The applicants note that iron is an essential element but that excessive iron accumulation leads to cell and organ dysfunction. Multiple tightly regulated mechanisms exist to meet the cellular need for iron and to remove iron from biological fluids. The applicants plan to continue their approach of using yeast genetics to identify genes involved in iron metabolism. They first plan to study multicopper oxidase-based iron-transport systems. Studies in humans and in yeast have identified the ferroxidase activity of multicopper-based iron-transport systems as responsible for selective high-affinity iron transport across the plasma membrane. The applicants propose to use a combined genetic and biochemical approach to determine the mechanisms of assembly of the active site of multicopper oxidases. Studies in yeast by the applicants have provided evidence that chloride provided by intracellular chloride channels is required to assemble copper on the yeast Fet3p. They now propose to determine how chloride is involved in copper assembly and whether chloride binds to the enzyme. In yeast, the ferroxidase is involved in iron uptake, while in humans ferroxidases are involved in iron export and import. The applicants plan to determine the physical relationship between multicopper oxidases and the direction of iron transport, testing their hypothesis that it is the iron carrier or transporter that determines the direction of iron transport. In addition, the applicants will determine if Hephaestrin, the gene responsible for the phenotype of defective iron transport in the sex-linked anemia (sla) mouse, is a multicopper oxidase. If so, they plan to examine its cellular location and assembly. Iron, as well as other transition metals, is transported across the membrane of the vesicular system (endoplasmic reticulum, Golgi and lysosomes). The function of vesicular metal transport is two-fold: i) to store metals within the vesicular system, thereby removing them from the cytosol; and ii) to assemble metallo-proteins that are either resident or secreted enzymes. Using yeast genetics, the applicants have identified iron and copper-regulated or dependent transport systems in the vesicular apparatus. They now plan to define the function of these transporters, determining whether these transporters are involved in metal storage or metallo-enzyme assembly. They propose genetic approaches to determine if zinc or iron is stored in the vacuole and to identify the responsible transporters. They plan to determine where the lysosomal iron enzyme tartrate-resistant acid phosphatase (TRAP) obtains its iron by expressing this molecule in yeast and, then using selection systems to define iron transporters.
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