Characterization of the FET4 Protein of Yeast

The low affinity Fe²⁺ uptake system of Saccharomyces cerevisiae requires theFET4 gene. In this report, we present evidence thatFET4 encodes the Fe²⁺ transporter protein of this system. Antibodies prepared against FET4 detected two distinct proteins with molecular masses of 63 and 68 kDa. In vitrosynthesis of FET4 suggested that the 68-kDa form is the primary translation product, and the 63-kDa form may be generated 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 with uptake activity over a broad range of expression levels and is itself regulated by iron. Furthermore, mutations inFET4 can alter the kinetic properties of the low affinity uptake system, suggesting a direct 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 low affinity system. These mutations may identify residues involved in Fe²⁺ binding during transport.