The Acrodermatitis Enteropathica Gene ZIP4 Encodes a Tissue-specific, Zinc-regulated Zinc Transporter in Mice
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m305000200
ISSN1083-351X
AutoresJodi Dufner‐Beattie, Fudi Wang, Yien–Ming Kuo, Jane Gitschier, David Eide, Glen K. Andrews,
Tópico(s)Heavy Metal Exposure and Toxicity
ResumoThe human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans. The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans. A long recognized disease of zinc metabolism is the human genetic disorder acrodermatitis enteropathica (AE) 1The abbreviations used are: AE, acrodermatitis enteropathica; EST, expressed sequence tag; IRT, iron-regulated transporter; MT-I, metallothionein-I; RT, reverse transcriptase; ZIP, ZRT/IRT-related proteins; ZnA, zinc-adequate; ZnD, zinc-deficient; ZnE, zinc-excess; ZRT, zinc-regulated transporter; CMV, cytomegalovirus. (1Lorincz A.L. Arch. Dermatol. 1967; 96: 736-737Crossref PubMed Scopus (4) Google Scholar, 2Desmons F. Walbaum R. Ann. Dermatol. Venereol. 1979; 106: 9-12PubMed Google Scholar). 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That gene was named hZIP4 (the Human Genome Organization Nomenclature Committee named this gene SLC39A4). ZIP4 was found to be expressed in enterocytes and to reside in the plasma membrane. Mutations in hZIP4 were detected in AE patients (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 18Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (425) Google Scholar), strongly suggesting that they cause this genetic disorder. The recently identified ZIP superfamily of metal ion uptake transporters (19Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (83) Google Scholar, 20Guerinot M.L. Eide D. Curr. Opin. Plant Biol. 1999; 2: 244-249Crossref PubMed Scopus (101) Google Scholar) are found in all eukaryotes, and many of its members mediate zinc uptake. In yeast, ZRT1 encodes the high affinity zinc transporter, and ZRT2 encodes the low affinity zinc transport system. The Arabidopsis iron-regulated transporter gene (IRT1) encodes a metal transporter that has remarkable sequence similarity with the yeast ZRTs and with other Arabidopsis zinc transporters (19Eide D. Curr. Opin. Cell Biol. 1997; 9: 573-577Crossref PubMed Scopus (83) Google Scholar, 21Eide D.J. Annu. Rev. Nutr. 1998; 18: 441-469Crossref PubMed Scopus (244) Google Scholar). Thus, the acronym ZIP was adopted to reflect ZRT/IRT-related proteins. Many members of the ZIP gene superfamily have now been detected based on sequence homology with yeast and Arabidopsis ZIP genes (22Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (209) Google Scholar, 23Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (891) Google Scholar). The ZIP proteins typically have eight membrane-spanning domains, and spanning domain four contains fully conserved histidyl and glycyl residues in an amphipathic α-helix. These proteins also often have a histidine-rich intracellular loop between spanners three and four. These structural motifs are hallmarks of the ZIP superfamily (22Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (209) Google Scholar, 23Guerinot M.L. Biochim. Biophys. Acta. 2000; 1465: 190-198Crossref PubMed Scopus (891) Google Scholar). Computer searches of the complete human genome sequence revealed ∼12 ZIP genes (24Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (433) Google Scholar). Three of these ZIP proteins (hZIP1–3) fall into a subfamily that shares a conserved 12-residue signature sequence. hZIP1 and hZIP2 function as zinc transporters, and hZIP1 is the major zinc uptake protein in K562 cells (25Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 26Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). ZIP4 and ZIP5 also comprise a ZIP subfamily (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar), but their metal transport properties have not yet been determined. ZIP genes that encode zinc transporters can also be regulated by zinc. The yeast ZRT1 and ZRT2 genes are up-regulated in response to zinc deficiency (27Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (453) Google Scholar). Transcription of these genes is controlled by the transcription factor Zap1p (28Zhao H. Eide D.J. Mol. Cell Biol. 1997; 17: 5044-5052Crossref PubMed Scopus (223) Google Scholar), and the activity of Zap1p is inhibited by zinc. In Arabidopsis, the ZIP1, ZIP3, and ZIP4 genes are zinc-regulated, consistent with a role in zinc uptake (e.g. in roots) (29Grotz 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 (560) Google Scholar). In mammals, dietary zinc also regulates zinc transport activities and zinc transporter gene expression in the intestines (30Cousins