Human NRAMP2/DMT1, Which Mediates Iron Transport across Endosomal Membranes, Is Localized to Late Endosomes and Lysosomes in HEp-2 Cells

Mitsuaki Tabuchi,Tamotsu Yoshimori,Kazuhito Yamaguchi,Tsutomu Yoshida,Fumio Kishi

doi:10.1074/jbc.m001478200

Abstract

NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent metal transporter 1) is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe(2+), Zn(2+), Mn(2+), Cu(2+), Cd(2+), Co(2+), Ni(2+), and Pb(2+), and mediates active proton-coupled transport. Recently, it has been shown that the microcytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2. These findings strongly suggested that NRAMP2 is the apical membrane iron transporter in intestinal epithelial cells and the endosomal iron transporter in transferrin cycle endosomes of other cells. To investigate the cellular functions of NRAMP2, we generated a polyclonal antibody against the N-terminal cytoplasmic domain of human NRAMP2. The affinity-purified anti-NRAMP2 N-terminal antibody recognized a 90-116-kDa membrane-associated protein, and this band was shifted to 50 kDa by deglycosylation with peptide N-glycosidase F. Subcellular fractionation revealed that NRAMP2 co-sedimented with the late endosomal and lysosomal membrane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not with the transferrin receptor in early endosomes. The intracellular localization of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-NRAMP2 was examined by immunofluorescence staining and by native fluorescence of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in vesicular structures and were colocalized with LAMP-2, but not with EEA1 (early endosome antigen 1) or the transferrin receptor. These results indicated that NRAMP2 is localized to the late endosomes and lysosomes, where NRAMP2 may function to transfer the endosomal free Fe(2+) into the cytoplasm in the transferrin cycle.

Highlights

NRAMP2/DMT1 is a divalent metal transporter conserved from prokaryotes to higher eukaryotes that exhibits an unusually broad substrate range, including Fe2؉, Zn2؉, Mn2؉, Cu2؉, Cd2؉, Co2؉, Ni2؉, and Pb2؉, and mediates active proton-coupled transport
It has been shown that the microcytic anemia mouse and the Belgrade (b) rat, which have inherited defects in iron transport that result in iron deficiency anemia, have the same missense mutation (G185R) in Nramp2
We produced a polyclonal antibody against the N-terminal cytoplasmic tail of human NRAMP2 by immunization with recombinant TrpENRAMP2-(1– 66) protein produced in E. coli and showed that this anti-NRAMP2 N antiserum recognized the functional NRAMP2 protein expressed in fission yeast [18]

Summary

The abbreviations used are

Transferrin; TfR, transferrin receptor; NRAMP, natural resistance-associated macrophage protein; DMT1, divalent metal transporter 1; pAb, polyclonal antibody; mAb, monoclonal antibody; GST, glutathione S-transferase; EEA1, early endosome antigen 1; LAMP, lysosome-associated membrane protein; PNS, postnuclear supernatant; PBS, phosphate-buffered saline; PNGase F, peptide N-glycosidase F; PFA, p-formaldehyde; GFP, green fluorescent protein. B animal models [11, 12] Both the mk mouse and the b rat have been shown to carry the same mutation at Nramp, a glycine-to-arginine substitution (G185R) in one of the predicted transmembrane domains (TM4) of the protein. Immunocytochemical analysis with protein-specific antibodies revealed that Nramp is expressed in the late endosomal and lysosomal membranes in macrophages and is recruited to the membrane of the phagosome upon phagocytosis (20 –22). We have reported both cDNA and genomic DNA structures of human NRAMP2 [26, 27], generated human NRAMP2 N-terminal domain-specific antiserum, and showed that this antiserum recognized functional recombinant NRAMP2 protein expressed in fission yeast [18]. We investigate the subcellular localization of NRAMP2 in cultured human cells with the affinity-purified anti-NRAMP2 Nterminal antibody and showed that NRAMP2 is localized to late endosomes and lysosomes

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION