CHARACTERIZATION OF THE FET4 PROTEIN OF YEAST - EVIDENCE FOR A DIRECTROLE IN THE TRANSPORT OF IRON

The low affinity Fe2+ uptake system of Saccharomyces cerevisiae requir es the FET4 gene. In this report, we present evidence that FET4 encode s the Fe2+ transporter protein of this system. Antibodies prepared aga inst FET4 detected two distinct proteins with molecular masses of 63 a nd 68 kDa. In vitro synthesis of FET4 suggested that the 68-kDa form i s the primary translation product, and the 63-kDa form may be generate d by proteolytic cleavage of the full-length protein. Consistent with its role as an Fe2+ transporter, FET4 is an integral membrane protein present in the plasma membrane. The level of FET4 closely correlated w ith uptake activity over a broad range of expression levels and is its elf regulated by iron. Furthermore, mutations in FET4 can alter the ki netic properties of the low affinity uptake system, suggesting a direc t interaction between FET4 and its Fe2+ substrate. Mutations affecting potential Fe2+ ligands located in the predicted transmembrane domains of FET4 significantly altered the apparent K-m and/or V-max of the lo w affinity system. These mutations may identify residues involved in F e2+ binding during transport.