In order to be incorporated into essential cellular enzymes, iron must first traverse various membrane barriers, and yet the molecular mediators of these transport processes have not been identified. We have utilized the genetically tractable eukaryote, Saccharomyces cerevisiae, as a model to identify genes required for the high-affinity transport of iron across the plasma membrane. These genes include CTR1, CCC2, FET3 and FTS3. The FET3 gene encodes an oxidase activity required for high-affinity iron uptake, and the amino acid sequence exhibits similarity to the multi-copper oxidases laccase, ascorbate oxidase and, to some extent, ceruloplasmin. Mutations in CTR1 or CCC2 result in the production of an inactive FET3 apoprotein due to failure to incorporate copper, and the defect in iron uptake in these mutants is a consequence of deficiency of FET3 oxidase activity. Genetic evidence of interaction of FET3 and FTS3 has been obtained: 1) overexpression of both genes is required to augment iron uptake in a wild-type strain, 2) deletion of FTS3 results in loss of iron uptake activity and FET3 oxidase function, and 3) specific mutant alleles of FTS3 abrogate iron uptake without effect on oxidase function. Thus, FET3 may act together with FTS3, which encodes a polytopic membrane protein, to form an iron transporting complex. A genetic approach has also enabled us to identify the iron regulator, AFT1, which mediates the homeostatic control of the iron uptake system. This is accomplished through interaction of the AFT1 protein with cis-acting elements in promoter DNA under conditions of iron deprivation, inducing transcription of the target genes in an iron dependent fashion.
