Eukaryotic iron metabolism involves two processes: redox cycling and trafficking. The transport of 'free' iron across eukaryotic plasma and some intracellular membranes is a paradigm of this metabolism. Thus, uptake of environmental Fe3+ involves first its reduction by a plasma membrane ferrireductase. The Fe2+ produced can be substrate for a multicopper oxidase - a ferroxidase - that couples the reduction of O2 to the production of 4Fe3+. This ferric iron is then ligand for an iron permease that transports the iron across the plasma membrane. High affinity iron uptake in the yeast, Saccharomyces cerevisiae, exhibits all of these features. The metalloreductase, Fre1p, produces the Fe2+ that is substrate for ferroxidation by Fet3p, a ceruloplasmin ortholog, with permeation facilitated by Ftr1p. In yeast, as in the intestinal epithelium, the ferroxidation and permeation steps are coupled in the strict metabolic sense: permeation requires ferroxidation. This coupling suggests a primary hypothesis of this research: in the Fet3p, Ftr1p system the ferric iron product of the Fet3p ferroxidase reaction is channeled to Ftr1p for subsequent transmembrane trafficking. A template for this model is the movement of iron into and out of the ferritin (Ft) core. This hypothesis requires that both Fet3p and Ftr1p possess amino acid residues that participate in this channeling process, in addition to those structural motifs required for ferroxidation and permeation per se. There also may be motifs associated with the coupling of these two processes. The objective of this research is a full and detailed structure-function analysis of the Fet3p, Ftr1 system using biochemical, biophysical, genetic and cell biology approaches. These include: kinetic, spectral and crystallographic studies of wild type and mutant Fet3 proteins; iron uptake kinetic analysis of Ftr1p iron trafficking mutants; biochemical, genetic and fluorescence analysis of the physical and functional interaction between Fet3p and Ftr1p; and kinetic and electrophysiologic analysis of the coupling of ferroxidation and uptake. This structure-function characterization of the Fet3p, Ftr1p system will provide significant new understanding of eukaryotic iron trafficking.
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