M. Tabuchi et al., Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells, J BIOL CHEM, 275(29), 2000, pp. 22220-22228
NRAMP2 (natural resistance-associated macrophage protein 2)/DMT1 (divalent
metal transporter 1) is a divalent metal transporter conserved from prokary
otes 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. Recently, it has been shown that the micr
ocytic anemia (mk) mouse and the Belgrade (b) rat, which have inherited def
ects in iron transport that result in iron deficiency anemia, have the same
missense mutation (G185R) in Nramp2. These findings strongly suggested tha
t NRAMP2 is the apical membrane iron transporter in intestinal epithelial c
ells and the endosomal iron transporter in transferrin cycle endosomes of o
ther cells. To investigate the cellular functions of NRAMP2, we generated a
polyclonal antibody against the N-terminal cytoplasmic domain of human NRA
MP2, 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 re
vealed that NRAMP2 co-sedimented with the late endosomal and lysosomal memb
rane proteins and LAMP-1 (lysosome-associated membrane protein 1), but not
with the transferrin receptor in early endosomes, The intracellular localiz
ation of endogenous NRAMP2 and recombinant green fluorescent protein (GFP)-
NRAMPS was examined by immunofluorescence staining and by native fluorescen
ce of GFP, respectively. Both endogenous and GFP-NRAMP2 were detected in ve
sicular structures and were colocalized with LAMP-S, but not with EEA1 (ear
ly 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 Fe2+ into the cytoplasm in the
transferrin cycle.