The human ZIP1 transporter mediates zinc uptake in human K562 erythroleukemia cells.

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined (65)Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent (65)Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous (65)Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.

The ZIP superfamily of transporters plays important roles in metal ion uptake in diverse organisms. There are 12 ZIP-encoding genes in humans, and we hypothesize that many of these proteins are zinc transporters. In this study, we addressed the role of one human ZIP gene, hZIP1, in zinc transport. First, we examined 65 Zn uptake activity in K562 erythroleukemia cells overexpressing hZIP1. These cells accumulated more zinc than control cells because of increased zinc influx. Moreover, consistent with its role in zinc uptake, hZIP1 protein was localized to the plasma membrane. Our results also demonstrated that hZIP1 is responsible for the endogenous zinc uptake activity in K562 cells. hZIP1 is expressed in untransfected K562 cells, and the increase in mRNA levels found in hZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent 65 Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous 65 Zn uptake activity. The observation that hZIP1 is the major zinc transporter in K562 cells, coupled with its expression in many normal cell types, indicates that hZIP1 plays an important role in zinc uptake in human tissues.
Zinc is an essential nutrient to all organisms because it is a required catalytic and/or structural cofactor for hundreds of zinc-dependent enzymes and other proteins such as transcription factors. Despite its importance, we knew little until recently about how eukaryotic cells take up zinc from their environment. The molecular insight into zinc uptake came from the identification of zinc transporters in fungi and plants. In yeast, the Zrt1 and Zrt2 proteins are zinc transporters involved in moving zinc from the extracellular medium across the plasma membrane into the cytoplasm (1,2). In plants, the Arabidopsis IRT1 protein transports iron from the soil into the root and is also capable of zinc, manganese, and cadmium transport (3)(4)(5). Zrt1, Zrt2, and IRT1 are the founding members of a superfamily of transporters referred to as the ZIP family (for Zrt/IRT-like proteins) (6). Other ZIP transporters have also been implicated in zinc uptake, including the Arabidopsis proteins ZIP1, ZIP2, ZIP3, and ZIP4 (7). The Znt1 protein of Thlaspi caerulescens has been shown to be involved in zinc uptake as well (8). Thus, many ZIP proteins are involved in zinc uptake. A notable exception is Zrt3 of Saccharomyces cerevisiae, a ZIP protein that transports stored zinc from the lumen of the vacuole into the cytoplasm (9). Thus, ZIP proteins can act in zinc uptake or intracellular zinc transport.
Members of the ZIP family are found at all phylogenetic levels, including archaebacteria, eubacteria, and eukaryotes. There are currently ϳ85 members reported in the sequence data bases, and these fall into four subfamilies based on their amino acid similarities (10). Most members are predicted to have eight transmembrane domains and share a predicted topology where the amino and carboxyl termini are extracytoplasmic. The greatest degree of conservation is found in transmembrane domains IV-VIII. Transmembrane domains IV and V are particularly amphipathic and contain conserved and functionally critical histidine residues flanked by equally important polar or charged amino acids (5). These residues are thought to line an aqueous cavity in the transporter through which the substrate moves (11). Notably, ZIP proteins do not contain ATP-binding sites or ATPase domains. Therefore, these proteins must function through either secondary active transport or facilitated diffusion.
There are 12 known ZIP members in the human genome, and five members have been found in the mouse (10). Three of the human proteins, hZIP1, 1 hZIP2, and hZIP3, are very closely related to the fungal and plant proteins known to be zinc uptake transporters. The recent studies of fungal and plant ZIP transporters indicated that the ZIP superfamily plays remarkably conserved roles in metal ion transport and especially zinc uptake. These observations suggested that the mammalian ZIP proteins play similar roles. To test this hypothesis, we expressed the hZIP2 protein in human K562 erythroleukemia cells and showed that hZIP2 localizes to the plasma membrane (12). Moreover, hZIP2 expression resulted in a novel zinc uptake activity not found in these cells. Thus, hZIP2 is a metal ion transporter capable of zinc uptake. In this report, we continue our characterization of mammalian ZIP transporters by functional expression of the hZIP1 protein. Our results demonstrate that hZIP1, like hZIP2, is a zinc transporter. We also found that hZIP1 is the endogenous zinc uptake transporter normally found in K562 cells. Given the ubiquitous expression of hZIP1 in human tissues, we propose that hZIP1 is the major zinc transporter for many human cell types.

