The long-term goals of this work is to understand the role of mitochondrial iron metabolism in regulating life span and cell death. Iron is an essential nutrient, however, in the presence of naturally produced reactive oxygen intermediates, iron augments free radical formation. Free radicals are suspected of causing cellular damage, for example, DNA fragmentation associated with aging, and high cellular iron concentration and sensitivity to the oxidizing potential of iron is associated with cell death observed in post- ischemic reperfusion injury and Friedreich's ataxia. In the cell, mitochondria are the major site of iron utilization and the major site of reactive oxygen intermediate formation implying that the ability of the mitochondria to manage iron may be extremely important to cell viability. Disruption of the gene for a previously undefined yeast traffic ATPase (mtATPase) that is suspected of regulating mitochondrial iron uptake and/or metabolism based on preliminary results, was shown to cause morphological defects similar to those observed during cell senescence. The overall aim of the proposed work is to confirm that this traffic ATPase is a mitochondrial iron uptake protein and determine if this traffick ATPase affects the overall cell cycle, as suspected based on preliminary data. Specifically the aims are: 1) to verify that the traffick ATPase is disrupted in a previously constructed mutant cell line and prove that a decrease in mtATPase expression causes the mutant phenotype by introducing an ATPase expression vector into mutant cells, 2) to prove that mtATPase is involved in mitochondrial iron metabolism by determining iron uptake and iron management activity of mtATPase disruption mutants and mtATPase over-expressing strains, and to verify that mtATPase is a mitochondrial protein by localizing the protein in situ using immunoassays, and 3) to prove that the mtATPase-1 cell line has an altered mitochondrial content and an altered cell cycle and life span by cytometry, fluorometry, and gene expression analysis. Determining the underlying mechanisms for mitochondrial iron handling is essential for future studies aimed at defining the role of iron aging, and for better understanding diseases associated with free radical damage and maladaptive iron metabolism. Since preliminary data indicates that a mammalian mtATPase homologue exists, a long term consequence of this work may be discovery of more effective treatment for iron related disorders and diseases.
