Identification of the N-terminal region of TjZNT2, a Zrt ⁄ Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity Sho Nishida 1 , Yasuhiro Morinaga 2 , Hitoshi Obata 1 and Takafumi Mizuno 1 1 Graduate School of Bioresources, Mie University, Japan 2 Faculty of Bioresources, Mie University, Japan Introduction Heavy metal elements, such as iron, zinc, manganese, copper, molybdenum, and nickel, are essential in plants as catalytic cofactors or as structural elements of numerous proteins. They are transported into cells across biomembranes by transporter proteins. Recently, several heavy metal transporter families (e.g. COPT, NRAMP, P-type ATPase, CDF, and MOT) have been identified in plants [1–3]. The Zrt ⁄ Irt-like proteins (ZIPs) constitute one of the most important metal transporter families. ZIP transporters are pre- dicted to have eight transmembrane (TM) domains, with the N-terminal and C-terminal regions exposed to the extracellular surface, and have been demonstrated to transport divalent heavy metal ions into the cyto- plasm [4,5]. To date, many ZIP genes have been iso- lated and characterized in plants [1,2,6]. Most ZIP transporters display selectivity for Zn 2+ and ⁄ or Fe 2+ , but several members are also selective for Cu 2+ or Co 2+ [7–9]. ZIP transporters play critical roles in the uptake of these essential metal elements and mainte- nance of their appropriate distribution in plants. Most ZIP transporters also transport toxic metal ions, e.g. Cd 2+ or Hg 2+ , and therefore mediate the accumulation of these elements in plants [10–13]. In Keywords ion selectivity; manganese; metal transporter; zinc; ZIP family Correspondence T. Mizuno, Mie University, Graduate School of Bioresources, Kurimamachiya-cho 1577, Tsu, Mie 514-8507, Japan Fax: +81 59 231 9684 Tel: +81 59 231 9607 E-mail: tmizuno@bio.mie-u.ac.jp (Received 20 April 2010, revised 11 December 2010, accepted 24 December 2010) doi:10.1111/j.1742-4658.2011.08003.x The Zrt ⁄ Irt-like protein (ZIP) family of transporter proteins is involved in the uptake of essential metal elements in plants. Two homologous ZIP genes from Thlaspi japonicum, TjZNT1 and TjZNT2, encode products that share high amino acid sequence similarity except at the N-terminus and the cytoplasmic loop between transmembrane domains III and IV, and that have been shown to be Zn 2+ and Mn 2+ transporters, respectively. To iden- tify the region that determines the ion selectivity of these transporters, we constructed a series of TjZNT1 and TjZNT2 chimeric genes and assayed for the Zn 2+ uptake of yeast cells expressing them. As a result, the extra- cellular N-terminal ends were identified as regions involved in Zn 2+ selec- tivity. TjZNT2 possesses a 36 amino acid hydrophilic extension at its N-terminus that is absent in native TjZNT1, and a mutant TjZNT2 lacking the N-terminal extension was shown to possess Zn 2+ uptake activity. This suggests that the extended N-terminal region inhibits Zn 2+ transport by TjZNT2. Further studies showed that it is the first 25 amino acid region of the N-terminus that is important for the inhibition of Zn 2+ transport. Fur- thermore, the N-terminal truncated TjZNT2 lacked Mn 2+ uptake activity. These findings suggest that the N-terminal region is a novel substrate selector in the ZIP family of transporters. Abbreviations HA, hemagglutinin; HRD, histidine-rich domain; LZM, low-zinc medium; sGFP, synthesized green fluorescent protein; TM, transmembrane; YNB, yeast nitrogen base; ZIP, Zrt ⁄ Irt-like protein. FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 851 recent years, the accumulation of toxic metals in crops has become a major issue for food safety and human health [14]. To develop new technologies for reducing toxic metal levels in crops, it is essential to understand the molecular mechanisms of metal selectivity in ZIP transporters. As a representative study in this area, Rogers et al. reported that the extracellular loop between TM II and TM III was involved in the ion selectivity