High level expression of Nramp1(G169) in RAW264.7 cell transfectants: analysis of intracellular iron transport

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 pathog ens. The causative defect was described as G169D within membrane spanning d omain 4 of a transporter. The biochemical activity of Nramp1 is implied fro m sequence conservation with Nramp2. Nramp2 encodes a divalent cation trans porter and is the carrier of a defect in models of microcytic anaemia, asso ciated with impaired intestinal iron uptake. Iron sequestration has been pr oposed as an antimicrobial mechanism. Therefore, such an activity for Nramp 1 is consistent with model systems. Here we showed that Nramp1 directs iron transport within the macrophage. We describe stable, high-level Nramp1(G16 9) allele-derived polypeptide expression in Balb/c Nramp1(D169) RAW264.7 ce lls. Transfectants express levels, comparable to those in Nramp1(G169)-resi stant macrophages, of a 90-100 x 10(3) MW Nramp1 polypeptide. Expression of the Nramp1 polypeptide correlates with lower cellular iron loads and a red uced 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 Nram p1(G169)-expressing cells we observed an increased iron flux into the cytop lasm from a calcein-inaccessible cellular location. These data suggest Nram p1, in resting macrophage cells, mobilizes iron, from an intracellular vesi cle, which is destined for cell secretion. We propose that under these cond itions Nramp1 plays a role in a salvage pathway of iron recycling.