Histidine pairing at the metal transport siteof mammalian ZnT transporters controlsZn2þ over Cd2þ selectivityEitan Hocha,1, Wei Linb,1, Jin Chaib, Michal Hershfinkelc, Dax Fub,2, and Israel Seklera,2

aDepartment of Physiology, Morphology, Faculty of Health Sciences, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel;cDepartment of Morphology, Ben-Gurion University of the Negev, P.O.B. 653, Beer-Sheva 84105, Israel; and bBrookhaven National Laboratory,Upton, NY 11973

Edited by H. Ronald Kaback, UCLA, Los Angeles, CA, and approved March 8, 2012 (received for review January 8, 2012)

Zinc and cadmium are similar metal ions, but though Zn2þ is anessential nutrient, Cd2þ is a toxic and common pollutant linked tomultiple disorders. Faster body turnover and ubiquitous distribu-tion of Zn2þ vs. Cd2þ suggest that a mammalian metal transporterdistinguishes between these metal ions. We show that the mam-malian metal transporters, ZnTs, mediate cytosolic and vesicularZn2þ transport, but reject Cd2þ, thus constituting the first mamma-lian metal transporter with a refined selectivity against Cd2þ.Remarkably, the bacterial ZnT ortholog, YiiP, does not discriminatebetween Zn2þ and Cd2þ. A phylogenetic comparison between thetetrahedral metal transport motif of YiiP and ZnTs identifies ahistidine at the mammalian site that is critical for metal selectivity.Residue swapping at this position abolished metal selectivity ofZnTs, and fully reconstituted selective Zn2þ transport of YiiP. Finally,we show thatmetal selectivity evolves through a reduction in bind-ing but not the translocation of Cd2þ by the transporter. Thus, ourresults identify a unique class of mammalian transporters and thestructural motif required to discriminate between Zn2þ and Cd2þ,and show that metal selectivity is tuned by a coordination-basedmechanism that raises the thermodynamic barrier to Cd2þ binding.

Cd transport ∣ metal binding site ∣ zinc ∣ Zn transporter ∣ Cd toxicity

Zn2þ and Cd2þ are both d10 closed shell metals with similarouter electronic structures. However, while Zn2þ is an essen-

tial micro nutrient (1), Cd2þ is a common environmental pollu-tant associated with severe toxicity and linked to many disorders,including hypertension, cancer, infertility, thyroid, renal, andbone diseases (2–4). Due to their chemical similarity, Cd2þcan exploit Zn2þ uptake routes to enter cells through, for exam-ple, Zn2þ influx transporters such as ZIPs (5–7). Similarly,Metallothioneins, the major cellular metal buffering proteins,bind both Cd2þ and Zn2þ, and the divalent metal transporter 1(DMT1) catalyzes Hþ cotransport of Cd2þ and Zn2þ as well asother heavy metals (8–10). In plants, most of the Heavy metalATPases (HMA) classified P-type ATPase share the same nonse-lective metal ions transport (11). Although bacterial metal pumpsHMA1 and plant OsHMA3 are selective for Zn2þ over Cd2þ andtheir selectivity is associated with transmembrane domains (12,13), the structural basis for their metal selectivity is not fullyunderstood.

Mammalian cells do not possess such a wide repertoire ofheavy metal pumps and only express the ATP7A and ATP7BP-type cupper selective pumps (14). Yet once inside mammaliantissues, Cd2þ is trapped with a retention half-time of more than30 y (15) in a tissue restricted manner whereas Zn2þ undergoesrapid bodily dissemination through a sequence of secretion andreabsorption processes (16). This difference in the extrusion ratesof Zn2þ and Cd2þ suggests that in addition to Cd2þ bufferingby metallothioneins (MTs), a mammalian class of metal transpor-ters may selectively mediate Zn2þ transport while leaving Cd2þtrapped.

