Assembly, activation, and trafficking of the Fet3p•Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae

The high affinity iron uptake complex in the yeast plasma membrane ( PM) consists of the ferroxidase, Fet3p, and the ferric iron permease, Ftr1p. We used a combination of yeast two- hybrid analysis, confocal fluorescence microscopy, and fluorescence resonance energy transfer ( FRET) quantification to delineate the motifs in the two proteins required for assembly and maturation into an uptake-competent complex. The cytoplasmic, carboxyl- terminal domain of each protein contains a four- residue motif adjacent to the cytoplasm-PM interface that supports an interaction between the proteins. This interaction has been quantified by two- hybrid analysis and is required for assembly and trafficking of the complex to the PM and for the similar to 13% maximum FRET efficiency determined. In contrast, the Fet3p transmembrane domain ( TM) can be exchanged with the TM domain from the vacuolar ferroxidase, Fet5p, with no loss of assembly and trafficking. A carboxyl- terminal interaction between the vacuolar proteins, Fet5p and Fth1p, also was quantified. As a measure of the specificity of interaction, no interaction between heterologous ferroxidase permease pairs was observed. Also, whereas FRET was quantified between fluorescent fusions of the copper permease ( monomers), Ctr1p, none was observed between Fet3p and Ctr1p. The results are consistent with a ( minimal) heterodimer model of the Fet3p center dot Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.