Arabidopsis thaliana MRP1 (AtABCC1) nucleotide binding domain contributes to arsenic stress tolerance with serine triad phosphorylation

Plant Physiology and Biochemistry 108 (2016) 109e120

Contents lists available at ScienceDirect

Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy

Research article

Arabidopsis thaliana MRP1 (AtABCC1) nucleotide binding domain contributes to arsenic stress tolerance with serine triad phosphorylation Ayan Raichaudhuri 1 Department of Biotechnology, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, West Bengal, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 5 June 2016 Accepted 7 July 2016 Available online 9 July 2016

Multidrug resistance protein AtMRPs belong to the ATP binding cassette (ABC) transporter super family. ABC proteins are membrane proteins involved in the transport of a broad range of amphipathic organic anions across membranes. MRPs (ABCCs) are one of the highly represented subfamilies of ABC transporters. Plant MRPs also transport various glutathione conjugates across membranes. Arabidopsis thaliana MRP1 is already known to be involved in vacuolar storage of folates. Using heterologously expressed AtMRP1 in yeast and its C-terminal nucleotide binding domain (NBD2) in Escherichia coli, it has been shown that Casein kinase II (CKII) mediated phosphorylation is a potential regulator of AtMRP1 function. AtMRP1 showed enhanced tolerance towards arsenite As(III) in yeast. CKIIII/CKII mediated phosphorylation of AtMRP1 was found to be involved in As(III) mediated signaling. AtMRP1-NBD2 and its serine mutants showed distinct change in secondary structure in the presence of arsenite and methotrexate (MTX) controlled by serine triad phosphorylation. Results showed that AtMRP1 is important for vacuolar accumulation of antifolates as well as tolerance against arsenic, both of which involved phosphorylation in the serine triads at the C terminal NBD of AtMRP1. The experiments provide an important insight into the role of AtMRP1 serine triad phosphorylation under AsIII stress conditions. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Arabidopsis thaliana ABC transporter Phosphorylation Arsenic stress Serine triad

1. Introduction ABC super families of transporters are expressed in microorganisms and mammals and are involved in the transport of chemically diverse compounds across membranes. ABCC (MRP) is a subfamily among the ABC transporters shown to have important functions across evolution. Mutations in ABCC1 (MRP1) cause drug resistance and immune response in humans. In plants, Arabidopsis thaliana ABCC1 is present in vacuolar membranes and is known to transport glutathione (GSH) conjugate and folates (Rea, 2007;

List of abbreviations used: ABC, ATP Binding Cassette; ABCC, ATP Binding Cassette subfamily C; NBD2, Nucleotide Binding Domain 2; CKII/CKI, Casein Kinase II/Casein Kinase I; As(III), sodium arsenite; As(V), sodium arsenate; MTX, methotrexate; GSH, Glutathione; NEM-GS, N-ethylmaleimide Glutathione; DNP-GS, S(2,4-dinitrophenyl)-Glutathione; PC, Phytochelatin; Ycf1, Yeast Cadmium Factor 1; AtMRP, Arabidopsis thaliana MRP; MES, 2-(N-morpholino)ethanesulfonic acid; MRP, multidrug resistance-associated protein; WS, Wassilewskia; Col-0, Columbia. E-mail address: [email protected]. 1 Present position: Scientist, Tea Board of India, Siliguri, 735135, West Bengal, India. http://dx.doi.org/10.1016/j.plaphy.2016.07.005 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved.

Raichaudhuri et al., 2009). Early studies with AtMRP1 showed MgATP-energized, vanadate-inhibitable, uncoupler-insensitive uptake of several glutathione (GSH) conjugates (GS-conjugates) like N-ethylmaleimide-GS (NEM-GS), S-(2,4-dinitrophenyl)-GS (DNP-GS), glutathionylated chloroacetamide herbicides, (metolachlor-GS), folates and antifolates (methotrexate MTX) mediated by this protein in isolated vacuoles (Martinoia et al., 1993) or in vacuolar membrane vesicles purified from plants (Li et al., 1995; Rea, 2007; Raichaudhuri et al., 2009). In addition, AtMRP1 was shown to have a role in tolerance and detoxification of heavy metal and metalloids (Rea, 2007; Song et al., 2010). MRP proteins are classified based on the presence of one or two ATP-binding cassettes or nucleotide binding domains (NBDs). Each NBD encompasses ~200 amino acid residues and contains three idiotypic sequence motifs. These are a Walker A motif (GX 4GK(ST)) and a Walker B motif ((RK)X 3GX 3L (hydrophobic)3), that are separated by ~120 amino acid residues (Walker et al., 1982), and an ABC signature (alias C motif, ((LIVMFY)S(SG)GX3(RKA)(LIVMYA)X(LIVFM)(AG)) (Bairoch, 1992). In addition to two NBDs, AtMRP transporters contain two or three hydrophobic integral membrane spanning domains (MSDs),

110

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

each of which contains multiple (usually four to six) transmembrane a-helices. The MSDs form the pathway for solute movement across the phospholipid bilayer. In south Asia, particularly in parts of Indian subcontinent, arsenic in ground water is a hazardous environmental pollutant. Arsenic content in water used for drinking and irrigation has been reported to cross the accepted limits of 10 mg/L proposed by the World Health Organization in several areas of South Asia. Arsenic is taken up by plant roots and is sequestered in plant vacuoles. Two transporters in Arabidopsis, from ABCC (MRP) family AtABCC1 and AtABCC2, were shown to contribute to arsenic tolerance via vacuolar sequestration of As (III)-phytochelatin conjugates. In Saccharomyces cerevisiae, Yeast Cadmium Factor 1 (Ycf1), an ABC transporter localized to the vacuolar membrane originally identified for its role in vacuolar cadmium sequestration, was shown to detoxify arsenic by transporting As(glutathione)3 complexes into vacuoles (Szczypka et al., 1994; Li et al., 1996; Ghosh et al., 1999). AtMRP1 is the Ycf1 homolog in A. thaliana. AtMRP1 is localized at the vacuolar membrane and transports substrates to the vacuolar lumen thereby effectively sequestering them from cytosol (Li et al., 1997; Rea et al., 1998). Over expression of Ycf1 (35S::ycf1) was shown to confer both accumulation and enhanced resistance to lead and cadmium in Arabidopsis plants (Song et al., 2003). The most common forms of arsenic available in the environment for uptake by plants are arsenate (AsV) and arsenite (AsIII). Arsenate is acquired by plant roots through endogenous transport systems for phosphate and is reduced to the thiol-reactive form As III inside the cell (Asher and Reay, 1979). At equimolar concentrations, AsIII is more toxic than AsV. Ycf1p (the yeast homolog of AtMRP1) shows several potential phosphorylation sites. Genetic evidence in yeast suggested that yeast casein kinase 2a (Cka1p) may regulate Ycf1p function through phosphorylation of Ser251 either directly or indirectly (Paumi et al., 2008). Ycf1p phosphorylation at Ser908 and Thr911, was found to be required for cadmium detoxification (Eraso et al., 2004), and phosphorylation at Ser251 negatively regulated transport activity (Paumi et al., 2008). Six additional sites of phosphorylation were identified within the linker region by studies aimed at examining the complete yeast phosphoproteome via highthroughput analysis (Li et al., 2007; Smolka et al., 2007; Albuquerque et al., 2008). The experiments described here were directed towards understanding the role of phosphorylation events controlling AtMRP1 transporter function both under AsIII stress as well as under antifolate treatment. There are several potential phosphorylation sites in AtMRP1. Using mutational analysis and heterologous expression in yeast, the role of phosphorylation, especially in the serine triad, has been investigated. The data presented here shows that AtMRP1 C-terminal NBD2 phosphorylation is important in the presence of As III and antifolate MTX. A potential signaling event in response to As III is also indicated. 2. Materials and methods 2.1. Chemicals ATP, histone mixture, imidazole, Hepes, glycerol, Triton X-100,