R.J. Adv. Exp. Med. Biol. 1989; 249: 3-12Crossref PubMed Scopus (28) Google Scholar, 31Hempe J.M. Carlson J.M. Cousins R.J. J. Nutr. 1991; 121: 1389-1396Crossref PubMed Scopus (30) Google Scholar, 32Cousins R.J. McMahon R.J. J. Nutr. 2000; 130: 1384S-1387SCrossref PubMed Google Scholar, 33Krebs N.F. J. Nutr. 2000; 130: 1374S-1377SCrossref PubMed Google Scholar, 34Cragg R.A. Christie G.R. Phillips S.R. Russi R.M. Kury S. Mathers J.C. Taylor P.M. Ford D. J. Biol. Chem. 2002; 277: 22789-22797Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 35Liuzzi J.P. Blanchard R.K. Cousins R.J. J. Nutr. 2001; 131: 46-52Crossref PubMed Scopus (203) Google Scholar). For example, the mouse, rat, and human ZnT1 genes, which encode members of the cation diffusion facilitator family of proteins, are regulated by zinc (34Cragg R.A. Christie G.R. Phillips S.R. Russi R.M. Kury S. Mathers J.C. Taylor P.M. Ford D. J. Biol. Chem. 2002; 277: 22789-22797Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 35Liuzzi J.P. Blanchard R.K. Cousins R.J. J. Nutr. 2001; 131: 46-52Crossref PubMed Scopus (203) Google Scholar, 36Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). There is little information regarding metalloregulation of ZIP family members in mammals. In support of the hypothesis that ZIP4 is the AE gene, the results presented herein demonstrate that mZIP4 encodes a zinc transporter, that this gene is expressed in intestine and embryonic visceral yolk sac, that ZIP4 protein localizes to the apical surface of enterocytes and visceral endoderm cells, and that the expression of this gene and its protein product is dynamically regulated by zinc. Animal Care and Use—All experiments involving mice were conducted in accordance with NIH guidelines for the care and use of experimental animals, and were approved by the Institutional Animal Care and Use Committee. CD-1 mice (48–60 days old) were purchased from Charles River Breeding Laboratories (Raleigh, NC). Mouse diets were purchased from Harlan Teklad (Madison, WI) and have been described in detail previously (37Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar). Zinc levels in the diets were as follows: zinc-deficient (ZnD), 1 ppm zinc; zinc-adequate (ZnA), 50 ppm zinc; and zinc-excess (ZnE), 50 ppm zinc plus 250 ppm zinc in the drinking water. These diets each contained ∼18 μg/g copper and are otherwise identical. To examine the tissue-specific expression of ZIP4, CD-1 female or male mice (6/group) maintained on ZnA feed were killed and the indicated tissues were harvested and snap-frozen in liquid nitrogen for subsequent extraction of RNA and Northern blot analysis. Pancreas RNA was extracted from fresh tissue. To examine the effects of zinc on ZIP4 expression, female mice were subjected to dietary zinc deficiency followed by either an injection of zinc or switching to ZnE conditions. Dietary zinc deficiency during pregnancy was induced as described previously (38Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (62) Google Scholar). CD-1 female mice were mated with CD-1 male mice and on day 1 (vaginal plug) of pregnancy mice were placed in pairs in cages with stainless steel false bottoms to reduce recycling of zinc (39Cook Mills J.M. Fraker P.J. Br. J. Nutr. 1993; 69: 835-848Crossref PubMed Scopus (53) Google Scholar). Mice were provided free access to the ZnA feed and deionized distilled water. Water bottles were washed in 4 m HCl and rinsed in deionized water to remove zinc (39Cook Mills J.M. Fraker P.J. Br. J. Nutr. 1993; 69: 835-848Crossref PubMed Scopus (53) Google Scholar). On day 8, the diet was changed to the ZnD diet (or, where indicated, mice were maintained on the ZnA diet). The visceral yolk sac and maternal small intestine were harvested on day 11 to day 15 (6 mice/group) and either fixed for immunohistochemistry or snap-frozen in liquid nitrogen for Northern blotting. Where indicated, zinc-deficient day 14 pregnant mice were either injected intraperitoneally with ZnCl2 (100 μmol/kg body weight) or switched to ZnE conditions, and the embryonic visceral yolk sacs and maternal small intestines were collected at the indicated times after zinc treatment. The maternal intestine was isolated as an intact tissue (not a mucosal scrape) but was subdivided as follows. The first three centimeters was considered the duodenum, and the remainder of the small intestine was divided into equal parts, which were considered the proximal (nearest the duodenum) and distal small intestine. Previous studies have documented that little anorexia occurs under these experimental conditions, and results using pair-fed controls do not differ from those obtained using mice allowed free access