EXPERIMENTAL PROCEDURES
DNA Manipulations-An hZIP1 cDNA clone was isolated from a prostate library using high throughput PCR screening (Genome Systems Inc.) and sequenced in its entirety. The hZIP1 ORF was amplified by PCR using the primers 5Ј-GGCCCAAGCTTGGGATGGGGCCCT-GGGGAGAGCC-3Ј and 5Ј-AAGGAAAAAAGCGGCCGCCTAGATTTG-GATGAAGAGCAG-3Ј and cloned into the HindIII and NotI sites of the pRc-CMV mammalian expression vector (Invitrogen). hZIP1 was epitope-tagged at its amino terminus with one copy of the hemagglutinin antigen (HA) by PCR to generate CMV-HA-hZIP1. This fragment was digested with HindIII and XbaI, cloned into the pRc-CMV vector, and confirmed by sequencing.
Cell Culture Methods-K562 erythroleukemia cells (ATCC CCL-243) were grown in complete RPMI 1640 medium (Life Technologies, Inc.) supplemented with 1% glutamine and 10% fetal bovine serum (Sigma). Cells were cultured in 10-cm 3 dishes, incubated in humidified 5% CO 2 isothermal incubators, and transfected by electroporation (Invitrogen) using 3 ϫ 10 6 cells and 20 g of purified plasmid DNA unless noted otherwise. Stable transfected cell lines were generated by the limiting dilution method. The stable cell lines were subsequently maintained in 350 g/ml G418. Cell numbers were determined with a hemocytometer, and cultures were examined weekly for mycoplasma contamination using Hoechst dye and fluorescence microscopy.

65
Zn Uptake Assays and Atomic Absorption Spectroscopy-Cells were grown to 50% confluence, harvested by centrifugation, and washed once in cold uptake buffer (15 mM HEPES, 100 mM glucose, and 150 mM KCl, pH 7.0). The cells were resuspended in uptake buffer and incubated for 10 min in a shaking 37°C water bath. The cells were then mixed with an equal volume of prewarmed uptake buffer containing the specified concentration of 65 ZnCl 2 (Amersham Pharmacia Biotech) and incubated for 15 min unless indicated otherwise. Assays were stopped by adding an equal volume of ice-cold uptake buffer supplemented with 1 mM EDTA (stop buffer). Cells were collected by filtration on glass-fiber filters (Schleicher & Schü ll) and washed three times in stop buffer (ϳ10 ml of total wash volume). Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650 ␥-counter. Chloride salt stock solutions (100 mM) of cadmium, copper, cobalt, magnesium, manganese, and nickel were prepared in distilled water. A ZnCl 2 stock was prepared at 100 mM in 0.02 N HCl, and an FeCl 3 stock was prepared at 50 mM in 0.1 N HCl. Sodium ascorbate (1 mM) was used to reduce Fe 3ϩ to Fe 2ϩ where indicated. Ascorbate treatment alone did not alter zinc uptake activity (data not shown). Sodium bicarbonate stocks (1 M) were prepared fresh before use. For experiments with complete medium as the uptake buffer, the 65 Zn was added to the medium and incubated at 20°C for 24 h before use to ensure equilibration of the 65 Zn with medium components. The statistical significance of differences in values was determined using STATVIEW software (Abacus Concepts, Inc., Berkeley, CA) and subjected to one-way analysis of variance followed by Scheffe's test. Michaelis-Menten kinetic constants were determined using Cleland Kinetic software (13). For atomic absorption spectroscopy, cells were harvested at 50% confluence and washed twice with equal volumes of PBS with or without 1 mM EDTA. The cells were resuspended in 6 M nitric acid and incubated at 95°C for 24 h. Acid-digested samples were then assayed for zinc content with a Varian Spectra AA-30 atomic absorption spectrophotometer, and the final values were normalized to cell number.
Northern Blot Analysis-Total mRNA was isolated from K562 cells using Trizol reagent (Life Technologies, Inc.). Twenty g of mRNA was run on a 2% agarose gel containing 6% formaldehyde and 1ϫ MOPS at 55 V for 5 h. The gel was transferred by capillary electrophoresis to nylon membranes (Biotrans, ICN) in 10ϫ SSC for 16 h. The blot was baked at 80°C for 30 min and then prehybridized at 42°C for 1 h in Super-Hyb solution (Sigma). 32 P-Labeled probes were generated using the random priming method (14). The denatured probe was added to the prehybridization solution and incubated at 42°C for 16 h. The blot was then washed three times with 2ϫ SSC for 5 min, twice with 1ϫ SSC and 1% SDS for 15 min, and twice with 0.1ϫ SSC and 0.1% SDS for 15 min at 42°C prior to autoradiography.
