The Human ZIP1 Transporter Mediates Zinc Uptake in Human K562 Erythroleukemia Cells
2001; Elsevier BV; Volume: 276; Issue: 25 Linguagem: Inglês
10.1074/jbc.m101772200
ISSN1083-351X
Autores Tópico(s)Iron Metabolism and Disorders
ResumoThe 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 65Zn 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 inhZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent 65Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous65Zn 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 65Zn 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 inhZIP1-overexpressing cells correlated with the increased zinc uptake activity. Furthermore, hZIP1-dependent 65Zn uptake was biochemically indistinguishable from the endogenous activity. Finally, inhibition of endogenous hZIP1 expression with antisense oligonucleotides caused a marked decrease in endogenous65Zn 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. human ZIP reverse transcription-polymerase chain reaction open reading frame cytomegalovirus hemagglutinin phosphate-buffered saline 4-morpholinepropanesulfonic acid green fluorescent protein fluorescence-activated cell sorting 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 (1Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (444) Google Scholar, 2Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In plants, the Arabidopsis IRT1 protein transports iron from the soil into the root and is also capable of zinc, manganese, and cadmium transport (3Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1032) Google Scholar, 4Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (596) Google Scholar, 5Rogers E.E. Eide D.J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12356-12360Crossref PubMed Scopus (362) Google Scholar). Zrt1, Zrt2, and IRT1 are the founding members of a superfamily of transporters referred to as the ZIP family (for Zrt/IRT-likeproteins) (6Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (836) Google Scholar). Other ZIP transporters have also been implicated in zinc uptake, including the Arabidopsisproteins ZIP1, ZIP2, ZIP3, and ZIP4 (7Grotz N. Fox T. Connolly E. Park W. Guerinot M.L. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7220-7224Crossref PubMed Scopus (538) Google Scholar). The Znt1 protein ofThlaspi caerulescens has been shown to be involved in zinc uptake as well (8Pence N.S. Larsen P.B. Ebbs S.D. Letham D.L. Lasat M.M. Garvin D.F. Eide D. Kochian L.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4956-4960Crossref PubMed Scopus (617) Google Scholar). 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 (9MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar). 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 ∼85 members reported in the sequence data bases, and these fall into four subfamilies based on their amino acid similarities (10Gaither L.A. Eide D.J. et al.Biometals. 2001; 30 (in press)Google Scholar). 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 (5Rogers E.E. Eide D.J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12356-12360Crossref PubMed Scopus (362) Google Scholar). These residues are thought to line an aqueous cavity in the transporter through which the substrate moves (11Eng B.H. Guerinot M.L. Eide D. Saier M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar). 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 (10Gaither L.A. Eide D.J. et al.Biometals. 2001; 30 (in press)Google Scholar). 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 (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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.DISCUSSIONIn a recent report, we used functional expression in K562 cells to characterize the biochemical properties of the hZIP2 zinc transporter (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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 detectablehZIP2 mRNA in K562 cells and (b) the clear differences in the biochemical properties of the hZIP2 and endogenous zinc transport systems. For example, HCO3− 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 thathZIP1 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 Km values and are sensitive to inhibition by an array of metal ions to almost precisely the same degree. Finally, we found that inhibition of endogenoushZIP1 expression in K562 cells with antisense oligonucleotides also inhibited zinc uptake activity. Although antisense oligonucleotides have been reported to give artifactual results (16Branch A.D. Trends Biochem. Sci. 1998; 23: 45-50Abstract Full Text PDF PubMed Scopus (167) Google Scholar), our control experiments indicate that 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 mixedhZIP1 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 (4Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (596) Google Scholar, 7Grotz N. Fox T. Connolly E. Park W. Guerinot M.L. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7220-7224Crossref PubMed Scopus (538) Google Scholar, 8Pence N.S. Larsen P.B. Ebbs S.D. Letham D.L. Lasat M.M. Garvin D.F. Eide D. Kochian L.