Here, we have focused on mammalian ZnTs, a major class ofZn2þ transporters that extrude Zn2þ out of the cytoplasm eitherdirectly to the extracellular medium or into intracellular vesiclesdestined to Zn2þ secretion (17, 18) and show that ZnTs candiscriminate between Zn2þ and Cd2þ. By carrying a functionalcomparative analysis of ZnTs with their bacterial ortholog YiiP,we further identify the structural motif and transport step re-quired for such metal selectivity.

ResultsTo investigate whether ZnTs can discriminate for Zn2þ againstCd2þ, we first compared the cytoplasmic efflux of Zn2þ or Cd2þin HEK293-Tcells expressing human ZnT5 (19). Consistent withprevious results (18), ZnT5 was expressed in the Golgi membranewhere it mediates robust Zn2þ efflux from the cytoplasm to theGolgi, as demonstrated by a decline of the fluorescence signalemitted by Flouzin-3AM, a cytoplasmic Zn2þ indicator (Fig. 1A).The rate of Zn2þ efflux by ZnT5 was fourfold higher than thebackground signal obtained for control cells transfected withan empty expression vector, pcDNA (Fig. 1C). Cytoplasmic Cd2þefflux was similarly examined (Fig. 1B) using Fura-2AM, aconventional Ca2þ indicator, which is effectively and frequentlyused to monitor intracellular Cd2þ because of its high Cd2þaffinity (20). In contrast to the robust Zn2þ efflux, no increase incytoplasmic Cd2þ efflux was observed in cells expressing ZnT5(Fig. 1C). The ZnT5-mediated Zn2þ efflux from the cytoplasmis coupled to vesicular uptake (18). We therefore monitoredvesicular uptake of Zn2þ and Cd2þ (Fig. 1D) using Zinpyr-1, adual Zn2þ and Cd2þ fluorescence indicator that is targeted tothe Golgi (21). A sevenfold increase in Zn2þ uptake was moni-tored in ZnT5 expressing cells, whereas no Cd2þ accumulationwas observed above the background level (Fig. 1E). Thus, thevesicular uptake data corroborated the cytoplasmic efflux results,showing that ZnT5 transports Zn2þ, but rejects Cd2þ. To furtherinvestigate whether other ZnT orthologs can discriminate be-tween Zn2þ and Cd2þ, we next examined Zn2þ and Cd2þ effluxand vesicular uptake in HEK293-T cells expressing human ZnT8(22, 23). No changes in Cd2þ transport were observed in cellsexpressing ZnT8 compared to control cells, although ZnT8expression led to a sixfold increase in both Zn2þ efflux and ve-sicular uptake (Fig. S1). Altogether, our experiments identifiedZnTs as a unique class of mammalian transporters with a refinedselectivity for Zn2þ against Cd2þ.

Author contributions: E.H., D.F., and I.S. designed research; E.H., W.L., and J.C. performedresearch; E.H., W.L., M.H., D.F., and I.S. analyzed data; and M.H., D.F., and I.S. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1E.H. and W.L. contributed equally to this work.2To whom correspondence may be addressed. E-mail: dax@bnl.gov or sekler@bgu.ac.il.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1200362109/-/DCSupplemental.

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The chemical similarity shared by Zn2þ and Cd2þ poses amajor catalytic challenge for the selection of Zn2þ against Cd2þby ZnTs. Intriguingly, the bacterial ZnTortholog, YiiP, transportsboth Zn2þ and Cd2þ with equal specificity (24). The crystal struc-ture of YiiP revealed a tetrahedral transport site with a DD–HDresidue composition (25) differing by a single residue from theHD–HD transport site (18) of mammalian ZnT orthologs(Fig. 2). We therefore examined how this single residue alterationcould affect transport specificity using an H-to-D mutation toconvert the ZnT transport site to a YiiP-like one. Expressionof either mutant, ZnT5H451D or ZnT8H106D, showed similar ex-pression patterns (Figs. S2 and S3), with cytoplasmic Zn2þ trans-port activity (Fig. 3B) and vesicular Zn2þ uptake (Fig. S4)unchanged when compared to either wild-type ZnT5 or ZnT8,respectively. However, in sharp contrast to leveled cytoplasmicCd2þ signals observed in cells expressing ZnT5 or ZnT8, expres-sion of the ZnT5H451D and ZnT8H106D mutants led to rapid Cd2þefflux from the cytoplasm (Fig. 3A) with a fivefold to sixfoldincrease respectively in the Cd2þ efflux rate above the basal levels(Fig. 3B). Mirroring the rapid efflux of cytoplasmic Cd2þ, a five-fold to fourfold increase in vesicular Cd2þ uptake was observed

