In this proposal, the genes and proteins involved in trafficking of iron between the plasma membrane and protein within mitochondria of Saccharomyces cerevisiae will be defined. A novel collection of yeast mutants will be studied that exhibit constitutively high cellular iron uptake, iron sensitivity and accumulation of iron within mitochondria. These mutants will be organized into complementation groups, the genes(s) will be cloned and the gene products(s) characterized. Mutants of this type, such as the ma15 mutant, accumulate iron as an iron-protein complex with mitochondria, and this complex will be purified and characterized in terms of its protein and iron composition. Furthermore, a specific iron protein isolated from mitochondria appeared deficient in the ma15 mutant (33 kDa iron protein). This protein will be purified and identified. The molecular defect in these mutants may be directly or indirectly related to this protein, and it will be important to ascertain if the lack of this iron protein represents a general defect in iron delivery to proteins within mitochondria or a more narrow defect. The second specific aim is to determine the biochemical features of iron import into mitochondria. The rates of import into mitochondria will be determined for living cells and/or isolated mitochondria, looking for abnormalities that might be specific for the mutants. Methods developed for measuring protein import into purified mitochondria will be used to aid in developments of these studies. Finally, specific aim 3 is aimed at determining the nature of iron toxicity in the ma15 mutant. The basis of the small colony phenotype and iron sensitivity will be evaluated and the evidence that mtDNA represents a target for toxic iron damage in these mutants will be evaluated using methods unique to yeast. The yeast mutants that accumulate iron in dense deposits within mitochondria while failing to properly synthesize iron proteins bear a resemblance to the erythroid progenitors of a group of human disorders called sideroblastic anemia. The acquired form of sideroblastic anemia often represents a preleukemic state, and the basis for the disease and for the transition to leukemia is unknown, Thus, clinical relevance of the yeast work will be sought by looking for conserved genes and function.
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