The goal of this proposal is to define mitochondrial iron homeostasis in eukaryotes at the molecular and biochemical level by studying yeast genes that are involved in mitochondrial iron transport. Using different selection systems the applicants have identified yeast genes that result in alterations in cellular and mitochondrial iron metabolism. A yeast gene, YFH, was identified by its ability to complement the low-iron phenotype of a mutant bm-8 defective in intracellular iron metabolism. YFH is the homologue of the Frataxin gene, responsible for the human disease Friedreich's ataxia, and encodes a mitochondrial protein. A deletion in YFH results in mitochondrial iron accumulation, increased sensitivity to oxygen-mediated damage and a respiratory deficit. The applicants propose to determine how YFH affects mitochondrial iron accumulation and to also determine if the mammalian Frataxin encodes a mitochondrial protein. Analysis of the function of the yeast Frataxin homologue should clarify the etiology of Friedreich's ataxia. The BM8 gene is essential and the applicants will determine whether it is a mitochondrial protein and if so, will define its function. The applicants have already identified a family of genes that affect intracellular iron levels. These genes encode proteins that appear to be transporters; they have extensive hydrophobic domains and clusters of histidine residues in the putative cytosolic loops. One of the genes has been localized to the mitochondria. Based on this localization the applicants have called the family Mitochondrial Metal Transporters (MMT). They propose to determine the localization of the other two members of this family. Through the use of deletion and overexpression constructs the applicants propose to examine the function of these genes. They have also developed a selection system which is designed to identify genes that encode mitochondrial iron transporters, particularly those that deliver iron to ferrochelatase. Through the use of their mutant strains and genetic constructs the applicants propose to examine the relationship between mitochondrial iron accumulation and heme biosynthesis. They will determine if defective heme biosynthesis results, as it does in reticulocytes, in excessive iron deposition in mitochondria. These studies will help define the regulation of mitochondrial iron metabolism.
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