Nramp 2 (DCT1/DMT1) Expressed at the Plasma Membrane Transports Iron and Other Divalent Cations into a Calcein-accessible Cytoplasmic Pool
2000; Elsevier BV; Volume: 275; Issue: 46 Linguagem: Inglês
10.1074/jbc.m005387200
ISSN1083-351X
AutoresVirginie Picard, Gregory Govoni, Nada Jabado, Philippe Gros,
Tópico(s)Trace Elements in Health
ResumoNramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7–TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7–TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co2+ and Cd2+ but not Mg2+ into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein. Nramp2, also known as DMT1 and DCT1, is a 12-transmembrane (TM) domain protein responsible for dietary iron uptake in the duodenum and iron acquisition from transferrin in peripheral tissues. Nramp2/DMT1 produces by alternative splicing two isoforms differing at their C terminus (isoforms I and II). The subcellular localization, mechanism of action, and destination of divalent cations transported by the two Nramp2 isoforms are not completely understood. Stable CHO transfectants expressing Nramp2 isoform II modified by addition of a hemaglutinin epitope in the loop defined by the TM7–TM8 interval were generated. Immunofluorescence with permeabilized and intact cells established that Nramp2 isoform II is expressed at the plasma membrane and demonstrated the predicted extracytoplasmic location of the TM7–TM8 loop. Using the fluorescent, metal-sensitive dye calcein, and a combination of membrane-permeant and -impermeant iron chelators, Nramp2 transport was measured and quantitated with respect to kinetic parameters and at steady state. Iron transport at the plasma membrane was time- and pH-dependent, saturable, and proportional to the amount of Nramp2 expression. Iron uptake by Nramp2 at the plasma membrane was into the nonferritin-bound, calcein-accessible so-called "labile iron pool." Ion selectivity experiments show that Nramp2 isoform II can also transport Co2+ and Cd2+ but not Mg2+ into the calcein-accessible pool. Parallel experiments with transfectants expressing the lysosomal Nramp1 homolog do not show any divalent cation transport activity, establishing major functional differences between Nramp1 and Nramp2. Monitoring the effect of Nramp2 on the calcein-sensisitve labile iron pool allows a simple, rapid, and nonisotopic approach to the functional study of this protein. transmembrane iron response element Chinese hamster ovary labile iron pool ferrous ammonium sulfate salicyladehyde isocotinoyl hydrazonye 6-desferrioxamine polymerase chain reaction hemagglutinin phosphate-buffered saline bovine serum albumin acetoxymethylester 4-morpholineethanesulfonic acid plasma membrane The Nramp2 gene (naturalresistance-associated macrophageprotein-2), also known as DCT1 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar) and DMT1 (2Fleming R.E. Migas M.C. Zhou X. Jiang J. Britton R.S. Brunt E.M. Tomatsu S. Waheed A. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3143-3148Crossref PubMed Scopus (257) Google Scholar), was first identified in mammals (3Gruenheid S. Cellier M. Vidal S. Gros P. Genomics. 1995; 25: 514-525Crossref PubMed Scopus (252) Google Scholar) and belongs to a large family of integral membrane proteins highly conserved throughout evolution, from bacteria to man (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar, 5Tabuchi M. Yoshida T. Takegawa K. Kishi F. Biochem. J. 1999; 344: 211-219Crossref PubMed Google Scholar, 6Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Crossref PubMed Scopus (177) Google Scholar, 7Agranoff D. Monahan I.M. Mangan J.A. Butcher P.D. Krishna S. J. Exp. Med. 1999; 190: 717-724Crossref PubMed Scopus (117) Google Scholar, 8Curie C. Alonso J.M. Le Jean M. Ecker J.R. Briat J.F. Biochem. J. 2000; 347: 749-755Crossref PubMed Scopus (416) Google Scholar). Structural similarity in the Nramp family translates into functional homology because several members have been shown to function as divalent metal transporters (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar, 8Curie C. Alonso J.M. Le Jean M. Ecker J.R. Briat J.F. Biochem. J. 2000; 347: 749-755Crossref PubMed Scopus (416) Google Scholar, 9Chen X.Z. Peng J.B. Cohen A. Nelson H. Nelson N. Hediger M.A. J. Biol. Chem. 1999; 274: 