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

2000; Elsevier BV; Volume: 275; Issue: 29 Linguagem: Inglês

10.1074/jbc.m001478200

1083-351X

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

Hemoglobinopathies and Related Disorders

NRAMP2 (naturalresistance-associated macrophageprotein 2)/DMT1 (divalentmetal 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 membraneprotein 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 endosomeantigen 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.