Indirect Immunofluorescence-Indirect immunofluorescence was performed as previously described (12) except for the use of a laser scanning confocal microscope.
Inhibition of hZIP1 Expression with Antisense Oligonucleotides-Antisense oligonucleotides were designed to bind to potentially singlestranded regions of the hZIP1 mRNA as determined with folding prediction algorithms (15). Six hZIP1-directed oligonucleotides were synthesized, and one oligonucleotide of random sequence was used as a control for nonspecific effects (see Table I). The oligonucleotides contained three phosphorothioate bonds at each end to slow their degradation in cells (16). K562 cells were grown to 50% confluence, harvested, and washed three times with PBS. The cells were resuspended in PBS and transfected with 5 or 15 g of the oligonucleotides and 10 g of pEGFP-N1 (CLONTECH) by electroporation. To compensate for the often low transfection efficiency of K562 cells, we used flow cytometry to enrich for transfected cells based on GFP fluorescence. Furthermore, the oligonucleotides were tagged at their 5Ј-ends with fluorescein to aid cell sorting. The transfected cells were added to prewarmed complete medium, grown for 24 h, and sorted by fluorescence-activated cell sorting (FACS) on a Becton Dickinson FACS Vantage TurboSort flow cytometer. After sorting, GFP/fluorescein-positive cells were grown for an additional 24 h before mRNA was isolated or 65 Zn uptake assays were performed.
Semiquantitative RT-PCR Analysis-Total mRNA was isolated from K562 cells using Trizol reagent. cDNA was synthesized with 0.75 g of total RNA using reverse transcriptase and random primers (GeneAmp ® RNA PCR, PerkinElmer Life Sciences). hZIP1 and glyceraldehyde-3phosphate dehydrogenase fragments were amplified from the cDNA templates using 35 cycles. These conditions were found to be in the quantitative range for detection of products based on input template concentration. The hZIP1 amplification primers used were 5Ј-TACAA-GGAGCAGTCAGGGCCGTCA-3Ј and 5Ј-CTAGATTTGGATGAAGAGC-TGGGCAGT-3Ј, and the glyceraldehyde-3-phosphate dehydrogenase primers used were 5Ј-CCACCATGGAGAAGGCTGGGGCTC-3Ј and 5Ј-AGTGATGGCATGGACTGTG-GTCAT-3Ј. Products were analyzed by agarose gel electrophoresis with ethidium bromide staining and photographed under UV light with a Eastman Kodak CCD camera. No products were detected without reverse transcriptase treatment, indicating the lack of contaminating genomic DNA.
Assessment of hZIP1 Expression in Human Tissues-The presence and level of hZIP1 mRNA in different human tissues were determined using Rapid Scan TM gene expression panels (Origene Technologies, Boston, MA). Each panel contains first-strand cDNA prepared from different human tissues that have been normalized to ␤-actin. The cDNAs are provided serially diluted over a 4-log range to allow semiquantitative assessment of expression levels. hZIP1 primers were used to amplify a 581-base pair fragment from within the hZIP1 ORF. The products were analyzed by agarose gel electrophoresis, stained with ethidium bromide, and photographed under UV light with the CCD camera. ␤-Actin primers were used to confirm similar levels of input cDNA in each PCR sample.