35089-35094Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 10Pinner E. Gruenheid S. Raymond M. Gros P. J. Biol. Chem. 1997; 272: 28933-28938Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 11D'Souza J. Cheah P.Y. Gros P. Chia W. Rodrigues V. J. Exp. Biol. 1999; 202: 1909-1915PubMed Google Scholar). Computer-assisted sequence analysis of Nramp2 protein predicts a polytopic membrane protein composed of 12 transmembrane (TM)1 segments, a glycosylated extracytoplasmic loop, a consensus transport signature (found in several prokaryotic and eukaryotic transport proteins), and precisely conserved charged amino acids in TM domains (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar). However, the membrane topology of Nramp2 has yet to be experimentally verified. TheNramp2 gene produces by alternative splicing of the 3′-terminal exon two distinct mRNAs that are distinguished by different C-terminal amino acid sequences and by the presence (isoform I) or absence (isoform II) of an iron response element (IRE) located in the 3′-untranslated region of the mRNA (12Lee P.L. Gelbart T. West C. Halloran C. Beutler E. Blood Cells Mol. Dis. 1998; 24: 199-215Crossref PubMed Scopus (273) Google Scholar). Nramp2 isoform I protein (13Moffett P. Dayo M. Reece M. McCormick M.K. Pelletier J. Genomics. 1996; 35: 144-155Crossref PubMed Scopus (29) Google Scholar) is expressed at the duodenum brush border where its expression is regulated by dietary iron (14Canonne-Hergaux F. Gruenheid S. Ponka P. Gros P. Blood. 1999; 93: 4406-4417Crossref PubMed Google Scholar). Mutations (G185R) atNramp2 in mice (mk) and rats (b) cause a severe form of iron deficiency and microcytic anemia, associated with impaired iron absorption at the intestinal mucosa (15Fleming M.D. Trenor 3rd, C.C. Su M.A. Foernzler D. Beier D.R. Dietrich W.F. Andrews N.C. Nat. Genet. 1997; 16: 383-386Crossref PubMed Scopus (1015) Google Scholar, 16Fleming M.D. Romano M.A. Su M.A. Garrick L.M. Garrick M.D. Andrews N.C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 1148-1153Crossref PubMed Scopus (801) Google Scholar, 17Russell E.S. Nash D.J. Bernstein S.E. Kent E.L. McFarland E.C. Matthews S.M. Norwood M.S. Blood. 1970; 35: 838-850Crossref PubMed Google Scholar, 18Edwards J.A. Hoke J.E. Proc. Soc. Exp. Biol. Med. 1972; 141: 81-84Crossref PubMed Scopus (61) Google Scholar, 19Farcich E.A. Morgan E.H. Am. J. Hematol. 1992; 39: 9-14Crossref PubMed Scopus (53) Google Scholar); together, these results have indicated that Nramp2 isoform I is responsible for iron transport from the duodenum lumen into the cytoplasm of epithelial cells. However, plasma membrane staining for Nramp2 has been difficult to ascertain (20Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (263) Google Scholar, 21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar) except at the intestinal brush border of iron-depleted animals (14Canonne-Hergaux F. Gruenheid S. Ponka P. Gros P. Blood. 1999; 93: 4406-4417Crossref PubMed Google Scholar). Subcellular localization studies of endogenous protein in Hep-2 cells, as well as studies using stably transfected CHO and RAW cells, show that Nramp2 is also expressed in a subcellular vesicular compartment identified as early (20Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (263) Google Scholar, 21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar) or late endosomes (22Tabuchi M. Yoshimori T. Yamaguchi K. Yoshida T. Kishi F. J. Biol. Chem. 2000; 275: 22220-22228Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar) or both. These findings have suggested that Nramp2, and more specifically its isoform II, may also be implicated in iron acquisition in peripheral tissues as well, transporting transferrin-bound iron across the membrane of acidified endosomes into the cytoplasm (20Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (263) Google Scholar, 21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar). This possible role for Nramp2 isoform II in iron transport at the plasma membrane or in acidified endosomes has yet to be explored.The mechanistic basis of transport has been analyzed inXenopus oocytes where Nramp2 (DCT1) isoform I transports a number of divalent cations such as Fe2+, Zn2+, Cd2+, Co2+, and Cu2+. This transport is pH-dependent, electrogenic, and associated with the symport