lifesciences. T4 DNA ligase, eukaryotic recombinant casein kinase II (CKII) and all enzymes were obtained from New England Biolabs. MTX drug (Biochem Pharmaceutical Industries Ltd., Mumbai, India) was purchased from a pharmacy and Alexa Fluor MTX was obtained from Invitrogen (U.S.A). Oligo nucleotides were obtained from Bioserve and Eurofins genomics. [g-32P] ATP was obtained from BRIT, Government of India. All other chemicals and biochemicals were of analytical grade. The nucleotide sequence for AtMRP1 genomic DNA is under GenBank Accession Number AF008125.1. The amino acid sequence of this protein can be accessed through NCBI Protein Database under NCBI Accession #AAB71832. 2.2. Cloning, expression, and purification of Arabidopsis MRP1 NBD2 polypeptide and other mutants AtMRP1 was PCR amplified using Primers 50 -ACGCGTCGACATAAAATTTGAGG ATGTTG-30 as forward and 50 -ATAGTTTAG CGGCCGCCATCTCGACATTGTCCC-30 as reverse primers and sub cloned in SalI and NotI sites of expression vector pET33b. The expressed polypeptide contains a N-terminal 6X-His tag. For mutant protein cloning PCR was done with forward and reverse primers (Table 3) and cloned in pET33b vector. Nucleotide sequence and in-frame ligation was checked for all the clones by DNA sequencing. The recombinant polypeptides were expressed in Escherichia coli BL21(DE3) by growing in the presence of 1 mM IPTG for 5 h at 37  C in the presence or absence of sodium arsenite salt (1 mM) and MTX drug (100 mM). 6X-His tagged soluble proteins were purified under non denaturing conditions using NiNTA (Qiagen) at 4  C according to manufacturer’s recommendations. The 47 kD recombinant proteins were analyzed for purity and integrity by SDS-PAGE (Laemmli, 1970). The protein concentrations were determined using Bradford method (Bradford, 1976). These purified proteins were used in all further work. 2.3. Heterologous expression and purification of vacuolar membrane enriched vesicles For studies of the transport capabilities of heterologously expressed AtMRP1, S. cerevisiae DYcf1 strain DTY168 (MATa his6 leu2-3,-112 ura 3e52 Ycf1::hisG) (Szczypka et al., 1994) was transformed with pYES3-AtMRP1 or empty pYES3 vector by the LiOAc/ polyethylene glycol method (Gietz and Schiestl, 1991) and selected for uracil prototroph by plating on AHC medium containing tryptophan (Kim et al., 1995; Lu et al., 1997). Vacuolar membraneenriched vesicles were purified from the transformants as described previously (Kim et al., 1995; Lu et al., 1997; Raichaudhuri et al., 2009). 2.4. Insertion of C-terminal 6X-His-tag in pYES3-AtMRP1 Insertion of 6X-His-tag in pYES3-AtMRP1 at 4836 position containing XbaI site was done by equal concentrations of 1X TE buffer dissolved primers 50 -CTAGACATCACCATCACCATCACT-30 and 50 -CTAGAGTGATGGTGATGGTGATGT-30 with 6X-His-tag complementary sequences in frame with XbaI overhang. They were annealed in the presence of 65 mM sodium chloride at 85  C for 5 min and slow cooling to room temperature. XbaI restriction

b-mercaptoethanol, aprotinin, leupeptin, IPTG, Q-sepharose, PMSF, alkaline phosphatase-conjugated anti-rabbit IgG, and Western blotting kit were obtained from Sigma. pET expression vectors were purchased from Merck Millipore (U.S.A.), nickel-agarose and antiGAPDH antibody were obtained from BioBharati Life Science (Kolkata, India) and anti-RGS-His antibody was obtained from Qiagen (Germany). Chromatography materials were obtained from Ge

Table 1 Binding affinities calculated from modified Stern-Volmer plot.

Kd fa

MTX

Alexa Fluor MTX

0.14 mM 0.95 mM

10.31 mM 0.66 mM

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

111

Table 2 As(III) dependent secondary structure change of NBD2 and mutants expressed in absence () and presence (þ) of As(III) salt (1 mM) by Far-UV CD spectra.

Alpha helix

(As (þAs (As (þAs

Beta sheet

III) III) III) III)

NBD2

Mut1

Mut2

Mut3

Mut4 (Results in %)

20.46 23.76 29.80 23.78

19.69 21.57 31.52 28.13

4.44 23.70 44.17 26.20

5.30 20.46 43.19 29.80

3.66 7.52 47.11 32.19

Error ±3%.

Table 3 Primers used for site directed mutagenesis. Mutant1 forward Mutant2 forward Mutant3 forward Mutant4 forward Reverse primer

primer primer primer primer

50 -ACgCgTCgACggATggCCAgCAgCTggAgCCATA-30 50 -ACgCgTCgACggATggCCAgCAgCTggATCCATA-30 50 -ACgCgTCgACggATggCCATCAgCTggAgCCATA-30 50 -ACgCgTCgACggATggCCAgCATCTggATCCATA-30 50 -ATAGTTTAGCGGCCGCCATCTCGACATTGTCCC-30

