Dictyostelium Nramp1, structurally and functionally close to mammalian DMT1 transporter, mediates phagosomal iron efflux

The Nramp (Slc11) protein family is widespread in bacteria and eucaryotes, and mediates transport of divalent metals across cellular membranes. The social amoeba Dictyostelium discoideum harbours two Nramp proteins. Nramp1, like its mammalian ortholog, is recruited to phagosomal and macropinosomal membranes, and confers resistance to pathogenic bacteria. Nramp2 is located exclusively in the contractile vacuole membrane and controls, synergistically with Nramp1, iron homeostasis. It has long been debated whether mammalian Nramp1 mediates iron import or export from phagosomes. By selectively loading the iron-chelating fluorochrome calcein in macropinosomes, we show that Dictyostelium Nramp1 mediates iron efflux from macropinosomes in vivo. To gain insight in ion selectivity and transport mechanism, the proteins were expressed in Xenopus oocytes. Using a novel assay with calcein, electrophysiological and radiochemical assays, we show that Nramp1, similarly to rat DMT1, transports iron(II) and manganese, not iron(III) or copper. Metal ion transport is electrogenic and proton-dependent. By contrast, Nramp2 transports only iron(II) in a non-electrogenic and proton-independent way. These differences reflect evolutionary divergence of the prototypical Nramp2 protein sequence compared to the archetypical Nramp1 and DMT1 proteins.