RESULTS
Characterization of the hZIP1 Zinc Transporter-A previous study showed that hZIP1 is expressed in human prostate cells (17). Therefore, we isolated an hZIP1 cDNA clone from a prostate cDNA library. This clone was judged to contain the fulllength hZIP1 ORF because of the presence of an 18-base poly(A) tail at its 3Ј-end and an in-frame stop codon in the 5Ј-untranslated region upstream of the presumptive initiation codon. Given that most of the characterized eukaryotic ZIP proteins, including hZIP2, have been implicated in zinc transport, we hypothesized that hZIP1 is also a zinc transporter. To test the ability of hZIP1 to transport zinc, the hZIP1 ORF was cloned into the mammalian expression vector pRc-CMV, allowing expression from the CMV promoter. This plasmid was transfected into human K562 erythroleukemia cells, and stable G418-resistant clonal cell lines were isolated. The hZIP1-expressing transfectants (hereafter designated as CMV-hZIP1) and stable vector-only transfectants (CMV) were assayed for accumulation of 65 Zn. These assays were performed in uptake buffer. As found previously (12), K562 cells had an endogenous zinc uptake activity when assayed under these conditions (Fig.  1A). Consistent with the ability of hZIP1 to transport zinc, CMV-hZIP1 cells accumulated almost 2-fold more 65 Zn over a 60-min period than did the endogenous activity in CMV control cells. No zinc accumulation was seen in either cell type at 4°C, indicating that zinc accumulation by both systems was temperature-dependent and therefore likely to be transporter-mediated rather than due to cell-surface binding.
To determine if the higher zinc accumulation observed in CMV-hZIP1 cells was due to increased zinc influx rather than decreased zinc efflux, we assayed initial rates of 65 Zn uptake over a shorter time period (0 -60 s) (Fig. 1B). hZIP1-expressing cells accumulated zinc at an initial rate of ϳ6 pmol/min/10 6 cells, whereas in CMV transformants, that rate was only 2 hZIP1 Mediates Zinc Uptake in Human Cells pmol/min/10 6 cells. Thus, expression of hZIP1 increased the initial rate of zinc influx 3-fold over control cells. As we reported previously (12), the endogenous zinc uptake activity in K562 cells is concentration-dependent and saturable. This system shows Michaelis-Menten kinetics, with an apparent K m for zinc of 3.5 M and a V max of 11 pmol/min/10 6 cells (Fig. 1C). We determined the concentration dependence of hZIP1 activity by measuring the 65 Zn uptake rate over a range of zinc concentrations in CMV-hZIP1 cells and subtracting the endogenous activity from those values. hZIP1-dependent uptake activity was also concentration-dependent and saturable. The apparent K m for 65 Zn uptake in CMV-hZIP1 cells was 3 M, and the V max was 23 pmol/min/10 6 cells. Therefore, expression of hZIP1 in K562 cells caused an increase in 65 Zn uptake activity that was time-, temperature-, and concentration-dependent and saturable. These results are consistent with our hypothesis that hZIP1 is a zinc transporter in human cells.
The experiments in Fig. 1 (A-C) indicate that hZIP1 is capable of transporting zinc in a buffer lacking any zinc-binding agents. This buffer condition is very different from what mammalian cells encounter in vivo. Although the concentration of total zinc in blood plasma is normally 10 -20 M, most of that zinc is bound to proteins such albumin and ␣ 2 -macroglobulin. A reasonable estimate of the free Zn 2ϩ concentration in plasma is ϳ0.2 nM (18), and the relatively high K m value of hZIP1 activity called into question the relevance of this transporter in zinc acquisition in vivo. Therefore, it was important to determine whether hZIP1 could function under more physiological conditions. To address this question, we first examined the effects of hZIP1 expression on zinc accumulation by cells grown in complete culture medium containing 10% fetal bovine serum. The total zinc content of this medium was determined by atomic absorption spectroscopy to be 11 M, and the albumin concentration was ϳ60 M. Cell-associated zinc levels were measured in CMV and CMV-hZIP1 cells grown to 50% confluence in complete medium. When extracellular zinc was removed prior to analysis by washing the cells with PBS, CMV-hZIP1 cells were found to have accumulated ϳ60% more zinc than control cells (Fig. 1D). When PBS plus 1 mM EDTA was used as a more stringent wash buffer for the removal of surface-bound zinc, cell-associated zinc was reduced in both cell types. However, hZIP1-expressing cells consistently accumulated more zinc than control cells. This conclusion was also supported by the analysis of uptake rates using complete medium as the assay buffer. The zinc uptake rate in CMV-hZIP1 cells was ϳ2-fold higher than in controls (Fig. 1E). As expected, these rates were 20 -30-fold lower than in synthetic buffer, probably due to decreased substrate availability in medium containing zincbinding agents such as albumin. This conclusion was supported by the inability to saturate the transport process in complete medium by adding zinc up to 60 M (Fig. 1F). Taken together, these results indicate that hZIP1 can function as a zinc transporter under physiological conditions as simulated by complete medium.