enzyme digested pYES3-AtMRP1 plasmid was next ligated with these annealed primers with T4 DNA ligase (NEB) using manufacturer’s instructions. Plasmid DNA was isolated from the transformed plates and positive clones were selected by PCR using forward primer 50 -ACGCGTCGACATAAAATTTGAGGATGTTG-30 and reverse primer 50 - CTAGAGTGATGGTG ATGGTGATGT-30 . Positive PCR with 1010 bp fragments were found and pYES3-AtMRP1 with C-terminal in frame 6X-His tag was isolated (see also Fig. S1. AtMRP1 Multiple sequence alignment). 2.5. Circular dichroism Experiments were performed using Jasco-720 spectropolarimeters. A bandwidth of 1 nm and a scan step size of 0.25 nm were employed with a 0.1 cm path length. The recombinant polypeptides expressed as described above with the presence and absence of sodium arsenite salt (1 mM) and MTX drug (100 mM) and the 6X-His proteins were purified under non denaturing conditions using nickel-nitrilotriacetic acid agarose (Qiagen) at 4  C. CD spectra were recorded at 298 K with 5 mM of the indicated polypeptides. Five scans were averaged for each sample, and the spectrum for the buffer was always subtracted. Secondary structure quantitation was determined using k2d2 software (Perez-Iratxeta and Andrade-Navarro, 2008). 2.6. Exposure to As III and cell growth measurements A culture of S. cerevisiae transformed with pYES3 was used as a control and another transformed with pYES3-AtMRP1 was used in all experiments. Cell growth was measured at 600 nm using spectrophotometer (Jasco, V530) and cell numbers were measured by hemocytometer. Equal amount liquid culture of yeast with 2.4  106/mL cells were serially 1:10 diluted (4 times) with SD-Ura (synthetic dextrose lacking uracil) medium and spotted on SD-Ura plates with absence and presence of 70 mMAs III. Sodium arsenite was also added to early log phase culture of Saccharomyces cerevisiae to the concentrations of 10, 30, 70 and 100 mM and the cultures were grown upto 14 h. One culture of Saccharomyces cerevisiae containing pYES3 and another containing pYES3-AtMRP1 was used as control in all experiments. Cell growth was measured at various time points and from there cell numbers at each concentration were calculated. In each case pYES3 cell number was subtracted from pYES3-AtMRP1 and plotted against time. Several measurements of Saccharomyces cerevisiae gave Cell/ OD600 relationship of 4e9  107 cells/ml per OD600 (chart from UC, Boulder, U.S.A.). According to Current Protocols: OD600 of 1.0 is roughly 3  107 cells/ml (Treco and Winston, 2008).

2.7. Immunological techniques Antibody against AtMRP1 C-terminal domain was raised in rabbits. The affinity-purified 6X-His-tag NBD2 polypeptide was subjected to SDS-PAGE. After mild staining of the gel, the appropriate band was excised, crushed, and used for immunization according to standard protocols (Wright, 1989).

2.8. SDS PAGE and western blot Vacuolar membrane preparation and immunoblot analysis SDSPAGE was performed using 10% (w/v) SDS-polyacrylamide gels. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane (Ge lifesciences, U.S.A.) and probed with anti-NBD2 antiserum. The immune complex was detected using an alkaline phosphatase-conjugated anti-rabbit IgG (Sigma, U.S.A.) and developed using premixed BCIP/NBT solution (Bio-Rad). Primary and secondary antisera were used at 1:1000 and 1:3000 dilutions, respectively. For loading controls anti-GAPDH antibody was used at 1:3000 dilutions and anti-rabbit secondary antisera was used as usual.

2.9. Pull-down assay Interaction of 6X-His-tagged NBD2 and all mutant polypeptides with NBD2 antibody and their serine phosphorylation status were investigated following the binding conditions described previously (Raichaudhuri et al., 2006). Binding with NBD2 antibody was done for 4 h at 4  C and proteins were immunoprecipitated with Protein A sepharose beads. The reaction mixture was analyzed in 10% SDSPAGE and western blotted with phospho-serine antibody (Qiagen) following manufacturer instructions. 2.10. Transport assay and fluorescence microscopy For laser scanning confocal microscopy (LSCM), the cells were visualized using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss Inc., Germany). Polylysine-coated slides were used for fluorescence study. pYES3 and pYES3-AtMRP1 containing DTY168 S. cerevisiae vacuolar membrane vesicles were treated with Alexa Fluor MTX. Reaction mixture contained 3 mM ATP, 3 mM MgSO4, 10 mM creatine phosphate, 16 units/ml creatine kinase, 50 mM KCl, 400 mM sorbitol and 25 mM Tris/MES buffer (pH 8.0). Uptake was terminated by the addition of ice-cold wash medium (400 mM sorbitol, 3 mM Tris/MES (pH 8.0)). Finally, the coverslips were mounted with anti-fade mounting media and evaluated using the 63 objective of a confocal microscope (Carl Zeiss Inc., Germany). For Alexa Fluor MTX excitations were performed at 491 nm with FITC filter set and emission at 518 nm respectively. Image acquisition and analysis was done using LSM 510 software. Each experiment was performed in triplicate. The images were corrected for possible crosstalk by sequential scanning in multiple channels using the multitrack configuration.

112

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

2.11. Drug binding and tryptophan fluorescence by NBD2 A heterogeneous population of fluorophore quenching data was fitted to a modified Stern-Volmer equation to calculate various quenching parameters. The work was done in the presence of 50 mM MTX drug and 50 mM Alexa Fluor MTX. Since NBD2 contained more than one Trp the heterogeneous nature of Trp was evaluated by Lehrer plot of F0/DF versus 1/[Q] according to equation F0/DF ¼ 1/(fa$Ksv$[Q]) þ 1/fa; where fa is the fractional maximum accessible protein fluorescence. For tryptophan fluorescence Hitachi 7000 fluorimeter was used with band pass 5/10, PMT voltage 600 V at 295 nm excitation and 340 nm emission with NBD2 protein concentration of 3.5 mg/mL and Tris-EDTA pH 7.5 buffer. The uptake of Alexa Fluor MTX by yeast vesicles were examined at 491 nm excitation and 518 nm emission. 2.12. Phosphostaining and densitometry analysis Pro-Q Diamond Phosphoprotein Gel Stain (Invitrogen, U.S.A.) was used for phosphostaining the SDS PAGE-run proteins according to manufacturer instruction. For in vivo work proteins were expressed in E. coli BL21(DE3) in the presence of 1 mM sodium arsenite added after 20 min of IPTG addition. With in vitro work sodium arsenite (1 mM) and CKII was added to NBD2 and mutant proteins and incubated for 15 min. All proteins were loaded in SDS PAGE as usual and phosphostained. The gels were scanned for signals in Typhoon TRIO Variable Mode Imager (GE Healthcare). For MTX work, same procedures were followed using 100 mM MTX. Densitometry analysis of phosphostained bands were performed using UN-SCAN-IT gel software. 2.13. In vitro phosphorylation assay Kinase assays with wild type AtMRP1 C terminal protein and the mutants were performed in a 50 mL reaction volume containing 50 mM Tris, pH 8.0, 10 mM MgCl2, 10 mM NaCl supplemented with 0.2 mM [g-32P]ATP (30,000 cpm/pmol) (BRIT, Government of India) as the substrate in a reaction volume. Reactions were initiated by the addition of CKII enzyme (0.16 mg/100 mL) (New England Biolabs) and carried out for varying durations of time (0, 1, 2, 5, 10 and 20 min at 30  C. For histone phosphorylation, reactions were performed using unfractionated whole histone (0.1 mg/mL) mixture (Sigma) and supplemented with 0.2 mM [g-32P] ATP (4000 cpm/ pmol) as substrate. The reactions were carried out for 20 min and stopped by SDS-PAGE sample buffer, loaded on 10% SDS-PAGE and autoradiography was done. 2.14. Statistical analysis All statistical analysis was done from graphs of MTX and Alexa Fluor MTX with plus and minus ATP and with vanadate. P values were obtained by Ky plot and GraphPad Prism software. 3. Results 3.1. Domain analysis of Arabidopsis thaliana MRP1 and its homologues AtMRPs generally contain different domains that are the hallmark of the protein. This includes two nucleotide binding domains (NBD1 and NBD2) or ATP binding cassettes, two membrane spanning domains (MSD1 and MSD2) and linker domains L0 and L1 (Fig. 1A). Sequence analysis of AtMRP1 with Phospho Motif Finder (http://www.hprd.org/PhosphoMotif_finder) showed that it contains a casein kinase II phosphorylation site with four amino acids