to feed (37Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar, 38Andrews G.K. Geiser J. J. Nutr. 1999; 129: 1643-1648Crossref PubMed Scopus (62) Google Scholar). Nonpregnant female CD-1 mice were fed ZnA or ZnD feed for 2 weeks and then injected intraperitoneally with zinc or switched to ZnE conditions, as described above. The duodenum and proximal small intestine were harvested at the indicated times after zinc treatment. Computer Analyses of Sequence Data—Multiple sequence alignments were performed using the Vector NTI Suite Program (Informax, Bethesda, MD). RNA Extraction and Northern Blot Hybridization—Tissue RNAs were isolated as described in detail previously (36Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 40Andrews G.K. Lee D.K. Ravindra R. Lichtlen P. Sirito M. Sawadogo M. Schaffner W. EMBO J. 2001; 20: 1114-1122Crossref PubMed Scopus (86) Google Scholar). Total RNA (3 μg) was size-fractionated by agarose-formaldehyde gel electrophoresis, transferred, and cross-linked to nylon membranes (41Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (251) Google Scholar). Northern blot membranes were hybridized and washed under stringent conditions as described (36Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 37Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar, 41Dalton T.P. Palmiter R.D. Andrews G.K. Nucleic Acids Res. 1994; 22: 5016-5023Crossref PubMed Scopus (251) Google Scholar). Hybrids were detected by autoradiography with intensifying screens at –70 °C. Although not shown, duplicate gels were stained with acridine orange or the same membrane was rehybridized with a β-actin probe to monitor RNA loading and integrity (36Langmade S.J. Ravindra R. Daniels P.J. Andrews G.K. J. Biol. Chem. 2000; 275: 34803-34809Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar). The mouse metallothionein-I (MT-I) and β-actin probes were as described (37Dalton T.P. Fu K. Palmiter R.D. Andrews G.K. J. Nutr. 1996; 126: 825-833Crossref PubMed Scopus (107) Google Scholar). The mouse ZIP4 cDNA was identified as described under "Results" (GenBank™ accession number AK005535). The protein coding region of ZIP4 mRNA was amplified by RT-PCR from mouse intestinal RNA using Improm-II reverse transcriptase (Promega) and Pfu polymerase (Stratagene, La Jolla, CA) for PCR. The sense primer was located at +86 and the antisense primer at +2155 in this cDNA. Each primer was 26 bp in length. The reverse transcription-polymerase chain reaction (RT-PCR) product was cloned and the DNA sequence confirmed. Probes were labeled using the Random Primers DNA labeling system according to the instructions from the manufacturer (Invitrogen, Carlsbad, CA). Probes had specific activities of ∼1–3 × 109 dpm/μg. RT-PCR Detection of ZIP4 Isoform mRNAs—RT-PCR was used to distinguish between mZIP4 mRNAs that encode the long versus the short isoforms of this protein. Total RNA (1 μg), isolated from the maternal small intestine or embryonic visceral yolk sac harvested from the zinc-deficient pregnant mice described above, was DNase I-treated according to the instructions from the manufacturer (Invitrogen). DNase I was inactivated by addition of EDTA to 2.5 mm, followed by a 10 min incubation at 65 °C. Reverse transcription was subsequently carried out using Improm-II reverse transcriptase (Promega). Samples were then amplified by PCR for 27, 30, or 33 cycles using Platinum Taq DNA polymerase (Invitrogen). The long isoform transcript was amplified using the primers mZIP4EX1(s) (5′-AGAAGTCAGCACCTCTACAAGGAACGC-3′) and mZIP4EX2(as) (5′-AGTAGCTGGCTCAGACCCAGGGTC-3′), whereas the short isoform transcript was amplified using the primers mZIP4INT1(s) (5′-AACATGACATAAGATAGCTGATAGAATCCATGC-3′) and mZIP4EX2(as). The RT-PCR products of the long and short isoform transcripts were 475 and 329 bp, respectively. Immunohistochemistry—The rabbit polyclonal antiserum against a mZIP4 peptide was described previously (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar). Immunohistochemistry was performed using the Histostain-SP kit (Zymed Laboratories Inc., San Francisco, CA) for rabbit primary antibody and AEC staining. Tissues were fixed overnight in 4% paraformaldehyde at 4 °C, embedded in paraffin, and sectioned. Sections were deparaffinized, treated with 1% peroxide for 10 min, blocked with 10% normal goat serum for 10 min, and then incubated for1hat room temperature with the mZIP4 antiserum at a 1:160 dilution. Where indicated, the mZIP4 antiserum was neutralized by incubation for 2 h at room temperature with 6 × 10–5m peptide before application to the tissue sections. Other controls included nonimmune serum and omission of primary antiserum. Expression