in cells expressing ZnT5H451D or ZnT8H106D respectively, but notin cells expressing ZnT5 or ZnT8 (Fig. 3 C and D). Hence, ourresults indicate that an H-to-D substitution in the transport sitesof human ZnT5 and ZnT8 abolished their selectivity againstCd2þ, but had no effect on Zn2þ transport.

To further examine the conservation of the metal-selectivitymechanism, we then asked if this single coordination residuewould be sufficient to fully reconstitute Zn2þ selectivity againstCd2þ in the bacterial ortholog YiiP. We introduced a D-to-H

Fig. 1. Zn2þ transport selectivity against Cd2þ (A) Normalized Fluozin-3fluorescence changes in response to Zn2þ loading and withdrawal as indi-cated. The blue and red trace is recorded from HEK293-T cells transfectedwith wild-type ZnT5 and pcDNA control vector, respectively. The Inset showsa rapid ZnT5-mediated Zn2þ efflux from the cytoplasm upon Zn2þ with-drawal. (B) Normalized Fura-2 fluorescence changes in response to Cd2þ load-ing and withdrawal. TheInset shows no difference in Cd2þ effluxes recordedfrom the ZnT5-expressing cells (blue trace) and the control (red trace). (C)Rates of ZnT5-mediated Zn2þ and Cd2þ efflux normalized to the control rateas indicated (mean� s:e:, n ¼ 3). (D) Zinpyr-1 fluorescence imaging of vesi-cular Zn2þ (Top Boxes) and Cd2þ (Lower Boxes) uptake in HEK293-T cellstransfected with ZnT5 (Right Box) and control vector (Left Box). (E) ZnT5-mediated vesicular uptake of Zn2þ and Cd2þ normalized to control(mean� s:e:, n ¼ 6). (*P ≤ 0.01).

Fig. 2. Zn2þ binding site. (A) Sequence alignment of YiiP and human ZnTsas indicated. The coordination residues of the transport site are highlightedin yellow for three invariant positions, and in green/purple for the fourthposition that alternates between a histidine and aspartate, respectively.(B) Comparison between the metal binding sites of mammalian ZnTs (18) andbacterial YiiP (25). Histidine residues are colored red and aspartic acid resi-dues are colored blue.

Fig. 3. A selectivity determinant residue for Zn2þ over Cd2þ. (A) Fura-2 fluor-escence in response to Cd2þ withdrawal as described in Fig 1B. The red andblue traces were recorded from HEK293-T cells expressing ZnT5 andZnT5H451D (Top Box) and ZnT8 and ZnT8H106D (Lower Box), respectively. (B)Relative Zn2þ and Cd2þ efflux rate (mean� s:e:, n ¼ 3) normalized to the raterecorded from HEK293-T cells transfected with control vector. (C) Zinpyr-1fluorescence imaging of vesicular Cd2þ uptake in HEK293-T cells transfectedwith ZnT5 (Top Left Box) and ZnT5H451D (Top Right Box) or ZnT8 (Lower LeftBox) and ZnT8H106D (Lower Right Box). (D) Vesicular Cd2þ uptake (mean�s:e.,n ¼ 6) normalized to the total Cd2þ uptake measured from HEK293-T cellstransfected with control vector. Note that a single H-to-D mutation in bothZnT5 and ZnT8 causes a loss of selectivity against Cd2þ. (*P ≤ 0.01).

