Drosophila fear of intimacy Encodes a Zrt/IRT-like Protein (ZIP) Family Zinc Transporter Functionally Related to Mammalian ZIP Proteins
2004; Elsevier BV; Volume: 280; Issue: 1 Linguagem: Inglês
10.1074/jbc.m411308200
ISSN1083-351X
AutoresW. Rodney Mathews, Fudi Wang, David Eide, Mark Van Doren,
Tópico(s)Heavy Metal Exposure and Toxicity
ResumoZinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm (1Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar); however, little is known about their in vivo roles. Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI. Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes. Zinc is essential for many cellular processes, and its concentration in the cell must be tightly controlled. The Zrt/IRT-like protein (ZIP) family of zinc transporters have recently been identified as the main regulators of zinc influx into the cytoplasm (1Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar); however, little is known about their in vivo roles. Previously, we have shown that fear of intimacy (foi) encodes a putative member of the ZIP family that is essential for development in Drosophila (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). Here we demonstrate that FOI can act as an ion transporter in both yeast and mammalian cell assays and is specific for zinc. We also provide insight into the mechanism of action of the ZIP family through membrane topology and structure-function analyses of FOI. Our work demonstrates that Drosophila FOI is closely related to mammalian ZIP proteins at the functional level and that Drosophila represents an ideal system for understanding the in vivo roles of this family. In addition, this work indicates that the control of zinc by ZIP transporters may play a critical role in regulating developmental processes. Zinc is an essential ion for many diverse cellular processes and can be a co-factor or structural component for many types of proteins. The regulation of cellular zinc concentration is crucial, because both zinc deficiency and excess can be detrimental to cells. In humans, a zinc deficiency can cause major health problems such as growth retardation, and defects in the immune system, central nervous system, and gastrointestinal tract function (3Hambidge M. J. Nutr. 2000; 130: 1344-1349Crossref PubMed Google Scholar). Ion transporters belonging to either of two protein families regulate cellular zinc levels: the Zrt/IRT-like protein (ZIP) 1The abbreviations used are: ZIP, Zrt/IRT-like protein; Endo H, endoglycosidase H; IRT, iron-responsive transporter; TM, transmembrane; Zrt, zinc-responsive transporter; HA, hemagglutinin; HEK, human embryonic kidney; PBS, phosphate-buffered saline; h, human; m, mouse; UAS, upstream activating sequence. 1The abbreviations used are: ZIP, Zrt/IRT-like protein; Endo H, endoglycosidase H; IRT, iron-responsive transporter; TM, transmembrane; Zrt, zinc-responsive transporter; HA, hemagglutinin; HEK, human embryonic kidney; PBS, phosphate-buffered saline; h, human; m, mouse; UAS, upstream activating sequence. family (1Eng B.H. Guerinot M.L. Eide D. Saier Jr., M.H. J. Membr. Biol. 1998; 166: 1-7Crossref PubMed Scopus (207) Google Scholar) and the cation diffusion facilitator (CDF) family (4Paulsen I.T. Saier Jr., M.H. J. Membr. Biol. 1997; 156: 99-103Crossref PubMed Scopus (295) Google Scholar). ZIP proteins are involved in zinc influx to the cytoplasm from the plasma membrane or intracellular organelles, whereas the complementary CDF proteins efflux zinc from the cytoplasm to membrane-bound compartments or outside the cell. The ZIP family was named after its first identified members Zrt1 (5Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (446) Google Scholar) and Zrt2 (6Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), the main zinc transporters in Saccharomyces cerevisiae, and IRT1 (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 (543) Google Scholar), the main iron transporter in roots of Arabidopsis, and is conserved from bacteria to humans. Several features define members of the ZIP family, including a conserved transmembrane structure and a highly conserved "signature sequence," also known as the HELP domain in some family members (see Fig. 1B). Some ZIP proteins have been shown to transport zinc specifically (5Zhao H. Eide D. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2454-2458Crossref PubMed Scopus (446) Google Scholar, 6Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar, 8Gaither L.A. Eide D.J. J. Biol. Chem. 2001; 276: 22258-22264Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 9Gaither L.A. Eide D.J. J. Biol. Chem. 2000; 275: 5560-5564Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 10Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 11Wang F. Kim B.E. Petris M.J. Eide D.J. J. Biol. Chem. 2004; 279: 51433-51441Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), whereas others, such as IRT1, have broader substrate specificity (12Korshunova Y.O. Eide D. Clark W.G. Guerinot M.L. Pakrasi H.B. Plant Mol. Biol. 1999; 40: 37-44Crossref PubMed Scopus (601) Google Scholar). Currently, little is known about the in vivo roles of these zinc transporters, particularly in the animal kingdom. Recent work indicates that careful regulation of zinc homeostasis is likely to be essential for proper embryonic development and in disease prevention. Human ZIP4 (SLC39A4) has been identified as the gene mutated in the genetic disorder acrodermatitis enteropathica (13Kury S. Dreno B. Bezieau S. Giraudet S. Kharfi M. Kamoun R. Moisan J.P. Nat. Genet. 2002; 31: 239-240Crossref PubMed Scopus (415) Google Scholar, 14Wang K. Zhou B. Kuo Y.M. Zemansky J. Gitschier J. Am. J. Hum. Genet. 2002; 71: 66-73Abstract Full Text Full Text PDF PubMed Scopus (397) Google Scholar), which results in epidermal lesions, gastrointestinal defects, and infant mortality. These symptoms mimic those associated with dietary zinc deficiencies and can be treated with high doses of dietary zinc. The mouse homolog mZIP4 can act as a zinc transporter (10Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), and when mutations found in acrodermatitis enteropathica patients are introduced into mZIP4 they affect the subcellular localization or transport function of the protein (15Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (119) Google Scholar). Thus, defective zinc transport by hZIP4 is likely the cause of acrodermatitis enteropathica, although it remains unclear how these defects in zinc transport contribute to the physiological symptoms observed in acrodermatitis enteropathica patients. The first ZIP family member identified as critical during development is encoded by the Drosophila gene, fear of intimacy (foi). foi is required for the proper formation of the embryonic gonad, which gives rise to either the testis or ovary, and the tracheal system, which forms the respiratory system of Drosophila larvae (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). In both of these processes, FOI is essential for establishment of proper tissue architecture but is not required for cell identity. Thus, during Drosophila development, foi appears to play a specific role in the morphogenesis of these tissues. Recently, another ZIP family member was also shown to play an essential developmental role; Zebrafish LIV-1 is essential for the proper cell movements during gastrulation (16Yamashita S. Miyagi C. Fukada T. Kagara N. Che Y.S. Hirano T. Nature. 2004; 429: 298-302Crossref PubMed Scopus (311) Google Scholar). Therefore, two known in vivo roles of ZIP family members involve the regulation of tissue morphogenesis during embryonic development. If ZIP family members in these model systems function similarly to their mammalian counterparts, they will provide an excellent resource for understanding how zinc transporters act in vivo to regulate development and physiology. To determine whether Drosophila FOI is a bona fide ZIP protein capable of transporting zinc, we conducted an extensive characterization of the FOI protein. In this paper, we investigate the membrane topology of FOI under conditions where we can verify that the protein retains full biological activity. We demonstrate that FOI can function as a zinc transporter in both yeast and mammalian cells and has similar ion specificity to its mammalian counterparts. Finally, we present a structure-function analysis of FOI that provides insight into the mechanism of action of the ZIP family. Our work indicates that Drosophila ZIP family members are likely to be highly similar in function to mammalian ZIP proteins. Plasmids—Yeast plasmids pFL61, pMC5 (pFL61-yZRT1), and pA6 (pFL61-aZIP1) are as in Ref. 