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4956-4960Crossref PubMed Scopus (617) Google Scholar), hZIP1 expression in yeast failed to complement the zrt1 zrt2mutant, 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 (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), 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 Fe2+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 Fe2+ uptake in cells overexpressing either hZIP1 or hZIP2 and found no effect. 2L. A. Gaither, unpublished observation. 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 (1Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (444) Google Scholar, 2Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In contrast, neither hZIP1 nor hZIP2 (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) 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 HCO3−levels, suggesting that zinc uptake occurs via a Zn2+/HCO3− symport mechanism (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). In contrast, hZIP1 activity was not affected by HCO3− levels in our experiments here, suggesting that this protein may use a different transport mechanism. Alternatively, sufficient levels of HCO3− may already be present in our standard assay conditions, through equilibration with atmospheric CO2, 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 (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar) and uterine (20Yamaguchi S. Kokubyo Gakkai Zasshi. 1995; 62: 78-93Crossref PubMed Scopus (10) Google Scholar) epithelial cells, suggesting that this protein plays a very specialized tissue-specific 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 Costelloet al. (17Costello L.C. Liu Y. Zou J. Franklin R.B. J. Biol. Chem. 1999; 274: 17499-17504Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar), 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 bothhZIP1 mRNA levels and zinc uptake activity. Finally, Lioumi et al. (19Lioumi M. Ferguson C.A. Sharpe P.T. Freeman T. Marenholz I. Mischke D. Heizmann C. Ragoussis J. Genomics. 1999; 62: 272-280Crossref PubMed Scopus (46) Google Scholar) observed expression of hZIP1mRNA 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 designatedZIRTL (for Zrt/IRTtransporter-like) by these authors. In an earlier report, Costello et al. (17Costello L.C. Liu Y. Zou J. Franklin R.B. J. Biol. Chem. 1999; 274: 17499-17504Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) named this genehZIP1, 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 Km value of ∼3 μm for free Zn2+ ions. SimilarKm values have been reported for zinc transporters in a large number of mammalian cell types (21Reyes J.G. Am. J. Physiol. 1996; 270: C401-C410Crossref PubMed Google Scholar). The paradox arises when we consider the free Zn2+ concentration in mammalian serum. Although the total zinc concentration of serum is ∼20 μm, very little metal is present in an unbound form (18Magneson G.R. Puvathingal J.M. Ray W.J. J. Biol. Chem. 1987; 262: 11140-11148Abstract Full Text PDF PubMed Google Scholar). In serum, ∼75% Zn2+ is bound to albumin, and 20% is bound to α2-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 Zn2+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 from considering the capacity of these transporters relative to the zinc requirements of the cell. Steady-state cell accumulation of zinc is ∼100 pmol/106 cells (Fig.1 D), which is equivalent to 1 × 108 atoms of zinc/cell. This value is similar to those obtained by others (22Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). With a doubling time of 24 h, the uptake rate required to maintain this level of zinc in growing cells is ∼0.1 pmol/min/106cells, i.e. a value almost identical to the uptake rate observed using complete medium as the assay buffer (Fig. 1 E) and far lower than the rate (11 pmol/min/106 cells) measured in buffer (Fig. 1 C). Thus, our studies demonstrated that the capacity (i.e. Vmax) 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 diffusionfacilitator) family, and like the ZIP proteins, members of this group have been implicated in zinc transport in organisms of all phylogenetic levels (10Gaither L.A. Eide D.J. et al.Biometals. 2001; 30 (in press)Google Scholar, 23Paulsen I.T. Saier M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (293) Google Scholar). One mammalian CDF protein, ZnT-1, is a zinc efflux protein that transports zinc out of the cell (22Palmiter R.D. Findley S.D. EMBO J. 1995; 14: 639-649Crossref PubMed Scopus (636) Google Scholar). 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 (24McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar, 25McMahon R.J. Cousins R.J. J. Nutr. 1998; 128: 667-670Crossref PubMed Scopus (180) Google Scholar). A second member of the CDF family, ZnT-2, compartmentalizes intracellular zinc in the late endosome of the cell (26Palmiter R.D. Cole T.B. Findley S.D. EMBO J. 1996; 15: 1784-1791Crossref PubMed Scopus (393) Google Scholar, 27Kobayashi T. Beuchat M. Lindsay M. Frias S. Palmiter R.D. Sakuraba H. Parton R.G. Gruenberg J. Nat. Cell Biol. 1999; 1: 113-118Crossref PubMed Scopus (247) Google Scholar). 