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mutation in the transport-site of YiiP. Both wild-type YiiPD45 andmutated YiiPD45H were overexpressed, purified, and reconsti-tuted into proteoliposomes with an encapsulated fluorescenceindicator, Fluozin-1 (26). Exposing proteoliposomes to 10 μMextravesicular Zn2þ evoked a rapid increase in Fluozin-1 fluor-escence for both YiiPD45 and YiiPD45H in an EDTA reversiblemanner (Fig. 4A, Fig. S5). The rates of Zn2þ uptake for YiiPD45

and YiiPD45H remained largely unchanged (Fig. 4C). In contrast,the rate of Cd2þ transport by the ZnT-like YiiPD45H was reducedby 10-fold, under otherwise identical conditions (Fig. 4 B and D).The gain of Zn2þ selectivity over Cd2þ by a D-to-H mutationreplicated the native selectivity of ZnT5 and ZnT8 in YiiP,indicating that a single histidine residue constitutes a determinantfor Zn2þ selectivity over Cd2þ. Thus, the on-and-off switchingof Cd2þ transport by a single residue substitution demonstratesremarkable functional fidelity of the selectivity determinant frombacteria to human.

The transport site revealed in the YiiP crystal structure istailored to rapid Zn2þ binding and release by completely detach-ing its four coordination residues from outer-shell interactions(27). In agreement with the structure of a standalone transportsite, switching on and off selectivity against Cd2þ by a D-to-H andH-to-D mutation suggested that these mutations are limited toaffect a direct ligand-metal interaction in the inner coordinationshell. In sharp contrast to the marked effect on Cd2þ transport,neither mutation caused detectable change to Zn2þ transport,underscoring the minimal mutagenic perturbation to the trans-port site. To evaluate the effect of a D-to-H substitution on thebinding properties of the transport site, isothermal titrationcalorimetry was used to directly measure Zn2þ and Cd2þ bindingto purified YiiPD45 and YiiPD45H (Fig. S6). As described pre-viously, titrations of wild-type YiiPD45 yielded exothermic-to-endothermic heat transitions for Zn2þ binding and a purelyexothermic reaction for Cd2þ binding (28). The binding iso-therms for YiiPD45H displayed similar heat reaction profiles. YiiPcontains at least two fully titratable binding sites. Accordingly, a

two-component model was used for data fitting and interpreta-tion (24). Component-2 was assigned to a cytoplasmic bindingreaction with very similar binding affinities, stoichiometry andenthalpy changes for YiiPD45 and YiiPD45H (Table S1). Compo-nent-1 of the Zn2þ binding isotherm resulted from a mixed heatreaction associated with the transport site and an unresolvedcytoplasmic site. Again, a comparison of the fitting parametersfor Component-1 show insignificant differences between YiiPD45

and YiiPD45H for Zn2þ binding (Table 1). On the other hand,Cd2þ binding to the transport site could be unambiguously as-signed to Component-1 (24), showing a 33-fold affinity decreasefor the D45H mutated site (Table 1). Cd2þ affinity for Compo-nent-2 remained unchanged, demonstrating the specific effect ofthe D-to-H mutation on the transport site (Table S1). Taken to-gether, the binding data indicated that Zn2þ could effectivelybind to both DD–HD and HD–HD transport-sites, but Cd2þ fa-vored the DD–HD over the HD–HD transport site. Thus it is thedistinct effect of an additional histidine residue on Zn2þ vs. Cd2þbinding that gives rise to a refined Zn2þ selectivity over Cd2þ.