of a single proton (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar). Nramp2-mediated iron transport was also demonstrated at the apical membrane of Caco-2 cells (23Tandy S. Williams M. Leggett A. Lopez-Jimenez M. Dedes M. Ramesh B. Srai S.K. Sharp P. J. Biol. Chem. 2000; 275: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). In addition, transient overexpression of the wild type but not G185R Nramp2 in HEK293T cells results in a robust stimulation of cellular55Fe2+ uptake (21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar). The cellular compartment to which iron is delivered by Nramp2 has not yet been established. Indeed, transport studies using 55Fe as a ligand monitor total cellular accumulation and do not distinguish between free iron, ferritin-bound iron, and iron sequestered in subcellular organelles. Although electrophysiological measurements in Xenopusoocytes are extremely useful to elucidate the bioenergetics and mechanism of transport, such experiments do not recreate the normal environment and subcellular compartments of mammalian cells.To gain further insight into the structure and function of Nramp2 (isoform II), including the site of transport and destination of the transported substrate, CHO cell clones that stably express an epitope tagged copy of Nramp2 (isoform II) were created. Nramp2 (isoform II) was expressed at the plasma membrane and the epitope tag inserted between TM7 and TM8 was found to be accessible from the medium, indicating that the corresponding loop is indeed extracellular. Transport studies using the metal-sensitive and fluorescent dye calcein demonstrate that Nramp2 (isoform II) can indeed function as a pH-dependent divalent cation transporter at the plasma membrane acting on Fe2+, Co2+, and Cd2+. Our results also show that the incoming iron transported by Nramp2 (isoform II) is delivered to the cytoplasm of CHO cells, within the so-called "labile iron pool" (LIP) (24Epsztejn S. Kakhlon O. Glickstein H. Breuer W. Cabantchik I. Anal. Biochem. 1997; 248: 31-40Crossref PubMed Scopus (325) Google Scholar).DISCUSSIONOne of the distinguishing features of the Nramp protein family is the presence of a 20–46-amino acid residues hydrophilic loop delineated by TM7 and TM8. The sequence of this loop is not conserved throughout evolution; for example mammalian Nramp1 and Nramp2 show 19 of 42 identical residues with nine conservative substitutions in this segment. However, this segment often contains N-linked glycosylation signals (NXS or NXT), suggesting that this loop would be glycosylated and extracytoplasmic (35van Geest M. Lolkema J.S. Microbiol. Mol. Biol. Rev. 2000; 64: 13-33Crossref PubMed Scopus (164) Google Scholar). This, together with the presence of a transport signature in the adjacent TM9–TM10 interval, which is conserved at the cytoplasmic face of several bacterial periplasmic permeases, was initially used to anchor the predicted topological arrangement of the 12 TM domains of the Nramp protein (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar). In this study, the insertion of an epitope tag in the TM8–TM9 loop did not affect protein stability, membrane targeting, or transport properties. In addition, immunofluorescence studies in nonpermeabilized Nramp2 CHO transfectants with an anti-tag antibody confirmed that this loop was indeed extracellular in Nramp2. These results provide a first validation of the initial topological model of the protein based on hydropathy profiling and suggest that this approach could be used for topology mapping of individual TM domains of Nramp2.The mechanism of transport of Nramp2 has so far been studied after expression in Xenopus laevis oocytes (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar), in nontransfected or in stably or transiently transfected cultured cells (21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar, 23Tandy S. Williams M. Leggett A. Lopez-Jimenez M. Dedes M. Ramesh B. Srai S.K. Sharp P. J. Biol. Chem. 2000; 275: 1023-1029Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar) and by the use of radioisotopes. Although those are sound technological approaches there are certain limitations. Studies in Xenopusoocytes require unique expertise and instrumentation and may not fully recreate the transport environment