The ATPase activity of the ASNA-1 protein was dependent on the presence of AsIII or SbIII.. elegans, encodes a functional ArsA ATPase whose activity is stimulated by AsIII and SbIII and
Trang 1Yuen-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 colihas 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.
Trang 2bacterial 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 reporpredic-ted
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 (LC50) 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 20 40 60 80 100
A
0 20 40 60 80 100
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.
Trang 3toxicity 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)1Æmg)1was 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)1Æmg)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 MgCl2 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 37C 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 37C, 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 Km 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 Vmax of 6.21 ± 0.24 nmolÆmin)1Æmg)1 was observed
0
20
40
60
80
100
120
Hours
A
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.
Trang 4for the basal ATPase activity, whereas Vmax for the
Sb(III)-stimulated ATPase activity was 28.87 ± 3.31
nmolÆmin)1Æmg)1 (Fig 4A) Similar Km 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 Vmax rather than to
increased affinity of the MBP–ASNA-1 fusion protein
for ATP In addition, similar Kmand Vmaxvalues were
observed in the presence of either 2.5 or 5.0 mm
MgCl2(Fig 4B)
Half-maximal stimulatory concentrations of Sb(III)
and As(III) on ATPase activity were determined at
ATP saturation (Fig 5) Apparent Km values for
Sb(III) and As(III) were found to be 0.04 ± 0.00
and 3.54 ± 0.33 mm, respectively The Vmax for
Sb(III)-stimulated ATPase activity was 19.62 ± 0.16
nmolÆmin)1Æmg)1, whereas the As(III)-stimulated
activ-ity had a Vmaxof 11.67 ± 0.48 nmolÆmin)1Æmg)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 5 10 15 20 25 30
[ATP] m M
[ATP] m M
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 m M MgCl2(A) and in the presence of 0.5 m M
Sb(III) containing either 2.5 m M MgCl 2 (n) or 5.0 m M 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.
0.0
0.1
0.2
0.3
0.4
ASNA-1 (µg)
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 MgCl2
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).
Trang 5mouse [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 L1 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 (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
5
10
15
20
25
[Sb(III)] m M
A
0
2
4
6
8
10
[As(III)] m M
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 lg of
purified ASNA-1 protein in the presence of 1 m M ATP The reaction
was initiated by the addition of 5 m M MgCl2after 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.
Trang 6and 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 20C using the
E coliOP50 strain as the food source
Metal ions toxicity analyses
One hundred and twenty L3-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 20C 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¢-CCGCTGCAGGAA
AAAACGCTAAAATGGA-3¢ and reverse 5¢-CGCAAG 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 37C This culture was then diluted in 500 mL of the same medium and incubated with shaking at 37C until D600¼ 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)1and 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 4C, 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 colcol-umn 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 4C 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 4C 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
Trang 7The 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 37C
Fifteen micrograms of MBP–ASNA-1 was preincubated at
37C in the reaction mixture for 10 min before the
reac-tion was initiated by the addireac-tion of MgCl2 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 J Bacteriol 177,
2050–2056
3 Chen CM, Misra T, Silver S & Rosen BP (1986)
Nucleotide sequence of the structural genes for an anion
pump The plasmid-encoded arsenical resistance operon
J Biol Chem 261, 15030–15038
4 Francisco MJ, Hope CL, Owolabi JB, Tisa LS & Rosen
BP (1990) Identification of the metalloregulatory
ele-ment of the plasmid-encoded arsenical resistance
operon Nucleic Acids Res 18, 619–624
5 Hsu CM & Rosen BP (1989) Characterization of the
catalytic subunit of an anion pump J Biol Chem 264,
17349–17354
6 Meng YL, Liu Z & Rosen BP (2004) As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli
J Biol Chem 279, 18334–18341
7 Zhou T, Radaev S, Rosen BP & Gatti DL (2000) Structure of the ArsA ATPase: the catalytic subunit
of a heavy metal resistance pump EMBO J 19, 4838–4845
8 Bhattacharjee H, Ho YS & Rosen BP (2001) Genomic organization and chromosomal localization of the Asna1 gene, a mouse homologue of a bacterial arsenic-translo-cating ATPase gene Gene 272, 291–299
9 Williams PL & Dusenbery DB (1990) Aquatic toxicity testing using the nematode Caenorhabditis elegans Environ Toxicol Chem 9, 1285–1290
10 Kurdi-Haidar B, Aebi S, Heath D, Enns RE, Naredi P, Hom DK & Howell SB (1996) Isolation of the ATP-binding human homolog of the arsA component of the bacterial arsenite transporter Genomics 36, 486– 491
11 Kurdi-Haidar B, Heath D, Aebi S & Howell SB (1998) Biochemical characterization of the human arsenite-stimulated ATPase (hASNA-I) J Biol Chem
273, 22173–22176
12 Mukhopadhyay R, Ho YS, Swiatek PJ, Rosen BP & Bhattacharjee H (2006) Targeted disruption of the mouse Asna1 gene results in embryonic lethality 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