Previous studies suggest that metal transport by the bacterialZnTortholog, YiiP (28, 29), similarly to other cation transporters(30), can be described by a two-step kinetic scheme, initiatingby metal binding to the transport site and triggering a transloca-tion of the bound metal ion across the membrane. To determineif the refined Zn2þ selectivity over Cd2þ is linked to metal ionbinding or also affects the ion translocation step, we comparedthe kinetics of metal translocation occurring at the DD–HD andHD–HD transport sites. Purified YiiPD45 or YiiPD45H were re-constituted into proteoliposomes encapsulated with Fluozin-1.To override the reduced Cd2þ binding of YiiPD45H, we carriedout stopped-flow measurements of the initial rate of metal trans-port performed at a concentration range (0.2–2 mM), far abovethe Cd2þ concentrations encountered in vivo (31). As such, bothYiiPD45 and YiiPD45H were found to mediate both rapid Zn2þand Cd2þ fluxes. A D-to-H mutation increased the steady-stateMichaelis—Menten constant Km value for Cd2þ by 6.3-fold, butcaused no change to the Km for Zn2þ within experimental errors(Fig. 5A). The changes inKm values were parallel to the effects ofthe D-to-H mutation on Cd2þ and Zn2þ binding as describedabove. Furthermore, the D-to-H mutation increased the maxi-mum velocity(Vmax) of Cd2þ transport by sixfold as compared tothat of Zn2þ transport (Fig. 5A), showing a correlation between afaster release of a bound Cd2þ to the trans-membrane side and aweakening affinity of Cd2þ binding from the cis-membrane side.The rate of the cis-to-trans accessibility flipping of the transportsite (or transport catalytic efficiency) is determined by Vmax∕Km(32). The concomitant increases of both Vmax and Km for Cd2þtransport yielded a largely unchanged Vmax∕Km for YiiPD45H, in-dicating that although Cd2þ binding was reduced, Cd2þ translo-cation efficiency remained intact. Thus, the D-to-H mutation hadno effect on Cd2þ translocation once a Cd2þ ion was pushed overthe thermodynamic barrier to enter the transport site.

The dissection of the two elementary steps of the transportreaction localized the effect of the D-to-H mutation to metalbinding only. The ensuing metal translocation step is driven by aglobal conformational change involving residues outside of thetransport site (25). The structural detachment of the transport

Fig. 4. Functional conservation of the selectivity determinant in YiiP. (A)Normalized Fluozin-1 fluorescence in response to extravesicular Zn2þ expo-sure and EDTA addition as indicated. Fluorescence signals recorded from YiiP(red trace) or YiiPD45H (blue trace) proteoliposomes were normalized to theirrespective basal levels to remove the contribution of small differences inproteoliposome-loading. (B) Normalized Fluozin-1 fluorescence in responseto extravesicular Cd2þ exposure and EDTA addition. (C) Zn2þ uptake rate ofYiiP and YiiPD45H (mean� s:e:, n ¼ 4), calculated using the fluorescence risingphase in Fig 3A. (D) Cd2þ uptake rate of YiiP and YiiPD45H (mean� s:e:,n ¼ 4). Note that a single D-to-H mutation confers Zn2þ selectivity over Cd2þ.(* relative to control; P ≤ 0.01, # relative to YiiP; P ≤ 0.01).

Table 1. Zn2þ and Cd2þ binding parameters, determined bycalorimetric metal titrations of purified YiiP and YiiPD45H

Component-1

Metal Protein N Ka (μM−1) ΔH (Kcal∕mol)

Zn2þ YiiP D45H 1.9 ± 0.1 0.7 ± 0.5 −4.0 ± 0.3YiiP D45 1.6 ± 0.1 1.7 ± 1.6 −6.1 ± 0.6

Cd2þ YiiP D45H 0.7 ± 0.1 152 ± 68 −1.7 ± 0.1YiiP D45 0.8 ± 0.1 4.6 ± 1.8 −0.6 ± 0.7

