Iron is an essential element for nearly all organisms in the biosphere, being required for the activity of a number of proteins in cells. Defects in iron acquisition, utilization, or storage lead to human illness, constituting some of the most common forms of human disease, including hemochromotosis, sideroblastic anemia with ataxia, Freidreich's Ataxia, and cancer. Aberrant iron metabolism has also been linked to neurologic disorders, such as Parkinson's Disease. Organisms utilize iron in a variety of chemical forms, including as elemental iron, heme, and combined with sulfur in iron-sulfur (FeS) clusters. FeS clusters are assembled into proteins through a protein mediated processes that is conserved through evolution. FeS cluster biogenesis itself is an essential process in living organisms, and in eukaryotes this process also plays a central role in cellular iron regulation. At least two systems for FeS cluster biogenesis exist in eukaryotic organisms;one is primarily mitochondrial and the other is primarily cytosolic. A link between these pathways exsits, but is not yet completely understood. The newly emerging cytosolic FeS cluster assembly (CIA) machinery currently consist of four proteins factors, called Cfd1p, Nbp35p, Nar1p, and Cia1p (using the yeast terminology). Each of the cytosolic CIA factors is essential for FeS cluster assembly in cytoplasmic and nuclear proteins, and for yeast viability. The broader goals of the studies outlined in this proposal are to define the functional roles of the CIA factors in FeS protein biogenesis and to determine their role in cellular iron regulation. To achieve these goals, we will pursue the following specific aims: 1) Determine the molecular mechanism through which CIA factors assist extra-mitochondrial FeS cluster assembly;2) Determine the relationship of cytosolic and nuclear FeS cluster assembly to cellular iron regulation;and 3) Decipher the protein-interaction network mediating cytosolic and nuclear FeS cluster assembly and cellular iron regulation. These aims will be achieved using a combination of molecular genetic, biochemical and proteomic approaches in the model organism Saccharomyces cerevisiae. Completion of the proposed studies will lead to a significant advance in our understanding of the essential process of FeS cluster assembly in eukaryotic organisms, and will illustrate how this process is integrated into overall cellular iron metabolism.