(Ser1238Ser1239Gly1240Ser1241) at the start of NBD2 (Fig. 1) (Roach, 1991; Marin et al., 2003). Alignment and phylogenetic tree of AtMRP1 with other Arabidopsis MRP proteins showed that theserine triad (SSGS) is predicted by PhosphoMotif finder as the site of CKII/CKI dependent phosphorylation in AtMRP1 and AtMRP2 (Fig. 1B). Other AtMRPs however contained a modified form of the triad. Blast analysis showed that NBD2 domains from other AtMRP1 homologs contained the same conserved serine triad (Fig. 1C) indicating its possible significance in protein functions. 3.2. Phospho-status of expressed proteins The C terminal Nucleotide binding site (NBD2) of AtMRP1 was used and mutations at the predicted triad site Ser1238Ser1239Gly1240Ser1241 were carried out. Mutant 1 contained S1238A, S1239A and S1241A mutations; mutant 2 contained S1238A and S1239A mutations; mutant 3 contained S1239A and S1241A and mutant 4 contained S1238A mutation. The mutants as well as the wild type proteins were expressed in E. coli. Expressed proteins of 47 kD were first checked with anti NBD2 antibody (Fig. 2A). Both induced (þI) and uninduced (I) lanes are shown and the procedure is explained in materials and methods. Expressed proteins were then checked for their phosphorylation status using manufacturer instructions. This indicated the phosphorylation state of three protein in vivo. The wild type NBD2 in vivo was in phosphorylated state (Fig. 2B) and to check the effect of As III on the phosphorylation status of the NBD2, E. coli cells were grown in the presence of sodium arsenite during protein expression. Mutant 3 and mutant 2 showed strong in vivo phosphorylation (Fig. 2B). This indicated that in the presence of AsIII, Ser1238 and Ser1241 residue were in phosphorylated form in vivo. To check the phosphorylation in these mutants in vitro, a phosphorylation assay was carried out using purified protein with [g-32P] ATP in presence or absence of As III in the reaction buffer. Mutant1 was found to be in a not phosphorylated state but mutant 2 and mutant 3 were in phosphorylated state in this assay (Fig. 2B). Mutant 2 in in vitro assay showed a higher phosphorylation state compared to mutant 3 (Fig. 2B). This indicated that both Ser1241 and Ser1238 were important for phosphorylation and that they compensated for each other. In mutant4 where Ser1238 was mutated phosphorylation was much diminished than mutant3. Most probably in vivo condition is compensating the change of phosphorylation in the absence or presence of As III which is happening in vitro. Densitometry analysis of phosphorylated stained gels was performed to check the proteins exhibiting significant changes in phosphorylation signals. NBD2 and all its mutants were next phosphorylated using casein kinase II in the presence of [g-32P] ATP and in absence or presence of sodium arsenite (1 mM). Results showed that mutant3 get strongly phosphorylated in the presence of As III along with mutant2 and mutant4, which also showed phosphorylation (Fig. 2C). This led to the conclusion that Ser1238 was an important site for As III dependent phosphorylation. However since mutant4 with Ser1238 mutation was also phosphorylated, in the presence of As III it indicated that Ser 1238 mutation was compensated by Ser1241 phosphorylation primarily, and also at Ser1239. These results indicated Ser1238 or Ser1241 was phosphorylated in the presence of As III. However, Ser1239 was responsible for basal level of phosphorylation. As reaction control, unfractionated whole histone mixture (Sigma) was used as substrate (Fig. 2C). To check if methotrexate, (MTX) a known substrate of AtMRP1, could also induce phosphorylation at the serine triad, MTX was added in E. coli cells expressing the NBD2 and its triad mutants or to purified proteins in the presence of [g-32P] ATP. In vivo and in vitro phosphostaining results indicated both mutant2 and mutant3 (Fig. 2D) was in

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

113

Fig. 1. Domain organization, phylogenetic tree and homology of a potion of plant transporters. A. Schematic model of AtMRP1 with membrane spanning domains (MSD), nucleotide binding domains (NBD) and linker domains (L). B. Phylogenetic tree of full length A. thaliana MRPs constructed using ClustalW programme. AtMRPs with similar sequences are clustered together and their similar serine triad phosphorylation site SSGS mentioned in AtMRP1 and AtMRP2. C. Blast analysis of full length AtMRP1 show other plant transporters of which AtMRP2 (GI:15226801), VvMRP (GI:297740795), TcMRP (GI:590681599) RcMRP (GI:255571320), MtMRP (GI:357447229), PtMRP (GI:566164721) conserved motif. Serine triad phosphorylation area of MRPs is shown in box. Only a portion of the plant transporters are shown here.

phosphorylated state, which showed that this serine triad was also phosphorylated in MTX stress. 3.3. Phospho-status identification of serine residues in expressed proteins Phospho status of serine mutation of the SSGS triad was checked using pull down assays followed by western blot with a phosphoserine antibody. Wild type NBD2 and all mutants of NBD2 were cloned and expressed as usual in E. coli in the absence or presence of 1 mM sodium arsenite and used for all assays. In absence of sodium arsenite mutant1 containing S1238A, S1239A and S1241A was found to be in the dephosphorylated form in pull down and phospho-serine western blots compared to wild type NBD2 (Fig. 3) showing importance of these residues in phospho regulation. Mutant2 with S1238A S1239A mutations and mutant3 with S1239A and S1241A mutations both indicated higher serine phospho status compared to mutant1 indicating that both Ser1238 and Ser1241 can be phosphorylated in absence of As III (Fig. 3; lane i and

lane j). Mutant4, which was in the phosphorylated state indicated phosphorylation at Ser1241 (Fig. 3; lane k). From these results it was concluded that both Ser1238 and Ser1241 were important for phosphorylation and this area was a regulatory domain in controlling the signaling pathway by phosphorylation. In the presence of As III however, mutant1 was found to be in phosphorylated state (Fig. 3; lane m). This indicated that in the presence of As III and in absence of the serine triad phosphorylation occurred at some distant serine. This site was not detected by either phosphostaining (in vivo) or the in vitro phosphorylation assay described in the previous section. 3.4. Binding and transport of MTX by AtMRP1 Earlier studies with AtMRP1 confirmed uptake of MTX inside vesicles using [3H] labeled MTX (Raichaudhuri et al., 2009). Vesicles were purified from pYES3-AtMRP1 transformed S. cerevisiae DYcf1 strain DTY168 following the procedure of (Raichaudhuri et al., 2009) and transport of Alexa Fluor 488 conjugated MTX (Invitrogen, U.S.A) was measured by confocal microscopy (Carl Zeiss Inc.,

114

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

115

Fig. 3. Pull down assay to identify phosphorylated status of serine using anti p-Ser antibody. NBD2 (lanes b, g, l), mutant1 (lanes c, h, m), mutant2 (lanes d, i, n), mutant3 (lanes e, j, o) and mutant4 (lanes f, k, p) expressed in E. coli in absence (lanes aek) and presence (lanes leo) of As III were pulled down with anti-NBD2 antibody and western blot done using phosphorylated serine antibody. Some of the data were acquired from separate gels.