of mammalian cells.55Fe2+ and 56Fe2+ are high energy emitters that require containment that limit their use. In addition, isotopic iron has a tendency to bind nonspecifically to various cellular components and proteins in live and dead cells, producing a relative high background in whole cells assays. Also, the rapid oxidation of Fe2+ (substrate) to Fe3+(nonsubstrate) at various degrees in aqueous solutions complicates analysis of transport assays. Finally, neither method provides information on the destination of the transported iron, because they cannot distinguish between intracellular iron complexed to ferritin, and the cytoplasmic pool of free Fe2+ (LIP). Another limitation of these methods is that the subcellular localization of the protein to a functionally relevant site is difficult to establish with certainty. Calcein binds to a number of divalent cations, Ca2+, Fe2+, Cd2+, Mg2+, and Co2+, and some of these (notably Fe2+ and Co2+) are potent quenchers of calcein fluorescence. In addition, only binding of Fe2+ but not Fe3+ to calcein results in fluorescence quenching (32Breuer W. Epsztejn S. Millgram P. Cabantchik I.Z. Am. J. Physiol. 1995; 268: C1354-C1361Crossref PubMed Google Scholar), alleviating the problems associated with change in valence of the iron atom during transport experiments. Calcein has been previously used to monitor in nontransfected cells the size of the ferritin-free, so-called labile iron pool (24Epsztejn S. Kakhlon O. Glickstein H. Breuer W. Cabantchik I. Anal. Biochem. 1997; 248: 31-40Crossref PubMed Scopus (325) Google Scholar, 36Picard V. Epsztejn S. Santambrogio P. Cabantchik Z.I. Beaumont C. J. Biol. Chem. 1998; 273: 15341-15382Abstract Full Text Full Text PDF Scopus (115) Google Scholar), thus suggesting that it could also be used to monitor the activity of Nramp2 in intact cells, and may also provide information on the status of iron transported by Nramp2. By using a combination of membrane-impermeant (HES-DFO) and membrane-permeant (SIH) Fe2+ chelators in calcein-loaded cells, we were able to accurately measure in a kinetic fashion the effect of Nramp2 expression on the size of the intracellular iron pool in transfected CHO cells. We observed robust influx of iron in Nramp2 transfectants that was related to the amount of Nramp2 protein expressed in these clones; over a 3-min loading period, Nramp2-expressing cells showed a 5-fold increase in the initial rate of fluorescence quenching and 3–4-fold increase in total SIH-sensitive fluorescence over control CHO cells (ΔF). In this assay, Nramp2 transport of Fe2+ is pH-dependent (optimum at pH 5.5–6.0) and is saturated at approximately 1 μm, a value in good agreement with that measured in Xenopus oocytes for Nramp2/DCT1-mediated (K 0.5 = 2 μm) and SMF1-mediated (K m = 2.2 μm) iron transport (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar, 9Chen X.Z. Peng J.B. Cohen A. Nelson H. Nelson N. Hediger M.A. J. Biol. Chem. 1999; 274: 35089-35094Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Finally, Nramp2 expressed at the plasma membrane is shown to transport Co2+ and Cd2+ but not Mg2+. The fate of iron transported at the plasma membrane by an iron transporter such as Nramp2 has been debated (reviewed in Ref. 37Ponka P. Kidney Int. 1999; 55: S2-S11Abstract Full Text Full Text PDF Google Scholar). It could become quickly complexed to ferritin, sequestered away in subcellular membrane compartments or organelles such as mitochondria or could be part of a free cytoplasmic pool previously identified as the LIP (24Epsztejn S. Kakhlon O. Glickstein H. Breuer W. Cabantchik I. Anal. Biochem. 1997; 248: 31-40Crossref PubMed Scopus (325) Google Scholar). Results from this study show that iron transport into CHO cells by Nramp2 is into the calcein-accessible cytoplasmic LIP. In addition, kinetic and saturation measurements show that most of the iron transported by Nramp2 during the monitoring period is into that pool.The Nramp2 gene produces by alternative splicing of the 3′-terminal exon two distinct proteins and mRNAs that are distinguished by different C-terminal amino acid sequences and the presence (isoform I) or absence (isoform II) of an IRE located in the 3′-untranslated region of the mRNA (12Lee P.L. Gelbart T. West C. Halloran C. Beutler E. Blood Cells Mol. Dis. 1998; 24: 199-215Crossref PubMed Scopus (273) Google Scholar). The isoform I protein (14Canonne-Hergaux F. Gruenheid S. Ponka P. Gros P. Blood. 