If hZIP1 is a transporter protein involved in zinc uptake across the plasma membrane, it should be localized on the plasma membrane. However, a previous report (19) described preliminary studies localizing a GFP-hZIP1 fusion protein to an intracellular compartment. These studies lacked evidence that the fusion protein retained function, leaving open the possibility that the GFP moiety may disrupt the protein's normal localization. To reexamine this question of hZIP1 localiza- hZIP1 Mediates Zinc Uptake in Human Cells tion, we tagged the amino terminus of the hZIP1 gene with one copy of the HA antigen and generated stable CMV-HA-hZIP1 transfectants. The epitope-tagged hZIP1 protein was functional as shown by a 2-fold increase in 65 Zn accumulation over vector controls (data not shown). Localization of the HA-hZIP1 protein was determined by indirect immunofluorescence. No fluorescence was observed in cells expressing the untagged hZIP1 protein (Fig. 2, A and C). In contrast, cells expressing the tagged allele showed a bright rim of fluorescence at the cell periphery only (Fig. 2, B and D). These data demonstrated that functional hZIP1 protein is located on the plasma membrane. Similar results were obtained with permeabilized and nonpermeabilized cells (data not shown), indicating that the amino terminus of the protein is extracellular, as predicted (12).
hZIP1 Is the Endogenous Zinc Uptake System in K562 Cells-It was intriguing that the K m of the endogenous zinc uptake system in K562 cells was similar to that observed for both hZIP1-and hZIP2-dependent activities (this report and Ref. 12). We showed previously that hZIP2 is not expressed in K562 cells and that its uptake activity is clearly distinguishable from the endogenous system (12). These observations suggested that perhaps hZIP1 is responsible for endogenous zinc uptake in K562 cells. To test this hypothesis, we assayed for hZIP1 mRNA expression in K562 cells by Northern blot analysis and found that it was expressed in these cells (Fig. 3A). Furthermore, cells expressing hZIP1 from the CMV promoter had ϳ2-fold more hZIP1 mRNA compared with controls. This 2-fold higher level of expression was also confirmed by RT-PCR (Fig. 4D) and was consistent with the 2-fold increase in zinc accumulation in CMV-hZIP1 cells (Fig. 1). As a further test, we determined if hZIP1 activity was distinguishable from the endogenous system. First, we compared the sensitivity of hZIP1, hZIP2, and the endogenous system to inhibition by other metals. Zinc uptake activity was measured in the absence or presence of a 6-fold molar excess of various divalent cations (Fig.  3B). Mg 2ϩ had no effect on any system. Although hZIP2 was strongly inhibited by Co 2ϩ and Mn 2ϩ , both hZIP1 and the endogenous system were unaffected. Similarly, Cu 2ϩ and Fe 2ϩ greatly inhibited hZIP2 activity, but had lesser effects on hZIP1 and the endogenous system. Although hZIP1 and the endogenous system were inhibited by Ni 2ϩ , hZIP2 activity was not altered. Thus, both hZIP1 and the endogenous uptake activity share remarkably similar profiles of inhibition by other metal ions that are distinct from those of hZIP2.
We demonstrated previously that HCO 3 Ϫ treatment stimulates hZIP2 uptake activity, but inhibits uptake by the endogenous system (12). Here, we found that hZIP1 and the endogenous system were not stimulated by HCO 3 Ϫ treatment and were inhibited to a similar degree (Fig. 3C). As a further comparison of hZIP1 and endogenous activities, we determined if zinc uptake by either system was energy-dependent. Treatment with several different electron transport/oxidative phosphorylation inhibitors (i.e. oligomycin, antimycin A, CN Ϫ , N 3 Ϫ , rotenone, and cyanide 3-chlorophenylhydrazone) did not decrease zinc uptake by either system despite causing marked decreases in ATP levels (i.e. Ͻ10% of normal levels) (data not shown). Thus, hZIP1 and the endogenous uptake system are both energy-independent. The similar properties of hZIP1-dependent and endogenous zinc uptake activities strongly suggest that endogenous hZIP1 activity in K562 cells is responsible for zinc uptake in these cells.