Germany). Bright Alexa Fluor fluorescence in pYES3-AtMRP1 was observed, which was absent in vacuolar membrane enriched vesicles purified from empty vector transformed S. cerevisiae (Fig. 4A). This uptake corresponded to previous results where [3H] MTX and [3H] PteGlu1 were transported into pYES3-AtMRP1 transformed S. cerevisiae Ycf1 strain DTY168 as described by Raichaudhuri et al. (Raichaudhuri et al., 2009).

3.5. Fluorescence of purified NBD2 was quenched with MTX to determine the accessibility of Trp to MTX Drug binding property with NBD2 protein was checked both with MTX and Alexa Fluor 488 conjugated MTX (Fig. 4B) and their dissociation constants were calculated by fluorometric analysis using tryptophan fluorescence measurement. From the graph, linearity of the modified Stern-Volmer Plot was interpreted as the occurrence of static quenching processes in the presence of tryptophan residues. The actual binding affinities expressed as Kd calculated from the modified Stern-Volmer plot of MTX and Alexa Fluor MTX are shown in Table 1, along with the percentages of accessible fluorophores. Also it was seen from the greater intercept for Alexa Fluor MTX that it quenches a smaller proportion of the fluorescing tryptophan than MTX, suggesting that this high molecular-mass Alexa Fluor MTX was not able to completely fill the binding site of the NBD2 protein. Change in Ksv values showed that tryptophan residues were more buried in the presence of MTX but more open in the presence of Alexa Fluor MTX. The dissociation constants for MTX and Alexa Fluor MTX were found to be 0.14 mM and 10.31 mM respectively (Fig. 4B). So the rate of association of Alexa Fluor MTX was ~70 fold less than MTX drug to AtMRP1 NBD2 domain (Table 1) and strong association of MTX drug with NBD2 was confirmed. fa data indicated that when MTX bound with NBD2, 95% of neighbor tryptophan was exposed, but in case of Alexa Fluor MTX only 66% of tryptophan was in exposed condition (Table 1), which indicated that in the presence of bulky Alexa Fluor MTX tryptophan was mainly buried in the protein. In NBD2 the fluorescing tryptophan residues that were buried within the protein gave rise to heterogeneous quenching.

Tryptophan that was involved in the binding site of MTX acted as a quencher and was available to quenching; other trypthophans which were not involved in binding were not quenched at all. Tryptophan fluorescence at 340 nm showed proteins with sodium arsenite or MTX were more solvent accessible than their wild type counterparts with marked diminution in fluorescence concomitant to the increase in phosphorylation in the neighboring serine.

3.6. Global fold of AtMRP1 changes with serine mutation and in the presence of arsenic To check the change in the secondary structure of NBD2 and its triad mutants in the presence of As III and MTX, far UV CD spectra of NBD2 protein was obtained with the minima at 209 nm and 222 nm (Fig. 5A). All proteins were expressed in E. coli grown in the presence or absence of As III and purified as usual by nickelnitrilotriacetic acid agarose (Qiagen). There was a major decrease in alpha-helical component of mutant2 and mutant3 compared to NBD2 in absence of As III. The helical component of mutant2 and mutant3 decreased and that of mutant1 increased compared to NBD2 in absence of As III which showed disordered structure in mutant2 and mutant3. Also there was a strong change in the spatial distribution from an ordered structure to a disordered structure in mutant2 and mutant3. In the presence of As III however the random coils decreased and helical components increased in the mutants leading towards more ordered structure. Mutant 3 and mutant 1 proteins expressed in the presence of As III showed decrease in the alpha helix compared to NBD2 but increase in beta sheet whereas in mutant2 the alpha helical component remains unchanged. This data showed the influence of the three serine residues on the overall secondary structure of NBD2 as well as indicated As III dependent secondary structure change in these proteins (Table 2) MTX also showed similar structural changes in NBD2 (Fig. 5B). The helical secondary structure was decreased and beta sheet increased compared to NBD2 in the presence of MTX (alpha helix 3.13%, beta sheet 49.32% with MTX compared to alpha helix 20.46%, beta sheet 29.80% in NBD2 protein).

Fig. 2. Phosphorylated status of AtMRP1 NBD2 serine triad motif (Ser1238Ser1239Gly1240Ser1241). A. Western blot of expressed WT and mutant1 (S1238A, S1239A and S1241A), mutant2 (S1238A and S1239A), mutant3 (S1239A and S1241A) and mutant4 (S1238A) proteins with Anti-NBD2 antibody. I is uninduced and þI is induced protein explained in materials and methods. B. Phosphostain of NBD2 and mutant1, mutant2, mutant3 and mutant4 proteins expressed in the absence or presence of sodium arsenite (1 mM). As III added during IPTG induction (In vivo) and As III added to purified proteins and incubated with CKII (in vitro). Densitometry analysis of phosphostained bands were performed using UN-SCAN-IT gel software and shown in bar diagrams. C. g-32P phosphorylation of NBD2 and mutant1, mutant2, mutant3 and mutant4 were done in absence and presence of sodium arsenite (1 mM) and CKII. D. In vivo and in vitro phosphostain of NBD2 and mutant1, mutant2, and mutant3 proteins in absence and presence of MTX (100 mM). Densitometry analysis of phosphostained bands were performed using UN-SCAN-IT gel software and presented in bar diagrams.

116

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

Fig. 4. AtMRP1 dependent MTX transport and binding of MTX with NBD2. A. Fluorescence of pYES3 and pYES3-AtMRP1 transformed S. cerevisiae vacuolar membrane vesicles visualized with (a) alexa fluor MTX (b) bright field (c) merge. All data were captured on a laser scanning confocal microscope (Carl Zeiss). Scale bar ¼ 10 mm. B. NBD2 binding with (a) MTX drug (50 mM) and (b) Alexa fluor MTX (50 mM) and tryptophan fluorescence were measured in absence and presence of ATP (1 mM) and in the presence of Vanadate (100 mM). Values were plotted using modified Stern volmer equation and dissociation constant (Kd) values with minus ATP and minus vanadate calculated were 0.14 mM for MTX and 10.31 mM for Alexa Fluor MTX respectively. With minus ATP, P-values obtained by Ky plot software for MTX equals * (P  0.05) Two-sided and Alexa Fluor MTX equals *** (P  0.001) Two-sided. With plus ATP (1 mM) for MTX equals * (P  0.05) Two-sided and with Alexa Fluor MTX equals *** (P  0.001) Two-sided. Using vanadate (100 mM) for MTX equals * (P  0.05) Two-sided and with Alexa Fluor MTX equals *** (P  0.001) Two-sided. By conventional criteria these differences are considered to be statistically significant.

