Abstract
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 approximately 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.Ftr1p complex that supports the trafficking of iron from Fet3p to Ftr1p for iron permeation across the yeast PM.
Highlights
Dues on both Fet3p and Ftr1p have been identified that are essential to one or more of these steps in iron uptake (8 –11)
Neither pair supported the development of a measurable -galactosidase activity in cell extracts; rather than indicating that the interaction between a ferroxidase and permease pair was intrinsically weak, this result was likely related to the use of the standard two-hybrid approach to assess an interaction between soluble chimeras of two proteins that normally are membrane-targeted
As a complement to the studies above that investigated the trafficking motifs in Fet3p and Ftr1p directly, we proposed that by characterizing the targeting and copper-dependent activation of chimeras composed of Fet3p and Fet5p domains, we would obtain some information about those motifs that were specific to Fet3p as a plasma membrane (PM)-localized yeast ferroxidase and its specific assembly with Ftr1p
Summary
Dues on both Fet3p and Ftr1p have been identified that are essential to one or more of these steps in iron uptake (8 –11). Fet3p produced in an ftr1⌬-containing yeast strain remains in perinuclear and vesicular compartments (8, 11) This interdependence in trafficking for both proteins has been vividly demonstrated using fluorescent fusions of Fet3p and Ftr1p. Interaction trap technology has shown an interaction between the Fet3p1⁄7Ftr1p paralogs in S. cerevisiae, Fet5p and Fth1p (12, 13) These two proteins form an iron permease complex in the membrane of the yeast vacuole. FET5 was isolated as a high copy suppressor of the ironuptake (respiratory) deficiency of a fet3⌬-containing strain indicating that Fet5p could functionally replace Fet3p in the Fet3p1⁄7Ftr1p complex This complementation, sufficient to support iron uptake, was kinetically limited, suggesting that Fet5p1⁄7Ftr1p complex formation, trafficking, and/or activity was driven by the overproduction of Fet5p and not by a native-like interaction between the two proteins (14). Fet3p produced in the presence of Ftr1p but in a copper-defi-
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