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

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.Iron is indispensable for life, serving as a metal cofactor for many enzymes, including both non-heme and heme proteins.The normal human adult has 35-45 mg of iron/kg of body weight. More than two-thirds of the body's iron content is incorporated into hemoglobin in developing erythroid precursors and mature red cells (1). Nutritional iron absorption (both heme and non-heme iron) occurs primarily in the intestine. Heme iron constitutes only a small fraction of the available dietary iron, but it is highly available for absorption. On the other hand, the absorption of non-heme iron is low and markedly regulated in the first part of the duodenum, in which the acidic pH promotes solubilization of iron transformed to Fe 2ϩ by ferrireductase and ascorbate. In non-intestinal cells, iron is taken into the cell by receptor-mediated endocytosis by transferrin (Tf).1 Specific receptors (Tf receptor (TfR)) on the outer face of the plasma membrane bind diferric Tf with high affinity (2). Once internalized into the cells, the Tf⅐TfR complex is delivered to endosomes, which are acidified to pH 5.5-6.0 through the action of an ATP-dependent proton pump. E...

Localization of the iron transport proteins mobilferrin and DMT‐1 in the duodenum: The surprising role of mucin

“…The amino acid sequence of DMT should result in a molecular weight of about 60 kD. Other investigators have identified the presence of a large molecular weight form, about 90 kD, and shown that this was due to extensive glycosylation [19,20]. Treatment of the DMT-1 bound to the mucin fraction with glycosidase-P (Fig.…”

Ferroportin-1 is a ‘nuclear'-negative acute-phase protein in rat liver: a comparison with other iron-transport proteins

“…4 DMT-1 has been detected also in the endosomal compartment of different cells, suggesting that it participates in the transport of transferrin (Tf)-bound iron across the cellular membrane of acidified endosomes, resulting in the release of iron into the cytoplasm. 5,6 After entering enterocytes, iron is used for essential metabolic purposes, stored in ferritin or translocated to …”

“…4 DMT-1 has been detected also in the endosomal compartment of different cells, suggesting that it participates in the transport of transferrin (Tf)-bound iron across the cellular membrane of acidified endosomes, resulting in the release of iron into the cytoplasm. 5,6 After entering enterocytes, iron is used for essential metabolic purposes, stored in ferritin or translocated to the portal blood by ferroportin-1 (Fpn-1). The plasma protein, Tf, binds to diferric iron (Fe 2 Tf) and transfers it through portal blood to the liver, where either the Tf receptor-1 (TfR1) 7 or Tf-independent mechanism mediates hepatic uptake of iron.…”

Manganese transport in eukaryotes: The role of DMT1

“…Although the two isoforms of DMT1 produced by alternative splicing are both present at the plasma membrane, the difference in the subcellular localization appears to be dependent upon cation needs. The +IRE isoform is found mainly at the apical membrane of epithelial cells Courville et al, 2006) and late endosomes and lysosomes within HEp-2 cells (Tabuchi et al, 2000;Lam-Yuk-Tseung et al, 2005b). On the other hand, -IRE isoforms are found predominantly in early and recycling endosomes Kannurpatti et al, 2000;Touret et al, 2003).…”