of the ZIP transporter AtIRT1, a high-affin- ity Arabidopsis thaliana iron transporter [15]. Previously, we cloned two ZIP genes, TjZNT1 and TjZNT2, from a nickel hyperaccumulator, Thlaspi japonicum. TjZNT1 and TjZNT2 have been identified as excess nickel resistance genes [16,17]. TjZNT1 and TjZNT2 share high sequence similarity (78% iden- tity), with the exception of the N-terminus and the TM III–IV loop. To examine the functions of TjZNT1 and TjZNT2, these transporters were tagged with hemagglutinin (HA) at their N-termini to verify their expression and subcellular localization by immu- noassay, and were expressed in Saccharomyces cerevi- siae. The TjZNT1 and TjZNT2 expressed were clearly different with regard to ion selectivity: TjZNT1 pos- sesses selectivity for both Zn 2+ and Mn 2+ , and TjZNT2 possesses selectivity for Mn 2+ [16,17]. This finding indicates that there are structural differences involved in the differences in ion specificity between TjZNT1 and TjZNT2. The TM III–IV loop is one of the least similar regions between the two proteins, and has histidine-rich domains (HRDs) that render it a potential metal-binding site of the ZIP transporter [18]. Indeed, our recent work showed that the HRDs of TjZNT1 may be involved in ion selectivity [19], and the sequences of the HRDs are apparently differ- ent between TjZNT1 and TjZNT2. Therefore, we speculated that the difference in ion specificity between TjZNT1 and TjZNT2 derived from the TM III–IV loop. We show here, however, that untag- ged TjZNT1 shows selectivity exclusively for Zn 2+ , whereas untagged TjZNT2 remains an Mn 2+ trans- porter, indicating that mutation of the N-terminus affects the ion selectivity of TjZNT1. This finding raises the possibility that differences in N-terminal structure are involved in the differential ion selectivity between TjZNT1 and TjZNT2. This study aimed to identify the regions involved in TjZNT1 and TjZNT2 ion selectivity. We mapped the regions of interest by use of a series of TjZNT1 and TjZNT2 chimeric proteins, and found that differences in Zn 2+ selectivity between the two proteins were asso- ciated with differences in the extracellular N-terminal structure. Furthermore, an N-terminal-truncated TjZNT2 was shown to have Zn 2+ selectivity. Our findings suggest that the N-terminal region is a novel ion selection site in the ZIP family of proteins. Results Subcellular localization of TjZNT1 and TjZNT2 in yeast Initially, differences in subcellular localization between TjZNT1 and TjZNT2 in yeast cells were studied. ZIP proteins are generally targeted to the plasma mem- brane [20,21], with a few exceptions [22,23]. TjZNT1 and TjZNT2 were shown to complement the functions of plasma membrane transporters of S. cerevisiae, ZRT1 and SMF1, respectively, indicating that TjZNT1 and TjZNT2 are localized in the plasma membrane. Protein subcellular localization prediction software, wolf psort [24], also indicated that TjZNT1 and TjZNT2 are likely to be targeted to the plasma mem- brane (data not shown). Previously, we have shown that TjZNT1 with synthesized green fluorescent protein (sGFP) fused to the C-terminus (TjZNT1::sGFP) local- izes not only at the plasma membrane but also at the endomembrane, owing to heterologous expression, in yeast cells [19]. To visually confirm the subcellular localization of TjZNT2 in yeast cells, the sGFP gene was fused to the 3¢-end of TjZNT2 and driven by the MET25 promoter in S. cerevisiae. Confocal laser scan- ning microscopy revealed that TjZNT2::sGFP was localized to the plasma membrane and endomembrane (Fig. 1), and showed similar fluorescence patterns to those of TjZNT1::sGFP. These results indicate that Fluorescence DIC Overlap TjZNT2 TjZNT1 sGFP Fig. 1. Subcellular localization of TjZNT1 and TjZNT2 in yeast cells. TjZNT1::sGFP, TjZNT2::sGFP and sGFP alone were expressed in BJ1824 cells, under control of the MET25 promoter. sGFP fluores- cence images (left), differential interference contrast (DIC) images (center) and overlapped