As a further test of the role of hZIP1 as the endogenous zinc uptake system in K562 cells, we used antisense oligonucleotides to inhibit hZIP1 mRNA accumulation. When transfected into cells, antisense oligonucleotides can bind to target mRNA and stimulate its degradation by providing a substrate for RNase H (16). Six antisense oligonucleotides were designed to hybridize to regions in the 5Ј-and 3Ј-untranslated regions of hZIP1 mRNA (Table I). Five g of each oligonucleotide was mixed with 10 g of a GFP expression plasmid and transfected into K562 cells. After 24 h, successfully transfected GFP-ex- All results are reported as the percent of the untreated controls, and the hZIP1-and hZIP2-dependent activities were determined as described in the legend to Fig. 1C. C, effects of HCO 3 Ϫ treatment on endogenous, hZIP1-dependent, and hZIP2-dependent zinc uptake. Each point represents the mean in a representative experiment (n ϭ 3), and the error bars indicate Ϯ1 S.D.

hZIP1 Mediates Zinc Uptake in Human Cells
pressing cells were isolated from the population by FACS. The efficiency of this sorting was aided by tagging the 5Ј-end of each oligonucleotide with fluorescein. Untransfected cells showed a peak below 10 fluorescence units (Fig. 4A). The profile of an unsorted population following transfection is shown in Fig. 4B. The sorting procedure enriched for transfected cells (gate M2) to almost 100% (data not shown). The transfected cells isolated in this manner were Ն80% viable as judged by their front and side light-scattering properties (Fig. 4C, gate R1). After sorting, the cells were cultured in complete medium for an additional 24 h prior to zinc uptake assays and mRNA analysis by RT-PCR.
Transfection with GFP alone had no effect on zinc uptake activity, nor did cotransfection with 5 g of a control oligonucleotide of randomly chosen sequence (see C in Fig. 4D). Anti-hZIP1 oligonucleotides O2, O4, and O5 had no effect on zinc uptake activity or hZIP1 expression (data not shown). How-ever, 5 g of anti-hZIP1 oligonucleotide O1, O3, or O6 decreased zinc uptake by 30 -40%. A mixture of 5 g each of oligonucleotides O1, O3, and O6 caused a 90% decrease in zinc uptake. This effect was not due to nonspecific toxicity of the oligonucleotide treatment because 15 g of the control oligonucleotide had no effect on zinc uptake activity. To determine if endogenous hZIP1 expression was inhibited by oligonucleotide treatment, total RNA was isolated from these cells and analyzed by quantitative RT-PCR. Neither 5 nor 15 g of control oligonucleotide affected the hZIP1 mRNA level. Oligonucleotides O1, O3, and O6 and the mixture were all found to decrease hZIP1 mRNA levels, with no effect on a control mRNA, glyceraldehyde-3-phosphate dehydrogenase. Thus, oligonucleotides that inhibited accumulation of endogenous hZIP1 mRNA also inhibited zinc uptake activity. These results strongly suggest that hZIP1 is the endogenous zinc transporter in K562 cells.
Inhibition of zinc uptake by the mixture of oligonucleotides reduced zinc uptake activity to only 10% of normal levels. This result suggests that hZIP1 is the major zinc transporter in at least one mammalian cell type, K562 erythroleukemia cells. To determine what tissues may use hZIP1 as a zinc transporter in vivo, we assayed hZIP1 mRNA expression in various human tissues by semiquantitative RT-PCR. cDNAs prepared from a number of different tissues contained detectable levels of hZIP1 mRNA (Fig. 5). These included small intestine, kidney, liver, pancreas, and prostate, i.e. tissues known to be important in zinc metabolism. Thus, we conclude that hZIP1 is widely expressed in mammalian tissues. 10-Fold dilution of these cDNA samples caused hZIP1 mRNA to be undetectable in all tissue types under these conditions (data not shown). This result indicates that hZIP1 is expressed at similar levels in all of these tissues. The ubiquitous expression of hZIP1 in human tissues suggests that the hZIP1 protein plays an important housekeeping function in many cell types.

DISCUSSION
In a recent report, we used functional expression in K562 cells to characterize the biochemical properties of the hZIP2 zinc transporter (12). In the course of that study, we concluded that hZIP2 is not responsible for the endogenous zinc uptake activity in K562 cells. This conclusion was based on (a) the lack of detectable hZIP2 mRNA in K562 cells and (b) the clear differences in the biochemical properties of the hZIP2 and endogenous zinc transport systems. For example, HCO 3 Ϫ stimulated hZIP2 zinc uptake, but had no such effect on the endogenous system. In this study, we used a similar functional expression approach to demonstrate that, like hZIP2, hZIP1 encodes a zinc transporter. Consistent with our hypothesis, overexpression of hZIP1 in K562 cells led to an increase in zinc uptake activity, and the hZIP1 protein localized exclusively to the plasma membrane.