3.7. Effect of As III on yeast expressing AtMRP1 To test the role of AtMRP1 on arsenic resistance, AtMRP1 was heterologously expressed in yeast strain DYcf1 DTY168 lacking an ABC transporter. When grown on arsenic-free control medium, no difference was observed between cells transformed with the empty vector pYES3 or expressing ABC transporter AtMRP1. Equal amount of yeast up to 2.4  106/mL cells was serially diluted on SD-Ura plates in absence or presence of 70 mMAs III (Fig. 6A). However, the yeast cells expressing AtMRP1 grew better compared to the control when exposed to As III (Fig. 6A). Difference of empty vector

and AtMRP1 growth, though small, is clearly visible in Fig. 6A and it indicates AtMRP1 confers arsenic resistance. DYcf1 yeast strain DTY168 was used in which AtMRP1 was heterologously expressed. When grown on As III free control medium no difference was observed between yeast transformed with empty vector or AtMRP1. However when the DTY168 yeast cells were grown in the presence of 70 mM As III and the cells expressing AtMRP1 grew better (Fig. 6A panel D and E). Without yeast transporter (DYcf1) DTY168 strain was used as heterologous system to understand the AtMRP1 function which indicates AtMRP1 can transport As III as displayed by difference in yeast cell growth.

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

117

Fig. 5. Far-UV CD spectra of NBD2. A. The spectra were developed for the NBD2 and mutant1, mutant2, mutant3, mutant4 proteins expressed in absence and presence of sodium arsenite (1 mM). B. Far-UV CD spectra of NBD2 in the presence of MTX (100 mM). Spectra were recorded with 5 mM protein in 20 mM Tris-HCl, pH 7.4. NBD2 spectrum was taken as reference for all the mutants.

Also Saccharomyces cerevisiae DTY168 transformed with pYES3AtMRP1 was grown in the presence of different sodium arsenite concentrations up to 14 h, cell growth was measured and from there cell numbers at each concentration were calculated. pYES3 transformed cells were also grown in the same procedure and these cell numbers were subtracted from pYES3-AtMRP1 cell numbers which gives the number of cells that can survive in the presence of high As III salt concentrations. Results indicate cells with pYES3AtMRP1 transformed are surviving better than the control cells over increased time of incubation in As(III)-containing medium (Fig. 6B). A polyclonal antibody against NBD2 domain confirmed heterologous expression of AtMRP1 protein in S. cerevisiae vacuolar membrane vesicles (Fig. 6B). Anti His antibody cross reacted with the 170 kD pYES3-AtMRP1 6X-His tagged protein in S. cerevisiae (Fig. 6B). This result implicated a positive role of AtMRP1 in arsenic salt stress tolerance.

4. Discussion AsIII has a propensity to bind to sulfhydryl groups and thus has significant detrimental effects on general protein functioning. AsIII enters plants through aquaporin-like transporters, specifically the so called aquaglyceroporins (Wallace et al., 2006; Bienert et al., 2008). Free AsIII is highly toxic as it reacts with vicinal dithiols of

proteins altering their structure or catalytic functions (Chen et al., 2010; Liu et al., 2012). Cytosolic free AsIII must be maintained at a low level to avoid toxicity which can be achieved by complex formation with phytochelatins (PCs) in most plant species (Liu et al., 2010). The AsIII-PC complexes are transported into vacuoles by ABCC transporters (Song et al., 2010). An exception to the thioldependent AsIII detoxification is the arsenic hyper accumulator Pteris vittata, which is able to store noncomplex AsIII in its vacuoles (Indriolo et al., 2010). To prevent arsenic accumulation and to survive in arsenic contaminated environments, several strategies have been developed by organisms. In plants, strategies usually include inhibition of arsenic uptake by roots (Bleeker et al., 2003), chelation by phytochelatins (PCs) and sequestration into intracellular compartments (Li et al., 2004), efflux from root cells (Xu et al., 2007) and transformation into less toxic organic arsenic compounds (Zhao et al., 2009). ArsB, an antiporter of AsIII, constitutes a major detoxification pathway for arsenicals in Escherichia coli (Meng et al., 2004). Also, expression of ScACR3 in rice could enhance AsIII efflux in rice roots and thus reduce arsenic accumulation in rice plants. This is the first report of CKII targeted As III dependent phosphorylation of AtMRP1. This study shows that the proposed site of NBD2 in AtMRP1 is unique for phosphorylation and is on serine residues. This serine triad containing site is conserved in A. thaliana AtMRP1 (ABCC1) and AtMRP2 (ABCC2) and also among some other

118

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

Fig. 6. AtMRP1 helps S. cerevisiae grow in the presence of sodium arsenite. A. Expression of AtMRP1 in DYcf1 DTY168 S. cerevisiae in serial dilution in SD-Ura (synthetic dextrose lacking uracil) plate in the absence and presence of 70 mM NaAsO2 for 2 days. EV, empty pYES3 vector control. For yeast suspension equal amount of 2.4  106/mL cells were initially spotted on the plates and 1:10 diluted for each panel (A to E). The experiment was repeated three times. B. Cell number of pYES3 transformed yeast were subtracted from cell number of pYES3-AtMRP1 transformed yeast and plotted against time. Cells were grown in the presence of increasing concentrations of sodium arsenite up to 14 h. Cell number shown are means of three sets. C. Anti NBD2 and anti His antibody on protein blot with pYES3 and pYES3-AtMRP1 vacuolar membrane vesicle protein. Anti-GAPDH antibody was used for loading control. The position of AtMRP1 (170 kD) and GAPDH (37 kD) are indicated.

plant species. Previous reports with AtMRP1 established that heterologously expressed AtMRP1 is competent in the MgATP dependent transport of GS-conjugates and monoglutamylated folates, which is also shared by vesicles derived from the vacuolar membrane of red beet storage root. AtMRP1 and its functional equivalent in the vacuolar membrane of red beet translocate folate monoglutamates and antifolates in vitro and the former contributes to antifolate tolerance in vivo. In this study a novel site for casein kinase II (CKII) mediated phosphorylation of A. thaliana MRP1

(ABCC1) was found. The work started with finding a CKII phosphorylation motif in the C terminal of AtMRP1 at the start of nucleotide binding domain 2 (NBD2). Results here indicated this site can be a CKII substrate motif. CKII phosphorylate protein substrates and reports suggest the minimum substrate specificity for CKII can be S/TXX S/T/Y(P) and S-X-X-E/S(P) which indicates that position n þ 3 from the target serine or threonine must be occupied by a side chain with a phosphorylated group (Roach, 1991; Marin et al., 2003). To confirm this in AtMRP1, phosphorylation status of