Plasmid Construction—Mouse ZIP4 cDNA encoding the long isoform was cloned into an expression vector that was used for zinc uptake studies in transiently transfected cells. To subclone pCMV-mZIP4, total RNA from mouse intestine (1 μg) was reverse transcribed using Improm-II reverse transcriptase (Promega, Madison, WI), followed by amplification of the cDNA using Pfu DNA polymerase (Stratagene) with primers mZIP4(S) (5′-CGGAATTCGAAGTCAGCACCTCTACAAGGAACGC-3′) and mZIP4(AS) (5′-GGACTAGTAGTCAACAGACAGGGACAAGGACTGG-3′). The amplification product was digested with EcoRI and SpeI and ligated into pCMVSport6 (Invitrogen). Cell Culture and Transient Transfection—HEK293 cells were cultured under 5% CO2 in high glucose Dulbecco's modified Eagle's medium (Invitrogen) containing 100 units/ml penicillin, 100 μg/ml streptomycin, 2 mml-glutamine, and 10% fetal bovine serum. Cells (2 × 105) were seeded in 24-well poly-l-lysine-coated plates and transfected with the pCMV-Sport6 vector or pCMV-Sport6 expressing the mouse mZIP4 cDNA (pCMV-mZIP4). Transfections were performed using LipofectAMINE 2000 (Invitrogen) according to the instructions from the manufacturer. Transfection efficiencies were typically ∼60%. Between 36 and 48 h after transfection, the cells were used for zinc uptake assays. 65Zn Uptake Assays—Zinc uptake assays were performed essentially as described previously (25Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (265) Google Scholar, 26Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Cells were washed once in uptake buffer (15 mm HEPES, 100 mm glucose, and 150 mm KCl, pH 7.0) and then prewarmed uptake buffer containing the specified concentration of 65ZnCl2 (PerkinElmer Life Sciences) was added. The cells were then incubated in a shaking 37 °C water bath for 15 min unless indicated otherwise. Assays were stopped by the addition of an equal volume of ice-cold uptake buffer supplemented with 1 mm EDTA (stop buffer). Cells were collected on glass fiber filters (Type A/E, Gelman Sciences) and washed three times in stop buffer (∼10 ml of total wash volume). Parallel experiments were conducted at 0 °C to measure cell surface 65Zn binding, which was subtracted from the values at 37 °C to obtain net zinc uptake values. Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650 γ-counter. Metal salts were obtained from Sigma. Stock solutions of the chloride salts of various metals (CoCl2, CuCl2, MgCl2, MnCl2, NiCl2, and CdCl2) and AgNO3 were prepared at 100 mm concentration in distilled water. A ZnCl2 stock was prepared at 100 mm in 0.02 n HCl, and an FeCl3 stock solution was prepared at 50 mm in 0.1 n HCl. Sodium ascorbate (1 mm) was used to reduce Fe3+ to Fe2+. Ascorbate treatment alone did not alter zinc uptake activity (data not shown). Cells grown in parallel to those used for uptake experiments were washed three times with ice-cold uptake buffer, resuspended in PBS buffer containing 0.1% SDS and 1% Triton X-100, for cell lysis, and then assayed for protein content using a Bradford assay kit (Bio-Rad). Zinc accumulation and uptake rates were normalized to protein concentrations of these cell lysates. Michaelis-Menten constants were determined by nonlinear interpolation of the data using Prism (version 3.0a for Macintosh, GraphPad Software, San Diego, CA). GenBank™ Accession Numbers—GenBank™ accession numbers for the mouse ZIP4 ESTs were AK005535, AI314527, and BB848544 for long isoform, and BY147218 and BY136150 for short isoform. AC074152 was the accession number for mouse ZIP4 gene. Identification of the Mouse ZIP4 Gene and mRNA and Evolutionary Conservation of the Predicted ZIP4 Peptide—The predicted human ZIP4 protein sequence (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 18Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (425) Google Scholar) was used to search the mouse translated non-redundant data base on the NBCI server, and a 2264-bp cDNA containing a 660-amino acid open reading frame homologous to the long isoform of hZIP4 was identified (Fig. 1). Two ESTs extended the 5′ end of this mZIP4 cDNA an additional 439 bp. However, the 5′ end of the vast majority of mZIP4 ESTs corresponded to the 2264-bp transcript (Fig. 1A). The mZIP4 gene was subsequently identified using this cDNA sequence to search the mouse high throughput genomic sequence data base on the NCBI server. Intron-exon structure was determined by comparing the cDNA and genomic sequences in conjunction with splice donor and acceptor consensus sequences (Fig. 1A). Like the human gene (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 18Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (425) Google Scholar), mouse ZIP4 consists of 12 exons separated by 11 introns and is quite compact, spanning only ∼5 kb of DNA. The ZIP4 gene in humans is located on chromosome 8q24.3, whereas the mZIP4 gene is located in the syntenic region on mouse chromosome 15E1. Alignment of the predicted amino acid sequences of the long isoform of mZIP4 and hZIP4 (17Wang K. Zhou B. Kuo Y.