1999; 93: 4406-4417Crossref PubMed Google Scholar) is expressed at the duodenum brush border where it is regulated by dietary iron and ultimately responsible for iron transport from the duodenum lumen into the cytoplasm of eptihelial cells. It is the isoform I of Nramp2 that has been used in transport assays inXenopus oocytes to demonstrate the iron transport by Nramp2 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar). Subcellular localization studies in intact cells (Hep-2), in transfected CHO cells, and in RAW mouse macrophages show that Nramp2 is also expressed in a subcellular vesicular compartment identified as early (20Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (263) Google Scholar, 21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar) or late endosomes (22Tabuchi M. Yoshimori T. Yamaguchi K. Yoshida T. Kishi F. J. Biol. Chem. 2000; 275: 22220-22228Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar). In several cell lines tested (murine erythroleukemia MEL cells and Sertoli TM4 cells), it appears that the majority of the protein expressed at that site is the isoform II of Nramp2. 2S. Gruenheid and F. Canonne-Hergaux, unpublished results.Co-localization studies with transferrin-fluorescein isothiocyanate (20Gruenheid S. Canonne-Hergaux F. Gauthier S. Hackam D.J. Grinstein S. Gros P. J. Exp. Med. 1999; 189: 831-841Crossref PubMed Scopus (263) Google Scholar, 21Su M.A. Trenor C.C. Fleming J.C. Fleming M.D. Andrews N.C. Blood. 1998; 92: 2157-2163Crossref PubMed Google Scholar) and in vivo studies in mk andb mutant animals support a role for Nramp2 in the transport of transferrin iron from acidified endosomes into the cytoplasm of peripheral tissues (38Orgad S. Nelson H. Segal D. Nelson N. J. Exp. Biol. 1998; 201: 115-120Crossref PubMed Google Scholar, 39Farcich E.A. Morgan E.H. Am. J. Physiol. 1992; 262: R220-R224PubMed Google Scholar). In the present report, we have been able to establish by immunofluorescence that the IRE negative isoform II of the protein (used in our expression construct) can be expressed at the plasma membrane. The strict pH dependence of transport together with the rapid fluorescence quenching kinetics observed in Nramp2transfectants indicates that Nramp2 isoform II can indeed function as a pH-dependent divalent cation transporter at the plasma membrane of these cells. The fact that both isoforms I and II can be targeted to the PM membranes in primary cells and in transfected cells would suggest that the membrane targeting information required for this process is not located in the extreme C terminus of the protein, which shows no sequence homology between isoforms I and II. Thus, it is interesting to speculate that NPXY and YSCF motifs identified by scrutiny of the N-terminal sequence of Nramp2 may be implicated in this process, because they have been identified as sorting motifs for other membrane proteins such as transferrin receptor, Lamp-1, CD3, and H,K-ATPase (40Bonifacio J.S. Dell Angelica E., C. J. Cell Biol. 1999; 145: 923-926Crossref PubMed Scopus (365) Google Scholar, 41Chen W.J. Goldstein J.L. Brown M.S. J. Biol. Chem. 1990; 265: 3116-3123Abstract Full Text PDF PubMed Google Scholar, 42Letourneur F. Klausner R.D. Cell. 1992; 69: 1143-1157Abstract Full Text PDF PubMed Scopus (459) Google Scholar).The fluorescence quenching method developed here to monitor Nramp2 transport offers several advantages over current methods. First, its does not rely on the use of radioisotopic derivatives of Nramp2 substrates, some of which are not commercially available. Second, it is carried out in intact mammalian cells and is not destructive, and cells analyzed in this fashion can be further put through other tests. Third, the use of two chelators allows one to easily distinguish nonspecific binding from transport and intracellular accumulation of iron. Fourth, quantitative and rapid kinetic data can be obtained for transport of three divalent cations by this assay. Finally, and most importantly, we show that divalent cations transport by Nramp2, including Fe2+ is into a free cytoplasmic pool that is accessible to calcein. This assay can now be used to identify structure/function relationships in Nramp2 variants generated by site-directed mutagenesis, including identification of residues underlying pH dependence and substrate specificity of the transporter. In addition, insertion of the HA tag in the predicted TM7–TM8 loop does not seem to affect transport activity, compared with Nramp2 marked with a c-Myc tag at the C terminus. Thus, membrane topology of Nramp2 could be determined by immunofluorescence in CHO cells expressing recombinant proteins engineered with epitope tags at different positions and tested for biological activity in this assay (28Kast C. Gros P. Biochemistry. 