FUNCTIONAL COMPLEMENTATION OF THE YEAST DIVALENT-CATION TRANSPORTER FAMILY SMF BY NRAMP2, A MEMBER OF THE MAMMALIAN NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN FAMILY

The mammalian NRAMP gene family has two members, NRAMP1 and NRAMP2 tha t encode integral membrane proteins, Nramp1 is expressed exclusively i n macrophages where it is found in the phagosomal membrane, and NRAMP1 mutations cause susceptibility to infection by abrogating the capacit y of macrophages to control intracellular microbial replication. Nramp 2 is highly similar to Nramp1, but is expressed in several tissues and cell types, The Nramp protein family is remarkably conserved througho ut evolution, and recent data suggest that the mammalian Nramp2 and th e yeast homologues Smf1 and Smf2 transport divalent cations, We tested whether structural similarity between the mammalian Nramp and the yea st Smf proteins results in functional complementation in yeast, Wild-t ype and mutant variants of the Nramp1 and Nramp2 proteins were express ed in a yeast mutant bearing null alleles at the SMF1 and SMF2 loci, a nd complementation of the phenotypes of this yeast mutant was investig ated, Nramp2, but not Nramp1, was found to complement hypersensitivity to EGTA of the smf1/smf2 mutant under oxidative stress conditions (me thyl viologen), We also observed that the smf1/smf2 double mutant is h ypersensitive to growth at alkaline pH (pH 7.9) and that Nramp2 could complement this phenotype as well, Complementation by Nramp2 was speci fic and required a functional protein as independent mutations in resi dues highly conserved in all members of the Nramp family abrogated Nra mp2 complementation. Since Mn2+ was the only divalent cation capable o f completely suppressing both the EGTA and pH phenotypes, our results suggest that Nramp2 can transport Mn2+ in yeast.