movement when probed with a platinum wire. Tests were performed between three and six times for each metal ion. Construction of the recombinant MBP–ASNA-1 fusion protein expressed in E. coli Translational fusion was constructed by directional cloning of the PCR-amplified cDNA of ASNA-1 into multiple clo- ning sites of the pMAL-c2X vector (New England Biolabs, Hertfordshire, UK). Plasmid yk747c10, kindly provided by Y. Kohara (National Institute of Genetics, Mishima, Japan), was used as a template for PCR to generate the DNA fragment. PCR primers were designed with either PstI (forward primer) or HindIII (reverse primer) recogni- tion sequence extensions (underlined). Sequences of PCR primers were as follows: forward 5¢-CCG CTGCAGGAA AAAACGCTAAAATGGA-3¢ and reverse 5¢-CGC AAG CTTAGAACAAATTAGTTTAGT-3¢. The amplified PCR product was purified using a QIAquick PCR purification kit (Qiagen, Hilden, Germany). Purified PCR-amplified DNA fragment was digested with both PstI and HindIII and then ligated with the pMAL-c2X expression vector that had been similarly digested with both enzymes. The result- ing construct, MBP–ASNA-1, contained the asna-1 gene cloned inframe with the sequence for an N-terminal MBP tag. The correct reading frame and DNA sequence were verified by DNA sequencing. The MBP–ASNA-1 plasmid was used to transform BL21 cells. Purification of the MBP–ASNA-1 protein For expression, cells of the BL21 E. coli strain harboring the MBP–ASNA-1 plasmid were grown overnight in 5 mL of Luria–Bertani medium containing 0.2% glucose and 100 lgÆmL )1 ampicillin at 37 °C. This culture was then diluted in 500 mL of the same medium and incubated with shaking at 37 ° C until D 600 ¼ 0.5 was reached. Iso- propyl thio-b-d-galactoside was added to a final concen- tration of 0.1 mm, and the culture was incubated for an additional 2 h and harvested by centrifugation. Cell pellets were washed once with column buffer (20 mm Tris ⁄ HCl pH 7.4, 200 mm NaCl, 1 mm EDTA, 1 mm sodium azide, and 10 mm b-mercaptoethanol). Lysozyme was added to a final concentration of 1 mgÆmL )1 and incubated on ice for 30 min. Cells were suspended in 10 mL of column buffer containing 1 mgÆmL )1 lysozyme. Cells were sonicated on ice using six 15-s bursts at 200–300 W with 15 s cooling off between each burst. The lysate was centrifuged at 10 000 g for 30 min at 4 °C, and then preincubated for 1 h with 2 mL of amylose resin (New England Biolabs). The supernatant and amylose resin solution were loaded onto a column at a flow rate of 0.5 mLÆmin )1 . The col- umn was washed with column buffer at a flow rate of 1.0 mLÆmin )1 . Finally, ASNA-1 was eluted with 15 mL of column buffer containing 10 mm maltose. ASNA-1-con- taining fractions were identified by SDS ⁄ PAGE. Purified ASNA-1 was either quickly frozen and stored at )80 °C or kept in small aliquots at 4 °C. The concentration of purified ASNA-1 was determined by the Bradford assay (BioRad, Hercules, CA). ATPase activity assays ATPase activity was estimated colorimetrically from the release of inorganic phosphate as described by Gawronski and Benson [16]. Freshly purified MBP–ASNA-1 protein maintained at 4 °C was used for biochemical characteriza- tion throughout this work. ATPase activity was measured spectrophotometrically at room temperature from a released inorganic phosphate concentration of 650 nm. Y Y. Tseng et al. Caenorhabditis elegans ASNA-1 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS 2571 The reaction was allowed to equilibrate for 15 min at room temperature before reading the absorbance at 650 nm. The assay mixture contained ATP with or without Sb(III) or As(III), and was prewarmed to 37 °C. Fifteen micrograms of MBP–ASNA-1 was preincubated at 37 °C in the reaction mixture for 10 min before the reac- tion was initiated by the addition of MgCl 2 . The concen- tration of purified MBP–ASNA-1 protein was determined using the Bradford assay. Data analysis Survival data were subjected to Kaplan–Meier survival curve analysis using prism 4.0 (GraphPad Software, San Diego, CA). A log-rank test was performed comparing wild-type with asan-1-mutant worms. Kinetic parameters were calculated with prism 4.0 using the nonlinear regres- sion of the Michaelis–Menten equation. Experiments were performed between three and nine times for standard error analyses. References 1 Owolabi JB & Rosen BP (1990) Differential mRNA sta- bility controls relative gene expression within the plas- mid-encoded arsenical resistance operon. J Bacteriol 172, 2367–2371. 2 Diorio C, Cai J, Marmor J, Shinder R & DuBow MS (1995) An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and conserved in gram-negative bacteria. 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FEBS Lett 580, 3889–3894. 13 Shen J, Hsu CM, Kang BK, Rosen BP & Bhattacharjee H (2003) The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance. Biometals 16, 369–378. 14 Kao G, Nordenson C, Still M, Ronnlund A, Tuck S & Naredi P (2007) ASNA-1 positively regulates insulin secretion in C. elegans and mammalian cells. Cell 128, 577–587. 15 Broeks A, Gerrard B, Allikmets R, Dean M & Plasterk HR (1996) Homologues of the human multidrug resis- tance genes MRP and MDR contribute to heavy metal resistance in the soil nematode Caenorhabditis elegans. EMBO J 15, 6132–6143. 16 Gawronski JD & Benson DR (2004) Microtiter assay for glutamine synthetase biosynthetic activity using inor- ganic phosphate detection. Anal Biochem 327, 114–118. Caenorhabditis elegans ASNA-1 Y Y. Tseng et al. 2572 FEBS Journal 274 (2007) 2566–2572 ª 2007 The Authors Journal compilation ª 2007 FEBS . were as follows: forward 5¢-CCG CTGCAGGAA AAAACGCTAAAATGGA-3¢ and reverse 5¢-CGC AAG CTTAGAACAAATTAGTTTAGT-3¢. The amplified PCR product was purified using a QIAquick PCR purification kit (Qiagen,. encoding a protein of 342 amino acids and exhibits 33% identity to E. coli ArsA ATPase. A BLAST search showed that eukaryotic ArsAs from Saccharomyces cerevisiae (Arr4p), human (hASNA-I), mouse (Asna1),. transporter, which serves as both a membrane anchor for the ArsA portion and a toxic oxyanion translocating pathway [7]. Homologs of bacterial ArsA ATPase have been found in nearly every organism