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site from its protein surrounding supports our findings that theD-to-H mutation is decoupled to influence Cd2þ translocation.This highly specific effect on the binding step of the transportreaction provided strong evidence that the four coordination re-sidues of the transport site are sufficient to dictate the selection ofa central metal ion in the tetrahedral complex. Thus, YiiP offereda valid model to investigate the general mechanism of transportspecificity for a broad spectrum of metal ions. To determine themetal transport specificity of the tetrahedral transport site, weexposed YiiP proteoliposomes to a panel of divalent metal ions,stored under reducing conditions to minimize their oxidation(described in Materials and Methods), each at an extravesicularconcentration of 0.2 mM (Fig. 5B). The steady-state measure-ments of metal uptake by inductively coupled plasma mass spec-troscopy (ICP-MS) showed that YiiP transported only Zn2þ andCd2þ, but rejected all transition metal ions in the fourth period(Fig. 5B). This finding is consistent with previous studies demon-strating Zn2þ and Cd2þ transport by YiiP and the strict transitionmetal selectivity of mammalian ZnTs (24, 33). Our data thereforeargues against a size-based selectivity mechanism used, for exam-ple, by the potassium channels to discriminate between Kþ andNaþ (34).

DiscussionOur study identifies a unique mammalian transporter with a re-fined Zn2þ selectivity against Cd2þ. The discrimination of thesetwo closely related posttransition metal ions is accomplished by amutational drift in the evolution of the transport site. Of the fourcoordination residues in the transport site, three are invariantfrom bacteria to human. The fourth residue alternates betweenan aspartate and a histidine in bacteria, but is converged to ahistidine in mammalian ZnT transporters (35). Zn2þ and Cd2þare different in their ionic radii, but they share a strong prefer-ence for tetrahedral coordination (36). This common chemicalproperty matches the tetrahedral configuration of the transportsite (27). Therefore, YiiP may exploit coordination chemistry toselect for Zn2þ and Cd2þ against other transition metal ions thatprefer octahedral coordination. Although coordination chemistryrepresents the first line of selection against chemically mis-matched metal ions, our data show that the selectivity could be

further refined to discriminate for Zn2þ over Cd2þ by a histidinedeterminant in the transport site. This remarkable tuneability de-monstrates the molecular precision of the coordination-basedmechanism in regulating metal selectivity. Notably, various plantZnT holomogous manifest less stringent metal selectivity andtransport other metals, for example Co2þ (37). Interestingly ZnTsmetal selectivity has been linked to a cytoplasmic His-rich do-main. Although the basis for this difference in metal selectivityof plant and mammalian ZnT homologues is unclear, it is tempt-ing to speculate that their cytosolic metal buffering domainmay increase the local metal concentration above the thresholdrequired for selective transport, thus forcing subsequent Co2þtranslocation.

A role for histidine residues in enhancing Zn2þ selectivity isconsistent with their role in metal-binding proteins. For example,mammalian MT isoforms that employ a four-Cysteine site bindCd2þ with affinities that are two orders of magnitude higher thanZn2þ, but the metal binding domains of some plant MTs, com-prised of one cysteine and two histidine residues, show enhancedselectivity to Zn2þ (38). Similarly, a two-histidine, two-cysteinesite in Zn2þ finger domains shows preference for Zn2þ bindingcompared to a nonselective four-cysteine site. We propose thatthe two aspartates and two histidine residues in the tetrahedralZnT transport site combine the properties of histidine pairingrequired for enhanced Zn2þ selectivity coupled with the acidicresidues that allow rapid cation dissociation and Hþ exchangeshared, for example, with Ca2þ-pumps (39).

In vivo, ZnTs operate under strict substrate-limiting conditionsas cytoplasmic Zn2þ and Cd2þ are heavily buffered and hencetheir free concentration is low. Thus, the histidine substitutionat the transport site specifically affects the binding step of the trans-port reaction, capitalizing on the in vivo metal limiting condition toachieve an effective selectivity for Zn2þ against Cd2þ. The refinedZnTselectivity entails that once Zn2þ and Cd2þ permeate throughthe nonselective ZIP or DMT pathway, Cd2þ will be rejectedby ZnTs and trapped, while Zn2þ will be delivered via the ZnT-mediated transepithelial or secretory pathways. This scenarioagrees well with a much narrower distribution and longer retentionof Cd2þ in mammalian tissues, compared to the rapid bodily turn-over of dietary Zn2þ (40). Insight gained from the mechanism ofCd2þ over Zn2þ selectivity may lead to development of strategiesfor Cd2þ detoxification in human or bioremediation of Cd2þ in theenvironment.