images (right) are shown. Images were acquired with a laser scanning confocal microscope. Bar: 5 lm. N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al. 852 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS TjZNT1 and TjZNT2 are both targeted to the plasma membrane in yeast cells, and the different ion specifici- ties do not therefore derive from different membrane localizations. Mapping of the regions responsible for differences in Zn 2+ selectivity between TjZNT1 and TjZNT2 To determine the regions responsible for Zn 2+ selectiv- ity, TjZNT1 and TjZNT2 were divided into five parts on the basis of their amino acid sequence alignment (Fig. 2), and a series of chimeric constructs composed of the two genes were made in a systematic manner with a PCR strategy. In this study, the constructs were not tagged, because the functions of TjZNT1 and TjZNT2 may be affected by terminal tagging. As a rapid approach, the Zn 2+ selectivity of these alleles was determined by their ability to rescue the yeast zrt1 mutant that lacks the Zn 2+ uptake of ZRT1 and can- not survive in low-zinc medium (LZM), where Zn 2+ is limited by EDTA [15]. We confirmed that the wild- type strain (BY4741) could grow in LZM plates sup- plemented with 600 lm ZnCl 2 but the zrt1 mutant could not, and that both strains grew in LZM supple- mented with 1000 lm ZnCl 2 (data not shown). TjZNT1 and TjZNT2 also have Cd 2+ uptake activity (Fig. S1), and the expression of these transporters increases the Cd 2+ sensitivity of yeast cells. All strains transformed with the chimeric transporter genes showed increased sensitivity to Cd 2+ , as did the strains expressing TjZNT1 or TjZNT2, confirming that these chimeric transporters were functionally expressed as metal transporters in yeast (Fig. 3). Complementation assays showed that a dramatic change in phenotype occurred only when the N-termi- nal regions of the two proteins were exchanged. An in- frame exchange between the extracellular N-termini (Na region) conferred the ability to complement the zrt1 mutant on TjZNT2 (Fig. 3), and TjZNT1 with an in-frame fusion of the TjZNT2 N-terminus maintained its ability to complement the mutant. Exchange of the TM III–IV loop (L region), one of the least similar regions between the two proteins, did not affect the Zn 2+ selectivity of TjZNT1 or TjZNT2. In-frame exchange of high-similarity regions (Nb,Ca or Cb - region) also yielded chimeras that showed no change in their Zn 2+ selectivity. Inhibition of TjZNT2 Zn 2+ transport by its N-terminal region As described above, differences in ion selectivity between TjZNT1 and TjZNT2 were speculated to be caused by differences in the N-terminal regions of these proteins. TjZNT2 possesses a 36 amino acid hydrophilic extension at the N-terminus (Fig. 2), and has a second methionine at position 37 that is in a similar position to the TjZNT1 start codon. To clarify the effect of the N-terminal length on Zn 2+ selectivity, we constructed the N-terminally truncated TjZNT2 mutant and lacked the first 36 amino acids (DN36), and investigated its Zn 2+ uptake activity. It was found that DN36 complemented the zrt1 mutant (Fig. 4A), T jZNT1 1: MASSPTKILCDAGESDLCRDDAAAFLLKFVAIASIL T jZNT2 1:MFFIDVLWKLFPLYLFGSERDYLSETESILKIVPETMAAASSLSILCDAGEPDLCRDDSAAFLLKLVAIASIF T jZNT1 37:LAGVAGVAIPLIGKNRRFLQTEGNLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPDYPWSKFPFPGFFA T jZNT2 74:LAGAAGVAIPLIGRNRRFLQTDGSLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPEFPWKKFPFPGFFA T jZNT1 110:MVAALITLIVDFMGTQYYESKQQRNEVAGGGEAADVVEPGREETS-SVVPVVVERGNDDSKVFGEEDGGGMHI T jZNT2 147:MVAALITLLVDFMGTQYYEKKQEREATTHSGEQP SSGPEQSLGIVVPVAGEEGNDE-KVFGEEDSGGIHI T jZNT1 182:VGIRAHAAHHRHSHSNGHGTCDGH AHGQSHGHVHVHGSHDVENGARHVVVSQILELGIVSHSIIIGLS T jZNT2 216:VGIHAHAAHHTHNHTQGQSSCDGHSKIDIGHAHGHGHGHSHGGLELGNGARHVVVSQVLELGIVSHSIIIGIS T jZNT1 250:LGVSQSPCTIRPLIAALSFHQFFEGFALGGCISQAQFKNKSAIIMACFFALTTPIGIGIGTAVASSFNSHSPG T jZNT2 289:LGVSQSPCTIRPLIAALSFHQFFEGFALGGCISQAQFKNKPATIMACFFALTTPISIGIGTAVASSFNAHSVG T jZNT1 