1998; 37: 2305-2313Crossref PubMed Scopus (76) Google Scholar).As opposed to their Nramp2 counterparts, CHO cells transfected with and expressing high levels of the macrophage-specific Nramp1 protein (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar) do not demonstrate increased divalent cations import in the calcein quenching assay. The high degree of sequence homology between mammalian Nramp1 and Nramp2 (78% sequence similarity), and the report that a loss-of-function mutation at the mvl locus can be corrected by Nramp1 in transgenic flies (11D'Souza J. Cheah P.Y. Gros P. Chia W. Rodrigues V. J. Exp. Biol. 1999; 202: 1909-1915PubMed Google Scholar) in a manner similar to that of increased dietary metals (38Orgad S. Nelson H. Segal D. Nelson N. J. Exp. Biol. 1998; 201: 115-120Crossref PubMed Google Scholar), together strongly suggest that Nramp1 can transport divalent cations as well. The lack of divalent cation transport reported here for Nramp1 CHO cells (Figs. 2 E and3 B) is likely to be the result of the absence of Nramp1 expression at the plasma membrane concomitant to strict expression in the lysosomal compartment of these cells (25Gruenheid S. Pinner E. Desjardins M. Gros P. J. Exp. Med. 1997; 185: 717-730Crossref PubMed Scopus (387) Google Scholar, 43Govoni G. Canonne-Hergaux F. Pfeifer C.G. Marcus S.L. Mills S.D. Hackam D.J. Grinstein S. Malo D. Finlay B.B. Gros P. Infect. Immun. 1999; 67: 2225-2233Crossref PubMed Google Scholar). The results obtained here with Nramp1 and Nramp2 CHO transfectants clearly suggest that the protein signals underlying targeting of the two proteins to distinct subcellular compartments can be identified in chimeric proteins expressed in CHO cells and analyzed by this assay.AcknowledgmentWe are grateful to Martine Brault for technical support during this work. The Nramp2 gene (naturalresistance-associated macrophageprotein-2), also known as DCT1 (1Gunshin H. Mackenzie B. Berger U.V. Gunshin Y. Romero M.F. Boron W.F. Nussberger S. Gollan J.L. Hediger M.A. Nature. 1997; 388: 482-488Crossref PubMed Scopus (2633) Google Scholar) and DMT1 (2Fleming R.E. Migas M.C. Zhou X. Jiang J. Britton R.S. Brunt E.M. Tomatsu S. Waheed A. Bacon B.R. Sly W.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3143-3148Crossref PubMed Scopus (257) Google Scholar), was first identified in mammals (3Gruenheid S. Cellier M. Vidal S. Gros P. Genomics. 1995; 25: 514-525Crossref PubMed Scopus (252) Google Scholar) and belongs to a large family of integral membrane proteins highly conserved throughout evolution, from bacteria to man (4Cellier M. Prive G. Belouchi A. Kwan T. Rodrigues V. Chia W. Gros P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10089-10093Crossref PubMed Scopus (290) Google Scholar, 5Tabuchi M. Yoshida T. Takegawa K. Kishi F. Biochem. J. 1999; 344: 211-219Crossref PubMed Google Scholar, 6Makui H. Roig E. Cole S.T. Helmann J.D. Gros P. Cellier M.F. Mol. Microbiol. 2000; 35: 1065-1078Crossref PubMed Scopus (177) Google Scholar, 7Agranoff D. Monahan I.M. Mangan J.A. Butcher P.D. Krishna S. J. Exp. Med. 1999; 190: 717-724Crossref PubMed Scopus (117) Google Scholar, 8Curie C. Alonso J.M. Le Jean M. 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Computer-assisted sequence analysis of Nramp2 protein predicts a polytopic membrane protein composed of 12 transmembrane (TM)1 segments, a glycosylated extracytoplasmic loop, a consensus transport signature (found in several prokaryotic and eukaryotic transport proteins), and precisely conserved charged amino acids in TM domains (4Cellier M. Prive G. Belouchi A. Kwan T. Rodr
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