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

NBD2 expressed in E. coli in absence and presence of sodium arsenite was determined, which showed NBD2 in vivo is always in phosphorylated state. Several mutants with one or more of the serine triad residues were analyzed by phosphostaining, which indicated Ser1241 and Ser1238 are compensating for each other. In the presence of As III mutant3 is in the phosphorylated state which indicates Ser1238 phosphorylation is As III dependent. Also, in mutant4 where Ser1238 is mutated phosphorylation is lower compared to mutant3. The phosphorylation in mutant3 could be due to phosphorylation at Ser1241 or Ser1239 which indicates this area is involved in phosphorylation. In vitro phosphorylation in the presence of g-32P ATP and CKII also confirms the phosphorylation here. Mutant1, mutant2 and mutant4 shows basal level in vitro phosphorylation which could be due to phosphorylation at other sites. Using MTX both in vivo and in vitro results indicated a similar phosphorylated state of Ser1238 and Ser1241. This indicates phosphorylated serine in this triad is important for both heavy metal As III and MTX. Transporters are often post-translationally modified by upstream kinases to control transport activity. The results indicate that the signaling pathways by MTX and AsIII on AtMRP1 are regulated by some common steps. Studies with Ycf1p earlier showed that Ser251 in L0 is a site of phosphorylation, and mutation of S251A results in increased transport function in vivo and in vitro (Smolka et al., 2007; Paumi et al., 2008). Further evidence suggested that Ycf1p function is negatively regulated by phosphorylation of Ser251 (Paumi et al., 2008). Also ABCC7 (CFTR) is regulated by CKII in vivo (Mehta, 2008). CKII has been shown to modulate ABC A1 transporter activity (Roosbeek et al., 2004). In the work presented here, AtMRP1 appeared to be a substrate of CKII under As III stress, suggesting a new underlying role of AtMRP1 in cellular resistance. CKII can interact with and phosphorylate serine in the triad, which supports the model that CKII regulates AtMRP1 function. Here As III or MTX treatment resulted in an apparent overall increase in phosphorylation signals with CKII treatment. Phosphorylation of one site is often known to directly and indirectly affect phosphorylation at other phosphorylation site(s) (Stolarczyk et al., 2011). Earlier results indicated presence of a calmodulin binding domain in this region (W1236-Y1250) (Geisler et al., 2004) covering this serine triad may also be important in downstream signaling in the presence of As III stress. 5. Conclusions The most interesting finding of this work is the discovery of a potentially new and exciting role of AtMRP1 in cellular response to arsenite stress. As III effects on plant growth are also confirmed and this is the first report of AsIII effect on mutants of atmrp1-1 and atmrp1-2. Results here show a much stronger phenotype on AtMRP1 in response to As III compared to earlier reports where sodium arsenate (AsV) Song et al. (2010) was used. AsV can be reduced to AsIII inside the cell which explains the difference in phenotypes. As different mutant lines vary in their sensitivity towards As III, atmrp1-2 used in this study shows a stronger phenotype than the one used by (Song et al., 2010). This finding is supported by previous studies that have demonstrated a role for CKII in cellular response to salt stress (Kanhonou et al., 2001; Hermosilla et al., 2005). Therefore, these results provide an important insight into the possible role of AtMRP1 transporter function under AsIII stress condition. Competing interests The author declares no competing interests.

119

Author’s contribution Author did all experiments, drafted, read and approved the manuscript. Acknowledgements I acknowledge Council of Scientific and Industrial Research (CSIR), Government of India for providing me CSIR Senior Research Associateship under Pool Scheme No. 13(8380)/Pool/2010 and Department of Biotechnology, Government of India. I also acknowledge Prof. Dhrubajyoti Chattopadhyay, Department of Biotechnology, University of Calcutta, India for his constant support and encouragement. Also I am indebted to Prof. Philip. A. Rea, Department of Biology, University of Pennsylvania, Philadelphia, U.S.A for providing me AtMRP1 clone. I thank all members of Prof. Dhrubajyoti Chattopadhyay’s laboratory for their all-time help. I thank Dr. Anindita Seal, Department of Biotechnology, University of Calcutta, India for reviewing the manuscript. My thanks to professors and researchers of Department of Biotechnology, Biochemistry and Microbiology, University of Calcutta, India for their help and support. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.plaphy.2016.07.005. References Albuquerque, C.P., Smolka, M.B., Payne, S.H., Bafna, V., Eng, J., Zhou, H., 2008. A multidimensional chromatography technology for in-depth phosphoproteome analysis. Mol. Cell Proteomics 7, 1389e1396. Asher, C., Reay, P., 1979. Arsenic uptake by barley seedlings. Funct. Plant Biol. 6, 459e466. Bairoch, A., 1992. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 20 (Suppl. 2013e2018). Bienert, G.P., Schussler, M.D., Jahn, T.P., 2008. Metalloids: essential, beneficial or toxic? Major intrinsic proteins sort it out. Trends Biochem. Sci. 33, 20e26. Bleeker, P.M., Schat, H., Vooijs, R., Verkleij, J.A.C., Ernst, W.H.O., 2003. Mechanisms of arsenate tolerance in Cytisus striatus. New Phytol. 157, 33e38. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Chen, W., Chi, Y., Taylor, N.L., Lambers, H., Finnegan, P.M., 2010. Disruption of ptLPD1 or ptLPD2, genes that encode isoforms of the plastidial lipoamide dehydrogenase, confers arsenate hypersensitivity in Arabidopsis. Plant Physiol. 153, 1385e1397. Eraso, P., Martinez-Burgos, M., Falcon-Perez, J.M., Portillo, F., Mazon, M.J., 2004. Ycf1-dependent cadmium detoxification by yeast requires phosphorylation of residues Ser908 and Thr911. FEBS Lett. 577, 322e326. Geisler, M., Girin, M., Brandt, S., Vincenzetti, V., Plaza, S., Paris, N., Kobae, Y., Maeshima, M., Billion, K., Kolukisaoglu, U.H., Schulz, B., Martinoia, E., 2004. Arabidopsis immunophilin-like TWD1 functionally interacts with vacuolar ABC transporters. Mol. Biol. Cell 15, 3393e3405. Ghosh, M., Shen, J., Rosen, B.P., 1999. Pathways of As(III) detoxification in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U. S. A. 96, 5001e5006. Gietz, R.D., Schiestl, R.H., 1991. Applications of high efficiency lithium acetate transformation of intact yeast cells using single-stranded nucleic acids as carrier. Yeast 7, 253e263. Hermosilla, G.H., Tapia, J.C., Allende, J.E., 2005. Minimal CK2 activity required for yeast growth. Mol. Cell Biochem. 274, 39e46. Indriolo, E., Na, G., Ellis, D., Salt, D.E., Banks, J.A., 2010. A vacuolar arsenite transporter necessary for arsenic tolerance in the arsenic hyperaccumulating fern Pteris vittata is missing in flowering plants. Plant Cell 22, 2045e2057. Kanhonou, R., Serrano, R., Palau, R.R., 2001. A catalytic subunit of the sugar beet protein kinase CK2 is induced by salt stress and increases NaCl tolerance in Saccharomyces cerevisiae. Plant Mol. Biol. 47, 571e579. Kim, E.J., Zhen, R.G., Rea, P.A., 1995. Site-directed mutagenesis of vacuolar H(þ)pyrophosphatase. Necessity of Cys634 for inhibition by maleimides but not catalysis. J. Biol. Chem. 270, 2630e2635. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680e685. Li, X., Gerber, S.A., Rudner, A.D., Beausoleil, S.A., Haas, W., Villen, J., Elias, J.E., Gygi, S.P., 2007. Large-scale phosphorylation analysis of alpha-factor-arrested