323:ALVTEGILDSLSAGILVYMALVDLIAADFLSKRMSCNLRLQVVSYVMLFLGAGLMSALAIWA T jZNT2 362:ALVTEGILDSLSAGILVYMALVDLIAADFLSKMMSCNFRLQIVSYLLLFLGSGLMSSLAIWT L Nα Nβ Cα Cβ Fig. 2. Sequence alignment between TjZNT1 and TjZNT2. The TM regions predicted by TOPPRED 2 (http://www.sbc.su.se/~erikw/toppred2/) are underlined. TjZNT1 and TjZNT2 were divided into five parts: Na1 ⁄ Na2 (1–7 amino acids ⁄ 1–44 amino acids), Nb1 ⁄ Nb2 (8–128 amino acids ⁄ 45–165 amino acids), L1 ⁄ L2 (129–223 amino acids ⁄ 166–266 amino acids), Ca1 ⁄ Ca2 (224–321 amino acids ⁄ 267–362 amino acids), and Cb1 ⁄ Cb2 (322–384 amino acids ⁄ 363–423 amino acids). These regions are shown as gray arrows above the sequences. S. Nishida et al. N-terminus of TjZNT2 is involved in ion selectivity FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 853 and measurement of the 65 Zn uptake of cells showed that the 65 Zn accumulation of cells expressing DN36 was 10-fold greater than that of control cells (Fig. 4B). These results indicate that truncated TjZNT2 possesses the ability to transport Zn 2+ , and that the extended N-terminal region inhibits that ability. To determine more precisely the region involved in Zn 2+ transport inhibition, three versions of the N-ter- minally truncated TjZNT2 mutants (DN10, DN15, and DN25) were constructed. DN10 was capable of comple- menting Zn 2+ uptake of the zrt1 mutant strain, although the cell growth observed under zinc-deficient conditions was considerably lower than that observed with cells expressing DN36 (Fig. 4A). The DN10 Zn 2+ uptake activity was 40% or less than that of DN36 (Fig. 4B). The strain expressing DN15 also showed sig- nificantly lower cell growth, and the Zn 2+ uptake activity was about 80% when compared with DN36. There was no significant difference in Zn 2+ uptake activity between DN25 and DN36. These results suggest that the first 25 amino acids are involved in inhibiting Zn 2+ transport. Involvement of the TjZNT2 N-terminus in Mn 2+ selectivity We examined the influence of N-terminal truncation of TjZNT2 on Mn 2+ selectivity. TjZNT2 complemented Mn 2+ uptake in the smf1 mutant, which lacks the Mn 2+ uptake of SMF1 and cannot survive in low-manganese medium [17], but DN36 did not show complementary Mn 2+ uptake (Fig. 5A). Manganese accumulation of an smf1 strain expressing TjZNT2 was significantly higher than that of the vector control cells, whereas a strain expressing DN36 showed no increase in manganese accumulation (Fig. 5B). The strain expressing DN36 had an increased zinc content pKT10 TjZNT1 TjZNT2 TjZNT1–L2 TjZNT2–L1 ZnCl 2 CdCl 2 1000 µM 600 µM 50 µM D 600 =10 –1 10 –2 10 –1 10 –2 10 –1 10 –2 TjZNT1–N 2 TjZNT2–N 1 TjZNT1–N 2 TjZNT2–N 1 TjZNT2–C 1 TjZNT1–C 2 TjZNT1–C 2 TjZNT2–C 1 Fig. 3. Mapping of the regions responsible for differences in Zn 2+ selectivity between TjZNT1 and TjZNT2. Chimera cDNAs were pro- duced with the overlap-PCR technique. The chimeric constructs are depicted as topology models; black and gray models represent TjZNT1 and TjZNT2, respectively. The pKT10 vector alone, TjZNT1, TjZNT2 or chimeric constructs were expressed in the zrt1 strain. Yeast cells were grown on LZM supplemented with 600 ⁄ 1000 l M ZnCl 2 or YNB medium supplemented with 50 lM CdCl 2 . Plates were incubated at 30 °C for 5 days. N36 N10 N15 N25 pKT10 ZnCl 2 CdCl 2 1000 µM 600 µM 50 µM D 600 = 10 –1 10 –2 10 –1 10 –2 10 –1 10 –2 TjZNT2 TjZNT1 N36 N25TjZNT2 65 Zn uptake (pmol 10 –6 cells·min –1 ) N10 N15pKT10 a a b b d c 1.0 0.8 0.6 0.4 0.2 0 1.2 1.4 1.6 TjZNT1 e AB Fig. 4. The effect of the TjZNT2 N-terminal truncation on Zn 2+ uptake activity. (A) TjZNT1, TjZNT2 or truncations (DN10, DN15, DN25, or DN36) were expressed in the zrt1 strain. Cells were grown on LZM supplemented with 600 ⁄ 1000 l M ZnCl 2 or YNB medium supplemented with 50 l M CdCl 2 . Plates were incubated at 30 °C for 5 days. (B) Yeast cell Zn 2+ uptake was measured with 65 Zn. Cells were incubated in LZM-EDTA containing 10 l M ZnCl 2 at 30 °C for 10 min. Data are means ± standard deviations