High level expression of Nramp1G169 in RAW264.7 cell transfectants: analysis of intracellular iron transport

Abstract

Nramp1 (natural resistance-associated macrophage protein) was positionally cloned as the defective biallelic locus in inbred mouse strains associated with uncontrolled proliferation of obligate intracellular macrophage pathogens. The causative defect was described as G169D within membrane spanning domain 4 of a transporter. The biochemical activity of Nramp1 is implied from sequence conservation with Nramp2. Nramp2 encodes a divalent cation transporter and is the carrier of a defect in models of microcytic anaemia, associated with impaired intestinal iron uptake. Iron sequestration has been proposed as an antimicrobial mechanism. Therefore, such an activity for Nramp1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high-level Nramp1G169 allele-derived polypeptide expression in Balb/c Nramp1D169 RAW264.7 cells. Transfectants express levels, comparable to those in Nramp1G169-resistant macrophages, of a 90-100x103 MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a reduced chelatable iron pool following challenge with iron: nitrilotriacetate. Pulse chase experiments support an enhanced iron flux in expressing cells. These data are supported using the fluorescent iron probe calcein. In Nramp1G169-expressing cells we observed an increased iron flux into the cytoplasm from a calcein-inaccessible cellular location. These data suggest Nramp1, in resting macrophage cells, mobilizes iron, from an intracellular vesicle, which is destined for cell secretion. We propose that under these conditions Nramp1 plays a role in a salvage pathway of iron recycling.

Figure 1

Nramp1 expression in Nramp1^D169 RAW264.7 macrophage cells and 90–100 MW×10³ Nramp1^G169 expression in stable RAW264.7 transfectant clones. RAW264.7 parental cell extracts (20 μg) were analysed for Nramp1 polypeptide expression by Western blotting. Extracts were prepared from untreated cells or following stimulation with: IFN-γ (10 U/ml), LPS (100 ng/ml) or both for 20 hr with or without boiling as indicated (a). (b) 20 μg of total cell extracts prepared from G418 drug resistance Nramp1 transfectant clones were analysed for Nramp1 expression without the standard boiling treatment and compared with an equivalent loading of Nramp1^G169 N11 cells. Of the sense orientation transfectant clones, R32, R37, R38 and R39 exhibited high molecular polypeptides displaying immunoreactivity to the anti-Nramp1 antisera of 90–100 MW×10³ and 65 MW×10³. Sense orientation clones R33 and R34 failed to exhibit immunoreactivty despite being resistant to G418.

Figure 2

Determination of endosomal iron efflux using the fluorescent dye calcein. RAW264.7 parental cells, sense (R37) or antisense orientation (R21). Nramp1 cDNA transfectants, as indicated, were loaded with calcein as described in the Materials and methods. Prior to the addition of iron chelate a fluorescent baseline was established of which the final 2 min are shown on the traces. Fe: NTA at (a) 10 μm or (b) 100 μm was added at the indicated time and the resulting decrease in calcein fluorescence monitored.