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 (543) Google Scholar. A 3× hemagglutinin-epitope (HA) tag was inserted into three positions in FOI (pKS2.4z, (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar)), the N terminus (after amino acid 186), between TM3 and TM4 (after amino acid 531), and the C terminus (after amino acid 704). These constructs were subcloned into pUAST using the EcoRI and NotI restriction sites for expression in Drosophila S2 cells and embryos. The C-terminal HA-FOI construct was subcloned into pCMV-sport6 (Invitrogen) using EcoRI and NotI for expression in mammalian cells and into pFL61 (17Minet M. Dufour M.E. Lacroute F. Plant J. 1992; 2: 417-422PubMed Google Scholar) via a PCR strategy to generate NotI restriction sites in FOI for expression in yeast. The following site-directed mutations were introduced into the C-terminal HA-FOI construct using QuikChange (Stratagene): D308A, H554A, E584A/E588A/D591A, and Y646A. The ΔN mutation was generated by introducing two unique AscI sites in the N terminus of FOI using QuikChange to delete amino acids Asp23-Asp254 (Q22GRAK255). All of the mutated versions of FOI were subcloned into pCMV-sport6 and pFL61 for expression in mammalian and yeast cells, respectively. All cloning strategies are available upon request. Fly Stocks and Transgenic Animals—ru st faf lacZ e ca flies were used as wild-type. Transgenic Drosophila were generated for each of the three UAS-HA-FOI constructs described above using standard genomic transformation methods (18Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2330) Google Scholar). UAS-HA-FOI constructs were expressed using the Gal4 transcriptional activator localized to either the mesoderm (twist-Gal4 (19Baylies M.K. Bate M. Science. 1996; 272: 1481-1484Crossref PubMed Scopus (279) Google Scholar)) or trachea (breathless-Gal4 (20Shiga Y. Tanaka-Matakatsu M. Hayashi S. Dev. Growth Differ. 1996; 38: 99-106Crossref Scopus (228) Google Scholar)). Rescue experiments were conducted with two independent UAS-HA-FOI transgenic lines in a genetic background lacking endogenous foi activity (foi20.71), as described previously (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). At least 40 hemi-embryos were analyzed for each genotype. Computer Analysis of Sequence Data—Multiple sequence alignments and a phylogenetic tree were constructed using Clustal in the Laser-gene software (DNAStar, Inc). We considered residues conserved within a subfamily or group if 80% of its members share an identical amino acid not found in proteins of the other subfamily or group. Transmembrane domain predictions were performed using the programs listed in Table I, N-linked glycosylation predictions were computed using PROSITE (21Sigrist C.J. Cerutti L. Hulo N. Gattiker A. Falquet L. Pagni M. Bairoch A. Bucher P. Brief Bioinform. 2002; 3: 265-274Crossref PubMed Scopus (668) Google Scholar), and signal sequence predictions were computed with SignalP V2.0 (22Nielsen H. Engelbrecht J. von Brunak S. Heijne G. Protein Eng. 1997; 10: 1-6Crossref PubMed Scopus (4923) Google Scholar) and PSORT II.Table ITransmembrane domain predictionsFOIhZIP-1TM domain prediction programsSignature sequenceTotalSignature sequenceTotalSUSUI (23Hirokawa T. Boon-Chieng S. Mitaku S. Bioinformatics. 1998; 14: 378-379Crossref PubMed Scopus (1561) Google Scholar)0628DAS (24Cserzo M. Wallin E. von Simon I. Heijne G. Elofsson A. Protein Eng. 1997; 10: 673-676Crossref PubMed Google Scholar)1828HMMTOP 2.0 (25Tusnady G.E. Simon I. J. Mol. Biol. 1998; 283: 489-506Crossref PubMed Scopus (946) Google Scholar, 26Tusnady G.E. Simon I. Bioinformatics. 2001; 17: 849-850Crossref PubMed Scopus (1551) Google Scholar)1628TMPred1728TMHMM 2.0 (28Krogh A. von Larsson B. Heijne G. Sonnhammer E.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (8983) Google Scholar)0606SMART (29Schultz J. Milpetz F. Bork P. Ponting C.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5857-5864Crossref PubMed Scopus (2995) Google Scholar, 30Letunic I. Copley R.R. Schmidt S. Ciccarelli F.D. Doerks T. Schultz J. Ponting C.P. Bork P. Nucleic Acids Res. 2004; 32: D142-D144Crossref PubMed Google Scholar)061 or 28 or 9TMAP (31Persson B. Argos P. J. Mol. Biol. 1994; 237: 182-192Crossref PubMed Scopus (423) Google Scholar, 32Persson B. Argos P. Protein Sci. 1996; 5: 363-371Crossref PubMed Scopus (125) Google Scholar)2827PSORT II0527PRED-TMR (34Pasquier C. Promponas V.J. Palaios G.A. Hamodrakas J.S. Hamodrakas S.J. Protein Eng. 1999; 12: 381-385Crossref PubMed Scopus (144) Google Scholar)1615Split 3.1 Split 3.1 (35Juretic D. Lee B. Trinajstic N. Williams R.W. Biopolymers. 