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PMID:10751401

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Citation

Tabuchi, M, Yoshimori, T, Yamaguchi, K, Yoshida, T and Kishi, F (2000) Human NRAMP2/DMT1, which mediates iron transport across endosomal membranes, is localized to late endosomes and lysosomes in HEp-2 cells. J. Biol. Chem. 275:22220-8

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.

Links

PubMed Online version:10.1074/jbc.M001478200

Keywords

Animals; Biological Transport; Carrier Proteins/metabolism; Cation Transport Proteins; Cell Line; DNA-Binding Proteins/metabolism; Endosomes/metabolism; Humans; Iron/metabolism; Iron-Binding Proteins; Lysosomes/metabolism; Membrane Proteins/metabolism; Mice; Rats; Transcription Factors/metabolism

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:NRAM2

located_in

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NRAM2

located_in

GO:0005765: lysosomal membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NRAM2

located_in

GO:0031902: late endosome membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NRAM2

GO:0005765: lysosomal membrane

ECO:0000314:

C

complete

HUMAN:NRAM2

GO:0031902: late endosome membrane

ECO:0000314:

C

complete

HUMAN:NRAM2

GO:0048471: perinuclear region of cytoplasm

ECO:0000314:

C

complete

HUMAN:NRAM2

part_of

GO:0005765: lysosomal membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NRAM2

part_of

GO:0031902: late endosome membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:NRAM2

part_of

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:Q8IUD7

part_of

GO:0048471: perinuclear region of cytoplasm

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:Q8IUD7

part_of

GO:0031902: late endosome membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

HUMAN:Q8IUD7

part_of

GO:0005765: lysosomal membrane

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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