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.