1993; 33: 255-273Crossref PubMed Scopus (34) Google Scholar)0628TM-Finder (36Deber C.M. Wang C. Liu L.P. Prior A.S. Agrawal S. Muskat B.L. Cuticchia A.J. Protein Sci. 2001; 10: 212-219Crossref PubMed Scopus (111) Google Scholar)1627TopPred 2 (37von Heijne G. J. Mol. Biol. 1992; 225: 487-494Crossref PubMed Scopus (1401) Google Scholar)2828 Open table in a new tab In Vitro Translation and Glycosylation Assays—pKS2.4z (FOI) and ΔN-FOI were transcribed and translated in vitro in rabbit reticulocyte lysate (Promega) in the presence or absence of canine microsomal membranes (Promega) according to manufacturer's instructions. The protein was labeled with [35S]Met (Amersham Biosciences) and run on SDS-polyacrylamide gels. Samples were treated with endoglycosidase H (Endo H) (Calbiochem), as done previously (38Tu L. Wang J. Helm A. Skach W.R. Deutsch C. Biochemistry. 2000; 39: 824-836Crossref PubMed Scopus (56) Google Scholar), to remove N-linked sugars. Briefly, 15 μl of lysate, 4 μl of 5× reaction buffer (250 mm sodium phosphate buffer, pH 7.0), and 1 μl of Denaturing solution (2% SDS, 1 m β-mercaptoethanol) were heated at 100 °C for 5 min and cooled to room temperature. 1 μl of Endo H was added prior to incubation overnight at 37 °C. S2 cell extracts were prepared by homogenizing the cells in 17 μl of denaturing buffer and heating at 100 °C for 5 min. Prior to incubating overnight at 37 °C, 2 μl of reaction buffer and 1 μl of Endo H were added. After spinning the lysate at 14,000 for 5 min, 30-35 μg of protein was loaded onto an SDS polyacrylamide gel, and a Western blot was performed essentially as described previously (39Baggett J.J. D'Aquino K.E. Wendland B. Genetics. 2003; 165: 1661-1674Crossref PubMed Google Scholar). Cell Culture and Transient Transfection—Drosophila S2 cells were cultured at room temperature in Schneider's medium (Invitrogen) containing 10% fetal bovine serum and transfected as described previously (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). Briefly, UAS-HA-FOI constructs were co-transfected with an actin-Gal 4 (gift from K. Howard) using Cellfectin (Invitrogen) according to the manufacturer's instructions. 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 FOI (wild-type or mutant). Transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Transfection efficiencies were typically 60%. Between 36 and 48 h after transfection, the cells were used for zinc uptake assays or Western blots (15Wang F. Kim B.E. Dufner-Beattie J. Petris M.J. Andrews G. Eide D.J. Hum. Mol. Genet. 2004; 13: 563-571Crossref PubMed Scopus (119) Google Scholar). Immunofluorescence Microscopy—Immunolabeling of S2 cells with permeabilization was done essentially as described previously (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). For immunolabeling of HEK293 with permeabilization, cells were fixed in 4% paraformaldehyde for 30 min at room temperature. After washing with 0.25% NH4Cl in PBS, the cells were permeabilized in PBS containing 0.1% Triton X-100. The cells were incubated with primary antibody in PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin for 1 h at room temperature, washed in PBS, and incubated with secondary antibody in PBS containing 0.1% Triton X-100 and 0.5% bovine serum albumin for 1 h at room temperature. For immunolabeling without permeabilization, S2 and HEK293 cells were incubated with primary antibody in PBS containing 0.1% bovine serum albumin for 30 min at 4 °C prior to fixation. Cells were then washed in PBS, and fixation and secondary antibody labeling was conducted as with permeabilized cells. Primary antibody and dyes used include mouse anti-HA (Roche Diagnostics, 0.8 μg/ml) and the nuclear dyes 4,6-diamidino-2-phenylindole (2 μg/ml) and OliGreen (Molecular Probes, 1:10,000). Alexa-Fluor-conjugated secondary antibodies (Molecular Probes) were used at a 1:500 dilution according to manufacturer's instructions. Cells were mounted in 70% glycerol containing 2.5% 1,4-diaza-bicyclo[2.2.2]octane (Sigma) and analyzed by either a Deltavision Deconvolution or Zeiss LSM 510 Meta Confocal microscope. Yeast Complementation Assays—The following yeast strains were used, ZHY3 (MATα zrt1::LEU2 zrt2::HIS3 ade6 can1 his3 leu2 trp1 ura3) and DY1459 (6Zhao H. Eide D. J. Biol. Chem. 1996; 271: 23203-23210Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar). Cultures were selectively grown overnight in liquid minimal medium to an A600 of 0.6-1.0 and diluted to an A600 of 0.25. Serial 1:5 dilutions were made in rich medium and were spotted onto selective minimal solid medium. Plates were incubated for 3 days at 30 °C prior to analysis. 