Materials and MethodsCell-Based Transport Assay. Cell Culture, plasmid transfection, and generationof mutated plasmids are described in SI Text: SI Materials andMethods. Fluor-escence detection of cytoplasmic metal efflux and fluorescence imagingof vesicular metal sequestration were described previously (18). Briefly, formonitoring Zn2þ or Cd2þ efflux, cells were loaded with 0.5 μM Fluozin-3AM (Invitrogen) or 3.5 μM Fura-2 AM (TEFLABS), superfused with Ca2þ freeRinger’s solution containing 1 μM of the ionophore, pyrithione, and 5 μMeither Zn2þ or Cd2þ. After loading Zn2þ or Cd2þ into the cytoplasm, cells weresuperfused with a metal-free and pyrithione-free Ringer’s solution to removeresidual metal ions and pyrithione. Fluozin-3 or Fura-2 fluorescence signalswere monitored using an imaging system described previously (41), duringthe time periods of metal loading and withdrawal, and normalized to themaximum fluorescence increase. Linear regression of the fluorescence signalin the metal withdrawal phase as a function of time yielded the rate of metalefflux from the cytoplasm. Relative rates of metal effluxes for cells expressingZnT5 and ZnT8 were obtained by comparing to those from cells transfectedwith an empty vector (pcDNA) as a control. Relative rates of metal effluxesfor cells expressing mutated ZnT5H451D or ZnT8H106D were compared either tocontrol or to the wild-type proteins. For monitoring vesicular metal seques-tration, cells were loaded with metal ions as described above, and thenstained with 5 μM ZinPyr-1 that preferentially accumulated within thetrans-Golgi compartment (21). All results shown are mean �s:e. of at leastthree individual experiments (n ≥ 3) with averaged responses of 40–100 cellsin each experiment.

Fig. 5. Transport specificity of YiiP. (A) Kinetics of Zn2þ and Cd2þ transportfor YiiP and YiiPD45H as indicated. The initial rate of Zn2þ or Cd2þ flux wasplotted as a function of themetal concentration. The rates (filled circles) werefitted to a hyperbolic equation (solid line) to yield kinetic parameter Km,Vmax

Vmax∕Km. (B) YiiP-mediated metal uptake. Reconstituted proteoliposomes orcontrol liposomes were briefly exposed to a panel of metal ions as indicated.Vesicles were washed free of extravesicular metal, and then subjected toICP-MS quantification. The background metal levels were subtracted fromthe total metal readings to yield the net metal uptakes for proteoliposomes(closed bar, mean� s:e:, n ¼ 4) and liposomes (open bars, mean� s:e:, n ¼ 4).(*P ≤ 0.01).

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Liposome-Based Transport Assay. YiiPD45 and YiiPD45H were overexpressed,purified, and reconstituted into proteoliposomes with encapsulatedFluozin-1 (20 μM) for monitoring Zn2þ and Cd2þ uptake. Liposomes were pre-pared as a control. The final protein concentration in the proteoliposomessample was 20 μM. Fluozin-1 fluorescence was measured on a photon count-ing spectrofluorometer with excitation and emission set at 495 and at515 nm, respectively. Metal uptake was initiated by exposing proteolipo-somes to 10 μM extravesicular ZnSO4 or CdSO4, followed by an additionof 100 μM EDTA to reverse metal uptake. The Zn2þ and Cd2þ concentrationsin the reaction mix were lower than the protein concentration, thus metalbinding was the limiting step of the transport reaction. This experimentalcondition was designed to mimic the in vivo condition where the availabilityof Zn2þ and perhaps Cd2þ are limited as a result of cellular metal homeostaticcontrols. The resulting fluorescence signal was normalized to the backgroundlevel, and a linear regression of the rising phase of the fluorescence change asa function of time yielded the uptake rate. Data were presented as mean�s:e. of four replicates (n ¼ 4).