120

A. Raichaudhuri / Plant Physiology and Biochemistry 108 (2016) 109e120

Saccharomyces cerevisiae. J. Proteome Res. 6, 1190e1197. Li, Y., Dhankher, O.P., Carreira, L., Lee, D., Chen, A., Schroeder, J.I., Balish, R.S., Meagher, R.B., 2004. Overexpression of phytochelatin synthase in Arabidopsis leads to enhanced arsenic tolerance and cadmium hypersensitivity. Plant Cell Physiol. 45, 1787e1797. Li, Z.S., Lu, Y.P., Zhen, R.G., Szczypka, M., Thiele, D.J., Rea, P.A., 1997. A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1catalyzed transport of bis(glutathionato)cadmium. Proc. Natl. Acad. Sci. U. S. A. 94, 42e47. Li, Z.S., Szczypka, M., Lu, Y.P., Thiele, D.J., Rea, P.A., 1996. The yeast cadmium factor protein (YCF1) is a vacuolar glutathione S-conjugate pump. J. Biol. Chem. 271, 6509e6517. Li, Z.S., Zhen, R.G., Rea, P.A., 1995. 1-Chloro-2,4-Dinitrobenzene-Elicited increase in vacuolar glutathione-S-conjugate transport activity. Plant Physiol. 109, 177e185. Liu, W., Schat, H., Bliek, M., Chen, Y., McGrath, S.P., George, G., Salt, D.E., Zhao, F.J., 2012. Knocking out ACR2 does not affect arsenic redox status in Arabidopsis thaliana: implications for as detoxification and accumulation in plants. PLoS One 7, e42408. Liu, W.J., Wood, B.A., Raab, A., McGrath, S.P., Zhao, F.J., Feldmann, J., 2010. Complexation of arsenite with phytochelatins reduces arsenite efflux and translocation from roots to shoots in Arabidopsis. Plant Physiol. 152, 2211e2221. Lu, Y.P., Li, Z.S., Rea, P.A., 1997. AtMRP1 gene of Arabidopsis encodes a glutathione Sconjugate pump: isolation and functional definition of a plant ATP-binding cassette transporter gene. Proc. Natl. Acad. Sci. U. S. A. 94, 8243e8248. Marin, O., Bustos, V.H., Cesaro, L., Meggio, F., Pagano, M.A., Antonelli, M., Allende, C.C., Pinna, L.A., Allende, J.E., 2003. A noncanonical sequence phosphorylated by casein kinase 1 in beta-catenin may play a role in casein kinase 1 targeting of important signaling proteins. Proc. Natl. Acad. Sci. U. S. A. 100, 10193e10200. Martinoia, E., Grill, E., Tommasini, R., Kreuz, K., Amrhein, N., 1993. ATP-dependent glutathione S-conjugate ‘export’ pump in the vacuolar membrane of plants. Nature 364, 247e249. Mehta, A., 2008. Cystic fibrosis as a bowel cancer syndrome and the potential role of CK2. Mol. Cell Biochem. 316, 169e175. Meng, Y.L., Liu, Z., Rosen, B.P., 2004. As(III) and Sb(III) uptake by GlpF and efflux by ArsB in Escherichia coli. J. Biol. Chem. 279, 18334e18341. Paumi, C.M., Chuk, M., Chevelev, I., Stagljar, I., Michaelis, S., 2008. Negative regulation of the yeast ABC transporter Ycf1p by phosphorylation within its N-terminal extension. J. Biol. Chem. 283, 27079e27088. Perez-Iratxeta, C., Andrade-Navarro, M.A., 2008. K2D2: estimation of protein secondary structure from circular dichroism spectra. BMC Struct. Biol. 8, 25. Raichaudhuri, A., Bhattacharyya, R., Chaudhuri, S., Chakrabarti, P., Dasgupta, M., 2006. Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains. J. Biol. Chem. 281, 10399e10409. Raichaudhuri, A., Peng, M., Naponelli, V., Chen, S., Sanchez-Fernandez, R., Gu, H., Gregory 3rd, J.F., Hanson, A.D., Rea, P.A., 2009. Plant vacuolar ATP-binding

cassette transporters that translocate folates and antifolates in vitro and contribute to antifolate tolerance in vivo. J. Biol. Chem. 284, 8449e8460. Rea, P.A., 2007. Plant ATP-binding cassette transporters. Annu. Rev. Plant Biol. 58, 347e375. Rea, P.A., Li, Z.S., Lu, Y.P., Drozdowicz, Y.M., Martinoia, E., 1998. From vacuolar GS-X pumps to multispecific ABC transporters. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 727e760. Roach, P.J., 1991. Multisite and hierarchal protein phosphorylation. J. Biol. Chem. 266, 14139e14142. Roosbeek, S., Peelman, F., Verhee, A., Labeur, C., Caster, H., Lensink, M.F., Cirulli, C., Grooten, J., Cochet, C., Vandekerckhove, J., Amoresano, A., Chimini, G., Tavernier, J., Rosseneu, M., 2004. Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter. J. Biol. Chem. 279, 37779e37788. Smolka, M.B., Albuquerque, C.P., Chen, S.H., Zhou, H., 2007. Proteome-wide identification of in vivo targets of DNA damage checkpoint kinases. Proc. Natl. Acad. Sci. U. S. A. 104, 10364e10369. Song, W.Y., Park, J., Mendoza-Cozatl, D.G., Suter-Grotemeyer, M., Shim, D., Hortensteiner, S., Geisler, M., Weder, B., Rea, P.A., Rentsch, D., Schroeder, J.I., Lee, Y., Martinoia, E., 2010. Arsenic tolerance in Arabidopsis is mediated by two ABCC-type phytochelatin transporters. Proc. Natl. Acad. Sci. U. S. A. 107, 21187e21192. Song, W.Y., Sohn, E.J., Martinoia, E., Lee, Y.J., Yang, Y.Y., Jasinski, M., Forestier, C., Hwang, I., Lee, Y., 2003. Engineering tolerance and accumulation of lead and cadmium in transgenic plants. Nat. Biotechnol. 21, 914e919. Stolarczyk, E.I., Reiling, C.J., Paumi, C.M., 2011. Regulation of ABC transporter function via phosphorylation by protein kinases. Curr. Pharm. Biotechnol. 12, 621e635. Szczypka, M.S., Wemmie, J.A., Moye-Rowley, W.S., Thiele, D.J., 1994. A yeast metal resistance protein similar to human cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance-associated protein. J. Biol. Chem. 269, 22853e22857. Treco, D.A., Winston, F., 2008. Growth and manipulation of yeast. Curr. Protoc. Mol. Biol. Chapter 13: Unit 13 12. Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J., 1982. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945e951. Wallace, I.S., Choi, W.G., Roberts, D.M., 2006. The structure, function and regulation of the nodulin 26-like intrinsic protein family of plant aquaglyceroporins. Biochim. Biophys. Acta 1758, 1165e1175. Wright, K., 1989. Antibodies a Laboratory Manual: by E Harlow and D Lane. Cold Spring Harbor Laboratory, p. 726, 1988. $50 ISBN 0-87969-314-2. Biochemical Education 17, 220e220. Xu, X.Y., McGrath, S.P., Zhao, F.J., 2007. Rapid reduction of arsenate in the medium mediated by plant roots. New Phytol. 176, 590e599. Zhao, F.J., Ma, J.F., Meharg, A.A., McGrath, S.P., 2009. Arsenic uptake and metabolism in plants. New Phytol. 181, 777e794.