-M. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar, 18Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (425) Google Scholar) revealed 76% amino acid similarity between these two proteins (Fig. 1B). The amino-terminal half of these proteins, which is predicted to be extracellular, is not as well conserved, although several conserved blocks of amino acids are present. The highest degree of similarity occurs in the carboxyl-terminal half of the protein, which contains the eight predicted membrane-spanning domains characteristic of ZIP proteins. The amino acid sequence within the predicted membrane-spanning regions is highly conserved; most of the amino acid differences between these proteins occur in the intervening loops. Interestingly, of the 11 amino acid changes found in various AE patients, 9 occur at residues that are also conserved in the mouse protein. These changes often convert an uncharged to a charged residue within the highly conserved transmembrane segments. In humans, two ZIP4 mRNAs have been detected which are predicted to encode either a long (647-residue) or short (622-residue) isoform of this protein (18Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (425) Google Scholar). These hZIP4 isoforms are identical in the carboxyl-terminal 583 residues, but the short form is predicted to have a 39-residue amino terminus encoded in intron 1 and to lack the 64 amino acids encoded in exon 1 (Fig. 1C). Similarly, two mZIP4 transcripts were identified in the mouse EST data base. The vast majority of mZIP4 ESTs correspond to the long isoform of this protein (Fig. 1B), and to date only two ESTs that correspond to a short form of mZIP4 have been entered into the EST data base. These mouse ESTs are predicted to encode a portion of a 613-residue protein with a unique 16-residue amino terminus (Fig. 1C). This amino terminus is encoded within intron 1, but the remaining 597 residues are identical between the long and short isoform of mZIP4. There is very little similarity between the amino-terminal amino acids of the short isoforms in human and mouse. The short form transcripts are predicted to originate from an alternate transcription start point within intron 1 (Fig. 1A), but are almost the same length as those that encode the long isoform. The functional significance of two isoforms of mZIP4 is unknown, but based on the relative abundance of these ESTs, the long isoform of mZIP4 is predicted to be far more abundant, and was therefore studied in more detail. Mouse ZIP4 Can Function as a Zinc Transporter—Because of its homology with the ZIP family of proteins and the presence of hZIP4 mutations in AE patients, mZIP4 is predicted to function as a zinc transporter. To assess the potential role of mZIP4 in zinc transport, the ZIP4 open reading frame encoding the more abundant long isoform was cloned into a mammalian expression vector, pCMV-Sport6, allowing high level expression from the CMV promoter. This plasmid (pCMV-mZIP4) and the vector alone were transiently transfected into HEK293 cells, and these transfected cells were then assayed for 65Zn uptake activity (Figs. 2 and 3). Consistent with an ability of mZIP4 to transport zinc, cells expressing mZIP4 from the CMV promoter accumulated ∼5-fold more 65Zn over a 60-min period than did the endogenous zinc transport activity assayed in cells transfected with the vector alone (Fig. 2A). Only low levels of zinc accumulation were detected when either of these transfected cell types were incubated with 65Zn at 0 °C. These results indicated that zinc accumulation by both the mZIP4-dependent activity and the endogenous system was temperature-dependent, and therefore likely to be transporter-mediated and not the result of zinc binding to the cell surface. Also consistent with zinc transport, mZIP4-dependent zinc accumulation was concentration-dependent and saturable. This activity showed Michaelis-Menten kinetics with an apparent Km of 1.6 ± 0.1 μm and a V max of 13.1 ± 0.2 pmol of zinc/min/mg of protein (Fig. 2B). The endogenous system in HEK293 cells had a similar apparent Km (2.1 ± 0.2 μm) but a far lower V max (4.1 ± 0.1 pmol of zinc/min/mg of protein).Fig. 3Characterization of metal specificity of uptake in cultured cells transiently transfected with an mZIP4 expression vecto
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