PMID:16522632

Citation	Singh, A, Severance, S, Kaur, N, Wiltsie, W and Kosman, DJ (2006) Assembly, activation, and trafficking of the Fet3p.Ftr1p high affinity iron permease complex in Saccharomyces cerevisiae. J. Biol. Chem. 281:13355-64
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.
Links	PubMed Online version:10.1074/jbc.M512042200
Keywords	Cell Membrane/metabolism; Ceruloplasmin/chemistry; Ceruloplasmin/genetics; Ceruloplasmin/metabolism; Copper/metabolism; Enzyme Activation; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Fungal; Iron/metabolism; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/genetics; Membrane Transport Proteins/metabolism; Mutation; Protein Transport; Recombinant Proteins; Saccharomyces cerevisiae/cytology; Saccharomyces cerevisiae/enzymology; Saccharomyces cerevisiae/metabolism; Saccharomyces cerevisiae Proteins/chemistry; Saccharomyces cerevisiae Proteins/genetics; Saccharomyces cerevisiae Proteins/metabolism
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