Metal Binding Assay. Calorimetric titrations of HPLC purified YiiPD45 andYiiPD45H were carried out on a Microcal MCS titration calorimeter at 25 °Cas described previously (28). Metal titrants (chloride salts of Zn2þ, Cd2þ) weredissolved in the HPLC mobile phase to a concentration of 2 mM. Titrationheats were measured by consecutively injecting a titrant into the proteinsample at a concentration of 0.3 mM. Identical injections in the absenceof proteins were made to obtain the heats of titrant dilution. The net reac-tion heat was calculated by subtracting the heat of dilution from the corre-sponding total heat of reaction. The titration data were fitted to a bindingmodel consisting of two sets of noninteracting binding sites using a nonlinearleast-squares algorithm provided by the Microcal Origin software. The bind-ing enthalpy change ΔH, association constant Ka and the binding stoichio-metry n were permitted to float during the least-squares minimizationand taken as the best fit values.

Stopped-Flow Kinetics. Proteoliposomes or liposomes loaded with 200 μMFluozin-1 were 1∶1 mixed on a stopped-flow apparatus (KinTek Corp.) withan assay buffer (20 mM BTM, 50 mM K2SO4, pH 6.8) containing ZnSO4 orCdSO4 with a concentration ranging from 0 to 2 mM. The resulting fluores-cence rise was normalized to the maximum fluorescence increase inducedby mixing proteoliposomes with 4 mM ZnSO4 or CdSO4 in the presence of

3% n-octyl-μ-D-glucoside to permeabilize the vesicles. Liposome traces wererecorded in parallel and subtracted from the proteoliposome traces for base-line corrections. The kinetic data were fitted to an exponential function usingthe data analysis software SigmaPlot to yield the initial rate. Least-squares fitof the initial rate as a function of metal concentration yielded Km and Vmax.

Transport Specificity Assay. Proteoliposomes or liposomes were deposited ona GS- nitrocellulose filter and overlaid with 100 μL of an assay buffer (20 mMBTM, 50 mM K2SO4, 20 mM CaSO4, pH 6.8) plus an indicated metal ion at aconcentration of 200 μM. To minimize oxidation, fresh metal chloride saltstock solution (100 mM) was made in 100 mM L-ascorbic acid in a N2-filedcontainer. The solution was diluted to 0.2 mM to a fully degased assay buffer,and then immediately applied onto the liposomes deposited on the filter.After a brief 15-s metal exposure, vesicles were immediately washed free ofexternal metal using a high pH assay buffer (20 mM Tris, 50 mM K2SO4,pH 9.1) to prevent metal backflow out of the vesicles. The sample was thenanalyzed on a PlasmaQuad 3 ICP-MS. Total metal levels were quantified inppm. Background metal levels on the filter were estimated using an identicalfilter without deposited vesicles. The net metal uptakes for proteoliposomesand liposomes were obtained by subtracting the background metal levelsfrom the total metal counts.

Statistical Analysis of Data. Data analysis was performed using the SPSSsoftware (version 17.0; SPSS Inc.). All results shown are the means �s:e. ofat least three individual experiments (n ≥ 3). The t test P values of≤0.05wereconsidered significant following Levene’s test for equality of variances.Significance of the results is indicated as follows: relative to control (*) orrelative to wild-type ZnT5, ZnT8 or YiiP (#). �P ≤ 0.01.

ACKNOWLEDGMENTS. The authors thank Dr. Kambe from Kyoto UniversityJapan for providing the plasmid and antibody for ZnT5 and Dr. ChimientifromMellitech, France for providing the ZnT8 plasmid. This work was fundedby the Israel Science Foundation (985/07 and 485/11) (to I.S.), NationalInstitute of Health (R01 GM065137 to D.F. for the kinetic analysis of zinctransporters) and Office of Basic Energy Sciences, Department of Energy(DOE KC0304000 to D.F for funding the binding analysis of zinc transporters).Brookhaven National Laboratory is managed by Brookhaven Science Associ-ates for the Department of Energy. E.H. is supported by a fellowship from theKreitman foundation.

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