Perhaps of even greater significance is our demonstration that hZIP1 is the endogenous zinc transporter in K562 cells. This conclusion is based on several independent observations. First, we found that hZIP1 is normally expressed in these cells, and a 2-fold increase in mRNA level generated by expression from the CMV promoter correlated closely with a 2-fold increase in zinc uptake activity. Second, the endogenous uptake system and hZIP1 were indistinguishable in a number of different tests. For example, these systems have similar apparent K m values and are sensitive to inhibition by an array of metal ions to almost precisely the same degree. Finally, we found that inhibition of endogenous hZIP1 expression in K562 cells with antisense oligonucleotides also inhibited zinc uptake activity. Although antisense oligonucleotides have been reported to give artifactual results (16), our control experiments indicate that hZIP1 Mediates Zinc Uptake in Human Cells 22262 the decrease in expression is not due to toxicity or a general decrease in mRNA levels and requires hZIP1-specific sequences to be effective. It is interesting to note that the mixed hZIP1 antisense oligonucleotide treatment reduced uptake activity to 10% of normal levels. These data argue that other zinc transporters, if present in K562 cells, play minor roles in zinc uptake.
As an alternative test of the ability of hZIP1 to transport zinc, we expressed the protein in a yeast zrt1 zrt2 mutant that is defective for zinc uptake. Although this approach was successful for the characterization of many plant ZIP proteins (4,7,8), hZIP1 expression in yeast failed to complement the zrt1 zrt2 mutant, and there was no detectable increase in zinc uptake activity (data not shown). A similar lack of effect was observed when hZIP2 was expressed in yeast (12), suggesting that the mammalian members of the family are not functional in the yeast cellular environment. Unlike hZIP2, however, hZIP1-expressing yeast cells did show a zinc-dependent phenotype; those cells were hypersensitive to the growth inhibitory effects of high zinc, suggesting that the protein was produced, but perhaps improperly localized in the cell. Although we have not examined this effect of hZIP1 expression in yeast further, the zinc dependence of the phenotype provides additional support to the hypothesis that hZIP1 is a zinc transporter.
The similarity of hZIP1 and hZIP2 to IRT1, an Fe 2ϩ transporter in Arabidopsis, suggests that the human proteins may also serve as iron transporters. This hypothesis is supported by the observation that iron can inhibit zinc uptake by these proteins. However, we have assayed Fe 2ϩ uptake in cells overexpressing either hZIP1 or hZIP2 and found no effect. 2 These results indicate that the human zinc transporters are not also involved in iron uptake.
The mechanism of transport used by ZIP proteins is still unresolved. This is largely because varied results have been obtained when the properties of different transporters have been analyzed. In yeast, for example, both Zrt1 and Zrt2 are dependent on energy for zinc transport (1,2). In contrast, neither hZIP1 nor hZIP2 (12) requires ATP for activity. This conclusion is based on the observation that metabolic inhibitors that reduce ATP levels to below 10% of normal levels (data not shown) had no effect on uptake activity of either of these proteins. We determined that zinc uptake by hZIP2 is stimulated by increased HCO 3 Ϫ levels, suggesting that zinc uptake occurs via a Zn 2ϩ /HCO 3 Ϫ symport mechanism (12). In contrast, hZIP1 activity was not affected by HCO 3 Ϫ levels in our experiments here, suggesting that this protein may use a different transport mechanism. Alternatively, sufficient levels of HCO 3 Ϫ may already be present in our standard assay conditions, through equilibration with atmospheric CO 2 , to saturate the hZIP1 transporter. Thus, it remains unresolved if hZIP1 and hZIP2 use the same or different transport mechanisms.