of four independent experiments. Different letters indicate statistically significant differences (P < 0.01) between the strains, based on ANOVA (Tukey’s HSD). N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al. 854 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS as compared with the vector control strain, whereas the iron content was not significantly different among the strains (Fig. 5C,D). These data suggest that the Mn 2+ selectivity of TjZNT2 is lost by N-terminal truncation. Discussion The focus of the present study was to identify a novel region involved in ion selectivity in the ZIP family of transporters by structurally comparing TjZNT1 and TjZNT2, which display differential ion selectivities. To determine the region responsible for differences in ion selectivity, TjZNT1 and TjZNT2 chimeric genes were generated. We confirmed that these chimeric transport- ers were functionally expressed in yeast cells through assay of their Cd 2+ uptake, although this result does not guarantee that the chimeras are properly folded and fully active. Thus, it should be noted that the results from these chimeras could reflect artefacts. However, the approach lead to the identification of the extracellu- lar N-terminus as a substrate selector in TjZNT2. On alignment of the amino acid sequences of reported ZIP transporters, the length and sequence of the N-terminal region varied between members (data not shown), suggesting a relationship between N-terminal structure and ion specificity in other ZIP transporters. To our knowledge, this is the first report showing involve- ment of the N-terminus in ion specificity in ZIP trans- porters. TjZNT2 possesses a 36 amino acid hydrophilic exten- sion at its extracellular N-terminus that is not present in the sequence of TjZNT1. The present work provides several pieces of evidence for the involvement of this N-terminal region in the ion selectivity of TjZNT2 and related proteins. First, truncation of the extended N-terminus confers the ability to transport Zn 2+ on TjZNT2, which is not a property of the native protein. The TjZNT2 N-terminus therefore behaves as an ion transport autoinhibitory domain. Further studies showed that it is the first 25 amino acids that are important for this inhibition. Second, native TjZNT2 can transport Mn 2+ and Cd 2+ , whereas the truncated TjZNT2 lacks Mn 2+ transport activity. This indicates that the N-terminal region does not inactivate TjZNT2, but affects the ion selectivity of the protein. Finally, tagging with HA at the N-terminus of TjZNT1 was found to alter the ion selectivity of the protein, suggest- ing that modification of the N-terminal sequence directly affects the conformation of the ion-selectivity domain in ZIP transporters clustered with TjZNT2. As the N-terminal region contains no known metal- binding motifs, we speculate that it either interacts with other ion-selectivity regions in the protein, altering their AB pKT10 pKT10 TjZNT1 TjZNT2 N36 Wild-type smf1 MnCl 2 0 µM 50 µM D 600 = 10 –1 10 –3 10 –2 10 –1 10 –3 10 –2 pKT10 TjZNT2 N36 Zn accumulation (ng· g –1 · DW) Mn accumulation (ng· g –1 · DW) Fe accumulation (ng· g –1 · DW) 350 300 200 100 0 a a b 50 150 250 CD 100 80 60 40 20 0 aaa 120 pKT10 TjZNT2 N36 pKT10 TjZNT2 N36 12 10 8 6 4 2 0 a b c Fig. 5. The effect of the TjZNT2 N-terminal truncation on Mn 2+ uptake activity. (A) Wild- type (BY4741) and smf1 strains were grown on YNB medium supplemented with 2% galactose, 20 m M EGTA, 0 ⁄ 50 lM MnCl 2 and 50 mM Mes at pH 6.0. Plates were incubated at 30 °C for 5 days. (B–D) Cells were cultured at 30 °C to exponential phase in YNB medium supplemented with 50 l M MnCl 2 and 50 mM Mes at pH 6.0. Data are means ± standard deviations of four inde- pendent experiments. Different letters indicate statistically significant differences (P < 0.01) between the strains, based on ANOVA (Tukey’s HSD). DW, dry weight. S. Nishida et al. N-terminus of TjZNT2 is involved in ion selectivity FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS 855 conformation, or it constitutes a part of the ion-selec- tivity domain with other neighboring regions. The lat- ter proposal is supported by the presence of several charged residues that could directly interact with the substrate in the 25 amino acid N-terminus. Fusion of the TjZNT2 N-terminal region with the N-terminus of TjZNT1 did not inhibit the latter’s ability to trans- port Zn 2+ (Fig. 3). This suggests that the N-terminus of TjZNT2 interacts specifically with other regions in the protein that are not present in TjZNT1. In addi- tion, the exchange of the Nb,L,Ca and Cb regions between TjZNT1 and TjZNT2 did not cancel the auto- inhibition of Zn 2+ transport of TjZNT2 suggesting that the N-terminus may interact with multiple regions. To obtain further evidence, we are presently assessing the alteration of ion selectivity by use of a synthetic peptide to mimic the extracellular N-terminus of TjZNT2 as a biological approach. Further studies are in progress to reveal the ion selection mechanism for the N-terminus in greater detail. Two further regions were considered as candidates for mediating the difference in ion selectivity between TjZNT1 and TjZNT2: the TM III–IV loop, one of the least similar regions; and the TM II–III loop, identified as an ion-selective region in AtIRT1. However, exchanging these regions between TjZNT1 and TjZNT2 did not affect their Zn 2+ selectivity. As described above, the difference in ion selectivity between TjZNT1 and TjZNT2 primarily derives from the N-terminus. Indeed, there are no differences in ion specificity between TjZNT1 and truncated TjZNT2, and we conclude that these loops do not play a major role in ion selectivity in the case of TjZNT1 and TjZNT2, although they may play secondary roles in combination with the N-terminal region. Here, we report that the ion selectivity of a ZIP transporter can be controlled by modification of the N-terminus. Our findings may have relevance in the development of techniques for the reduction of toxic metal levels in crops, or the generation of plants that accumulate specific toxic metals for phytoremediation. We plan to confirm the generality of N-terminus involvement in the ion selectivity of ZIP transporters. The present study has also provided important clues regarding the function of TjZNT2. TjZNT1 and TjZNT2 are closely clustered with Thlaspi caerulescens TcZNT1 and TcZNT2, as well as Arabidopsis halleri ZIP4 and IRT3, which have all been suggested to play a critical role in metal accumulation in hyperaccumula- tor species [25–30]. It has also been suggested that TjZNT2 is involved in nickel hyperaccumulation in T. japonicum [17], but the true function of TjZNT2 in T. japonicum remains unclear. Currently, we are trying to determine the function of TjZNT2 through the study of transcript regulation as a molecular biological approach and the determination of the N-terminal sequence as a biochemical approach. Experimental procedures DNA manipulations The primers used in this study are summarized in Table S1. TjZNT1 and TjZNT2 chimeras were produced by a stan- dard overlap extension technique. PCR products were cloned into the pTAII vector (Toyobo Co., Ltd, Osaka, Japan), and subcloned into the pKT10-Gal-HA-BS yeast expression vector at the EcoRI–SalI sites to eliminate HA tags. Without a stop codon, TjZNT2 was subcloned into pSNM4 [19] at the EcoRI–PvuII site to express protein fused with sGFP at the C-terminus. All PCR-derived DNA clones were sequenced. Escherichia coli and yeast transfor- mations were performed with standard methods. Yeast strains and growth conditions The strains used in the metal uptake assays were: BY4741 (MATa his3 leu2 met15 ura3), zrt1 (BY4741 zrt1::KanMX4), smf1 (BY4741 smf1::kanMX4), and the BJ1824 strain (MATa leu2 ura3 trp1 pep4 cir + ). The BY4741 series was obtained from the European S. cerevisiae Archive for Func- tional Analysis (Frankfurt, Germany). The strains were grown in yeast nitrogen base (YNB) medium (0.67% yeast