While iron is an essential element, 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. Malregulation of iron transport can result in tissue injury, either from iron deprivation or iron overload. We propose to examine the mechanism and regulation of cellular iron transport. Our goal is to isolate mammalian transferrin-independent iron transport systems. Our studies suggest that these transport systems involve a reductase which converts ferric to ferrous iron, and a transmembrane ferrous iron transporter. A wide variety of species use reductase/ transporter systems to accumulate iron. One of our approaches to identifying transporter genes is to use yeast to create mutants in iron transport and metabolism, and then use complementation analysis to identify and clone yeast genes essential for iron metabolism. We have already cloned two genes involved in iron metabolism. We have termed these genes FET (Ferrous Transport). One of the genes FET3 is absolutely required for high affinity inducible iron transport. The other, FET4 , is necessary for low affinity iron transport. We propose to identify other yeast genes important in Fe(II) transport by using selection systems which take advantage of the iron dependent toxicity of streptonigrin. Mutants that are unable to transport iron are resistant to the effects of this agent. We also plan to use our existing mutants to obtain new mutants by looking for second site suppressors. Yeast genes and yeast mutants will be used to isolate mammalian genes involved in iron transport. The streptonigrin selection system will be used with cultured cells to isolate mammalian cells defective in iron transport and metabolism. We plan to study the biochemistry of the yeast Fet3 protein. The Fet3 amino acid sequence indicates that this gene is a member of the rare family of multicopper oxidases and we have hypothesized that it functions as a ferroxidase. We propose to isolate the enzyme and examine its copper content. Site specific mutagenesis will be used to determine if type I copper ligands are critical for its role in iron metabolism. To determine if a mammalian equivalent of Fet3 exists we propose, in addition to genetic screens, to examine the effect of intracellular copper depletion on high affinity iron uptake and on the incorporation of iron into heme and ferritin. As part of our study of intracellular iron transport, we predict that a mammalian lysosomal Fe(II) transport system exists. Using both in vivo and in vitro assays we propose to determine if lysosomes, isolated from cultured cells, have a Fe(II) transport system, and whether the lysosomal transport system is similar to the cell surface iron transport system. These studies will test our hypothesis that there exists a family of cell surface and organelle transmembrane ferrous-transporters. Our studies will clarify the mechanisms that regulate cellular iron content and the role of iron in normal and pathological processes.