An important unanswered question is what role these proteins play in zinc transport in vivo. hZIP2 expression has been detected only in prostate (12) and uterine (20) epithelial cells, suggesting that this protein plays a very specialized tissuespecific function. In marked contrast, hZIP1 is expressed in all 24 human tissues we examined. This observation, coupled with our results from K562 cells, suggests that hZIP1 may be the major endogenous zinc uptake transporter in many cells in the body. This conclusion is supported by Costello et al. (17), who previously provided evidence that hZIP1 is responsible for zinc uptake in prostate cells; treatment of those cells with prolactin and testosterone causes an increase in both hZIP1 mRNA levels and zinc uptake activity. Finally, Lioumi et al. (19) observed expression of hZIP1 mRNA in intestinal enterocytes. This location of expression suggests that hZIP1 may be involved in the uptake of dietary zinc from the intestine. It should be noted that hZIP1 was designated ZIRTL (for Zrt/IRT transporter-like) by these authors. In an earlier report, Costello et al. (17) named this gene hZIP1, and we have retained that nomenclature here.
One paradox that arose from our studies of hZIP1 and hZIP2 is that these transporters have a surprisingly low affinity for their substrate. Both transporters have a K m value of ϳ3 M for free Zn 2ϩ ions. Similar K m values have been reported for zinc transporters in a large number of mammalian cell types (21). The paradox arises when we consider the free Zn 2ϩ concentration in mammalian serum. Although the total zinc concentration of serum is ϳ20 M, very little metal is present in an unbound form (18). In serum, ϳ75% Zn 2ϩ is bound to albumin, and 20% is bound to ␣ 2 -macroglobulin. Much of the remaining zinc is complexed with amino acids such as histidine and cysteine. Because of the high chelation capacity of serum, the free Zn 2ϩ concentration in serum is calculated to be in the low nanomolar range. Given this extremely low concentration of substrate, it was initially unclear how these transporters could contribute to zinc accumulation by mammalian cells under physiological conditions. The solution to this paradox comes T*A*G*CTCCAG*T*G*A hZIP1 Mediates Zinc Uptake in Human Cells from considering the capacity of these transporters relative to the zinc requirements of the cell. Steady-state cell accumulation of zinc is ϳ100 pmol/10 6 cells (Fig. 1D), which is equivalent to 1 ϫ 10 8 atoms of zinc/cell. This value is similar to those obtained by others (22). With a doubling time of 24 h, the uptake rate required to maintain this level of zinc in growing cells is ϳ0.1 pmol/min/10 6 cells, i.e. a value almost identical to the uptake rate observed using complete medium as the assay buffer (Fig. 1E) and far lower than the rate (11 pmol/min/10 6 cells) measured in buffer (Fig. 1C). Thus, our studies demonstrated that the capacity (i.e. V max ) for zinc uptake is so high relative to the cellular demand for zinc that sufficient levels can be obtained despite the chelation capacity of serum and the apparent low affinity of the transporters.
With the characterization of hZIP1 and hZIP2, our understanding of zinc homeostasis in mammalian cells is greatly improving. A second family of zinc transporters has also been identified in mammals. This family is called the CDF (for cation diffusion facilitator) family, and like the ZIP proteins, members of this group have been implicated in zinc transport in organisms of all phylogenetic levels (10,23). One mammalian CDF protein, ZnT-1, is a zinc efflux protein that transports zinc out of the cell (22). ZnT-1 may play a role in removing excess zinc from cells and may also serve to transport zinc across the basolateral membrane of the intestinal enterocyte during zinc absorption (24,25). A second member of the CDF family, ZnT-2, compartmentalizes intracellular zinc in the late endosome of the cell (26,27). This zinc sequestration reduces the toxicity of intracellular zinc. Cellular zinc status is likely to be controlled by regulation of many of these transporters. Expression of both ZnT-1 and ZnT-2 has been shown to be induced in zinc-treated cells (28) or in animals fed zinc-rich diets (24,29). The zinc-responsive transcription factor MTF-1 was found to regulate ZnT-1 (28), and it seems likely that MTF-1 also regulates ZnT-2 expression. Thus, zinc treatment increases the cell's capacity to both export and sequester excess zinc. It is not yet clear if the uptake transporters are also regulated in response to zinc status. Many ZIP genes in yeast and plants are expressed at higher levels under zinc-limiting conditions (1,7,9). In yeast, this up-regulation was shown to occur at a transcriptional level and is mediated by the zinc-responsive Zap1 transcription factor (30). It is therefore intriguing that hZIP1 mRNA levels decrease in prostate-derived PC-3 cells treated with higher than normal levels of zinc (17). We are currently examining the regulation of hZIP1 and hZIP2 in response to zinc availability to determine if such a mechanism contributes to mammalian zinc homeostasis. Furthermore, the potential roles of the other human ZIP genes remain to be addressed.