Functional analysis of the human NRAMP family expressed in fission yeast

The Bcg/Ity/Lsh locus in the mouse genome regulates macrophage activation f or antimicrobial activity against intracellular pathogens, and the position al cloning of this locus identified the Nramp1 (natural resistance-associat ed macrophage protein) gene. Nramp2 was initially isolated as a homologue o f Nramp1. Recently, the rat divalent metal transporter DMT1 was identified electrophysiologically, and was found to be an isoform of Nramp2, a mutatio n which was subsequently identified in rats suffering from hereditary iron- deficiency anaemia. Despite the 64% amino acid sequence identity of Nramp1 and Nramp2, no divalent metal transport activity has yet been detected from Nramp1, and the function of Nramp1 on the molecular level is still unclear . To investigate the divalent metal transport activity of NRAMP molecules, we constructed four chimeric NRAMP genes by swapping the domains of human N RAMP1 and NRAMP2 with each other. The functional characteristics of wild-ty pe NRAMP1, NRAMP2 and their chimeras were determined by expression in the d ivalent metal transporter-disrupted strain of fission yeast, pclt1 Delta, a nd we analysed the divalent metal transport activity by complementation of the EGTA- and pH-sensitive phenotype of pdt1 Delta. Replacement of the N-te rminal cytoplasmic domain of NRAMP2 with the NRAMP1 counterpart resulted in inactive chimeras, indicating that the functional difference between NRAMP 1 and NRAMP2 is located in this region. However, results obtained with the reverse construct and other chimeras indicated that these regions are not s olely responsible for the differences in EGTA- and pH-sensitivity of NRAMP1 and NRAMP2, These findings indicate that NRAMP1 itself cannot represent th e divalent metal transport activity in S. pombe and the additional protein segments of the molecules located elsewhere in NRAMP1 are also functionally distinct from their NRAMP2 counterparts.