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 (28Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar) or in animals fed zinc-rich diets (24McMahon R.J. Cousins R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4841-4846Crossref PubMed Scopus (255) Google Scholar, 29Liuzzi J.P. Blanchard R.K. Cousins R.J. J. Nutr. 2001; 131: 46-52Crossref PubMed Scopus (200) Google Scholar). The zinc-responsive transcription factor MTF-1 was found to regulate ZnT-1 (28Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar), 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 (1Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (444) Google Scholar, 7Grotz N. Fox T. Connolly E. Park W. Guerinot M.L. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7220-7224Crossref PubMed Scopus (538) Google Scholar, 9MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar). In yeast, this up-regulation was shown to occur at a transcriptional level and is mediated by the zinc-responsive Zap1 transcription factor (30Zhao H. Eide D.J. Mol. Cell. Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (216) Google Scholar). It is therefore intriguing thathZIP1 mRNA levels decrease in prostate-derived PC-3 cells treated with higher than normal levels of zinc (17Costello L.C. Liu Y. Zou J. Franklin R.B. J. Biol. Chem. 1999; 274: 17499-17504Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). We are currently examining the regulation of hZIP1 andhZIP2 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. 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 (1Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (444) Google Scholar, 2Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). In plants, the Arabidopsis IRT1 protein transports iron from the soil into the root and is also capable of zinc, manganese, and cadmium transport (3Eide D. Broderius M. Fett J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5624-5628Crossref PubMed Scopus (1032) Google Scholar, 4Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (596) Google Scholar, 5Rogers E.E. Eide D.J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12356-12360Crossref PubMed Scopus (362) Google Scholar). Zrt1, Zrt2, and IRT1 are the founding members of a superfamily of transporters referred to as the ZIP family (for Zrt/IRT-likeproteins) (6Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (836) Google Scholar). Other ZIP transporters have also been implicated in zinc uptake, including the Arabidopsisproteins ZIP1, ZIP2, ZIP3, and ZIP4 (7Grotz N. Fox T. Connolly E. Park W. Guerinot M.L. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7220-7224Crossref PubMed Scopus (538) Google Scholar). The Znt1 protein ofThlaspi caerulescens has been shown to be involved in zinc uptake as well (8Pence N.S. Larsen P.B. Ebbs S.D. Letham D.L. Lasat M.M. Garvin D.F. Eide D. Kochian L.V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4956-4960Crossref PubMed Scopus (617) Google Scholar). 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 (9MacDiarmid C.W. Gaither L.A. Eide D. EMBO J. 2000; 19: 2845-2855Crossref PubMed Scopus (301) Google Scholar). 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 ∼85 members reported in the sequence data bases, and these fall into four subfamilies based on their amino acid similarities (10Gaither L.A. Eide D.J. et al.Biometals. 2001; 30 (in press)Google Scholar). 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 (5Rogers E.E. Eide D.J. Guerinot M.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12356-12360Crossref PubMed Scopus (362) Google Scholar). These residues are thought to line an aqueous cavity in the transporter through which the substrate moves (11Eng B.H. Guerinot M.L. Eide D. Saier M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar). 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 (10Gaither L.A. Eide D.J. et al.Biometals. 2001; 30 (in press)Google Scholar). 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 (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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. DISCUSSIONIn a recent report, we used functional expression in K562 cells to characterize the biochemical properties of the hZIP2 zinc transporter (12Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). 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 detectablehZIP2 mRNA in K562 cells and (b) the clear differences in the biochemical properties of the hZIP2 and endogenous zinc transport systems. For example, HCO3− 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 thathZIP1 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 Km values and are sensitive to inhibition by an array of metal ions to almost precisely the same degree. Finally, we found that inhibition of endogenoushZIP1 expression in K562 cells with antisense oligonucleotides also inhibited zinc uptake activity. Although antisense oligonucleotides have been reported to give artifactual results (16Branch A.D. Trends Biochem. Sci. 1998; 23: 45-50Abstract Full Text PDF PubMed Scopus (167) Google Scholar), our control experiments indicate that 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 mixedhZIP1 antisense oligonucleotide treatment re
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