nitrogen base without amino acids) supplemented with appropriate amino acids. Complementation tests of metal uptake mutants and Cd 2+ sensitivity test For the complementation test for the zrt1 mutant, LZM [31] supplemented with ZnCl 2 (0 or 600 lm) and adjusted to pH 6.0 was used. The complementation test for the smf1 mutant was performed following the methods of Thomine et al. [32], using medium containing 20 lm EGTA, MnCl 2 (0 or 50 lm), and 50 mm Mes (pH 6.0). For the Cd 2+ sen- sitivity test, YNB medium supplemented with 50 mm Mes and 50 lm CdCl 2 at pH 6.0 was used. Yeast cultures at the exponential phase were diluted and then spotted onto each assay plate. Cells were observed following a 5–7-day incu- bation at 30 °C. All solid plate media contained 2% agar. All assay media contained 2% galactose to induce Gal1 promoter expression. sGFP observation Strains transformed with TjZNT1-pSNM4 and the pSNM4 empty vector were generated as described previously [19]. N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al. 856 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS The strains were cultured to an exponential phase in YNB medium at 30 °C, and imaged with a laser scanning confo- cal microscope (FV 1000; Olympus, Tokyo, Japan). Metal accumulation assay The Cd 2+ accumulation assay was performed as described in our previous report [19], with the exception that the Cd 2+ concentration in the cells was not corrected for dry weight but for cell number. For the Mn 2+ accumulation assay, yeast cells were cultured to exponential phase (D 600 nm of 0.8–0.9) in YNB medium supplemented with 50 lm MnCl 2 and 50 mm Mes at pH 6.0. The Mn 2+ con- centration was determined as described previously [16]. Measurements of mineral concentrations were made by inductively coupled plasma atomic emission spectrometry (ICPS-7500; Shimadzu Corp., Kyoto, Japan). 65 Zn uptake assay The 65 Zn uptake assay was conducted following the method of Eide et al. [33], except that 65 ZnCl 2 and LZM-EDTA (LZM without EDTA) were substituted for 59 FeCl 3 and low-iron medium–EDTA, respectively. LZM-EDTA was supplemented with 2% galactose and 10 lm ZnCl 2 , and adjusted to pH 6.0. Cells in the YNB medium (2% galac- tose) at the exponential phase (D 600 nm of 0.8–0.9) were harvested, washed twice, and resuspended in an ice-cold assay buffer (LZM-EDTA). Then, the attenuance of the cell suspensions was measured. Cells were incubated in the assay buffer containing 65 ZnCl 2 for 10 min at 30 °C and for 2 min on ice, collected on Ultrafree MC (pore size, 0.5 lm; Millipore, Billerica, MA, USA), and washed with ice-cold SSW (1 mm EDTA, 20 mm Na 3 -citrate, pH 4.2, 1mm KH 2 PO 4 ,1mm CaCl 2 ,5mm MgSO 4 , and 1 mm NaCl). 65 Zn concentration was determined on a c-counter. Acknowledgements We are grateful to D. R. Fernando (University of Mel- bourne) for critical reading of the manuscript. 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Supporting information The following supplementary material is available: Fig. S1. Cadmium accumulation of yeast strains expressing TjZNT1 and TjZNT2. Table S1. Primers used for the PCR amplifications. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. N-terminus of TjZNT2 is involved in ion selectivity S. Nishida et al. 858 FEBS Journal 278 (2011) 851–858 ª 2011 The Authors Journal compilation ª 2011 FEBS . Identification of the N-terminal region of TjZNT2, a Zrt ⁄ Irt-like protein family metal transporter, as a novel functional region involved in metal ion selectivity Sho Nishida 1 , Yasuhiro. This indicates that the N-terminal region does not inactivate TjZNT2, but affects the ion selectivity of the protein. Finally, tagging with HA at the N-terminus of TjZNT1 was found to alter the ion. 1:MFFIDVLWKLFPLYLFGSERDYLSETESILKIVPETMAAASSLSILCDAGEPDLCRDDSAAFLLKLVAIASIF T jZNT1 37:LAGVAGVAIPLIGKNRRFLQTEGNLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPDYPWSKFPFPGFFA T jZNT2 74:LAGAAGVAIPLIGRNRRFLQTDGSLFVAAKAFAAGVILATGFVHMLAGGTEALTNPCLPEFPWKKFPFPGFFA T jZNT1