65Zinc Uptake Assays—Zinc assays were performed essentially as described previously (10Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Parallel experiments were conducted with empty vector (pCMV-sport6) to measure endogenous 65Zn uptake rate, which was subtracted from the rate of 65Zn uptake in cells expressing FOI (wild-type or mutant) to obtain net zinc uptake values. Cell-associated radioactivity was measured with a Packard Auto-Gamma 5650-counter. 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 the protein concentrations of these cell lysates. Metal salts were obtained from Sigma and prepared as described previously (10Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). Michaelis-Menten constants were determined by nonlinear interpolation of the data, using Prism (version 4.0a for Windows, GraphPad Software, San Diego, CA). FOI Is a Member of the ZIP Family of Ion Transporters—To determine the relationship of FOI to the ZIP family, we compared the FOI protein sequence to other family members. Eukaryotic members of the ZIP family can be divided into the ZIP I/II and LIV-1 subfamilies (Fig. 1A (40Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (424) Google Scholar)) based on several conserved features, including the extended, histidine-rich N terminus of the LIV-1 subfamily (Fig. 1B). These subfamilies have further diverged into individual groups, the ZIP I/II subfamily into the ZIP I and ZIP II groups (40Gaither L.A. Eide D.J. Biometals. 2001; 14: 251-270Crossref PubMed Scopus (424) Google Scholar) and the LIV-1 subfamily into the LIV-1A and LIV-1B groups (Fig. 1A). These divergences are most obvious within the signature sequence domain, also referred to as the HELP domain in the LIV-1 subfamily, where individual groups can be defined by conservation of characteristic amino acids (Fig. 1C). Additional group-specific residues are found in other domains, such as in transmembrane domains (TM) 2 and 7 (Fig. 1C). Interestingly, Drosophila ZIP family members are found within each group that contains a mammalian ZIP family member (Fig. 1A). Individual Drosophila proteins are more closely related to their mammalian homologs than to other Drosophila ZIP family members (Fig. 1, A and B). This indicates that the distinct groups within the ZIP family existed prior to the divergence of vertebrate and invertebrate lineages. FOI is a member of the LIV-1A group within the LIV-1 subfamily, and is closely related to the human and mouse LIV-1 and ZIP4 proteins. Divergence in the signature sequence domain observed between the two ZIP subfamilies leads to differences in the predicted membrane topology in this region. Although computer algorithms generally agree about the predicted TM character of TM1-3 and TM6-8 throughout the ZIP family (Table I), the topology of the signature sequence remains unclear. This domain is variably predicted to form 0, 1, or 2 TM domains, depending on the protein and prediction program used (for consistency with the literature, we have counted this domain as TM4 and TM5). Generally, members of the ZIP I/II subfamily are predicted to have 2 TM domains within their signature sequence domain. In human ZIP1, this region is calculated to form 2 TM domains by ten of twelve computer algorithms used (Table I). TM predictions in this region are much less consistent for members of the LIV-1 subfamily, such as hLIV-1 (41Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar) and FOI. For FOI, five algorithms predict 0 TM domains, five predict 1 TM domain, and two predict 2 TM domains in this region. Thus, it is critical to determine the actual membrane topology of ZIP family members in vivo. FOI Has Extracellular N and C Termini—We wanted to experimentally address the membrane topology of FOI using a full-length functional protein. To do this, a 3× HA-epitope tag was inserted into the N terminus, middle (after TM3), or C terminus of FOI (Fig. 1B), and these constructs were transfected into cultured Drosophila S2 cells. Immunolabeling with an anti-HA antibody revealed that N-terminal HA-FOI could be detected in both permeabilized and non-permeabilized cells (Fig. 2A). Because only extracellular epitopes can be immunolabeled in non-permeabilized cells, this indicates that the N terminus of FOI is extracellular. The middle region of FOI was detected only