DESCRIPTION (adapted from the application) Macrophages are central to maintenance of iron homeostasis, acting as a major iron storage site in the body (1-3). Iron is also a major factor in macrophage anti-microbial defense, acting as a catalyst in the generation of reactive hydroxyl radicals (4-6). The Nramp1 gene in mice (9-11) controls natural resistance to infection by a number of intracellular pathogens. The gene exists as two allelic forms that determine resistance or susceptibility to growth of these pathogens within macrophages during the early stages of infection. The protein is expressed in endocytic vesicles of macrophages that traffic to the phagosome. The susceptible allele differs from the resistant allele by a single glycine to aspartic acid substitution in the fourth transmembrane domain. Our laboratory has investigated the role of Nramp1 in macrophage antimicrobial activity and in iron metabolism. We have shown that Nramp1 transports iron into phagosomes where the iron serves to catalyze the Haber-Weiss reaction. The resulting production of reactive hydroxyl radical then inhibits the growth of the phagocytized bacteria. Our observations that Nramp serves as a regulator of antimicrobial activity by transporting iron into phagosomes serves as the basis of this application to determine the factors that regulate Nramp1 mediated iron transport. Our specific aims are: 1. To determine regulation of Nramp1 mediated iron transport; 2. To determine if iron transport by Nramp1 requires protein phosphorylation and 3. To determine, by site directed mutagenesis, the domains within the Nramp1 protein that affect iron transport.