after permeabilization, confirming that this large, unconserved loop is cytoplasmic (Fig. 2A). The predictions for the localization of the C terminus of FOI vary depending on the number of TMs in the signature sequence. If this region contains a single TM, and TM6-8 are as predicted, the C terminus of FOI should be intracellular. If the signature sequence contains 0 or 2 TMs, the C terminus should be extracellular. We found that the C-terminal HA-FOI construct could be detected in both permeabilized and non-permeabilized cells, indicating that it is extracellular. Thus, the signature sequence is unlikely to have a single TM and likely has either 0 or 2 TM domains (Fig. 2B). To confirm that the HA-tagged versions of FOI retain biological activity, we tested the ability of each of these constructs to supply FOI function in vivo in a transgenic rescue assay (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar). foi mutant embryos have defects in gonad coalescence, in which the cells of the gonad fail to properly associate with one another, and in trachea formation, in which some tracheal branches fail to connect correctly with neighboring branches (2Van Doren M. Mathews W.R. Samuels M. Moore L.A. Broihier H.T. Lehmann R. Development. 2003; 130: 2355-2364Crossref PubMed Scopus (78) Google Scholar, 42Jenkins A.B. McCaffery J.M. Van Doren M. Development. 2003; 130: 4417-4426Crossref PubMed Scopus (64) Google Scholar). Expression of each HA-tagged version of FOI in the appropriate tissue was able to fully rescue the gonad and tracheal defects normally observed in foi mutants (Fig. 2, C and D). Thus, insertion of the HA-tag in the N terminus, middle, or C terminus of FOI does not disrupt the ability of the protein to function in vivo, indicating that the tagged proteins accurately represent the membrane topology of the wild-type FOI protein. The N Terminus of FOI Is Glycosylated—FOI contains eight consensus N-glycosylation sites, six on the N terminus, one between TM3 and TM4, and one in the signature sequence domain (Fig. 1B). To determine which, if any, of these sites are glycosylated, we translated FOI in vitro in the presence or absence of canine microsomal membranes, which allow for co-translational processing of the protein. In the presence of microsomes, FOI was detected at both the predicted, unprocessed size and at a higher molecular mass (Fig. 3A). The larger protein was Endo H-sensitive (Fig. 3A) indicating that FOI can be glycosylated in vitro. To confirm that FOI is a glycoprotein in vivo, we expressed HA-tagged FOI in Drosophila S2 cells. Again, FOI was observed at two molecular masses, the larger of which is Endo H-sensitive (Fig. 3B) confirming that FOI is a glycoprotein (in contrast to mammalian cells, mature glycoproteins in insects are often Endo H-sensitive (43Tomiya N. Betenbaugh M.J. Lee Y.C. Acc. Chem. Res. 2003; 36: 613-620Crossref PubMed Scopus (49) Google Scholar)). To determine whether glycosylation occurs on the N terminus of FOI, we deleted the entire N terminus (ΔN) and translated the resulting protein in vitro. The ΔN protein was detected at approximately the same molecular mass in both the presence and absence of microsomes and was not Endo H-sensitive (Fig. 3C). These data suggest that the N terminus is the only region of FOI that is glycosylated, further indicating that this domain is extracellular and that the unglycosylated region of the protein between TM3 and TM4 is cytoplasmic. FOI Functions as a Zinc Transporter in Both Yeast and Mammalian Cells—We next wanted to determine whether FOI can function as a zinc transporter and is therefore a true member of the ZIP family. Mammalian LIV-1 subfamily members have been shown to affect intracellular zinc accumulation (41Taylor K.M. Morgan H.E. Johnson A. Hadley L.J. Nicholson R.I. Biochem. J. 2003; 375: 51-59Crossref PubMed Scopus (134) Google Scholar, 44Begum N.A. Kobayashi M. Moriwaki Y. Matsumoto M. Toyoshima K. Seya T. Genomics. 2002; 80: 630-645Crossref PubMed Scopus (118) Google Scholar), and for mZIP4 and mZIP5, this has been demonstrated to be due to an increased rate of zinc uptake (10Dufner-Beattie J. Wang F. Kuo Y.M. Gitschier J. Eide D. Andrews G.K. J. Biol. Chem. 2003; 278: 33474-33481Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar, 11Wang F. Kim B.E. Petris M.J. Eide D.J. J. Biol. Chem. 2004; 279: 51433-51441Abstract Full Text Full Text PDF Pub
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