The long-term objective of this proposal is to characterize the mechanisms of zinc metabolism and the regulation of Zn2+-homeostasis in multicellular animals. Zn2+ metabolism has important implications for human health because zinc deficiencies caused by inadequate diet or inborn errors of metabolism result in many pathologies. Zn2+ also regulates important processes such as cell proliferation, and Zn2+ metabolism may affect diseases such as cancer. The first specific aim is to characterize the role of the C. elegans cdf-1 gene in Zn2+ metabolism in an intact animal. cdf-1 encodes a cation diffusion facilitator protein that promotes Zn2+ efflux across the plasma membrane. The research design and methods include developing assays of zinc content, distribution, uptake and excretion in nematode worms. These assays will be used to determine how changes in dietary zinc affect Zn2+-homeostasis. The role of cdf-1 in Zn2+ metabolism will be determined by analyzing mutants that lack CDF-1 activity, overexpress CDF-1, or express CDF-1 in specific tissues. The regulation of CDF-1 activity will be characterized by analyzing cdf-1 mRNA and protein in intact animals and determining how these products are regulated by dietary zinC. The final part of the first specific aim is to characterize the biochemical mechanism of action of CDF-1 in promoting Zn2+ transport across the plasma membrane. The research design and methods include developing assays of Zn2+ transport using purified components or cellular systems and characterizing the role of CDF-1 in Zn 2+ transport. Proteins that bind to CDF-1 will be identified using the yeast two-hybrid system and the role of CDF-l-interacting proteins in Zn2+ transport will be analyzed. The role of CDF-1-interacting proteins in intact animals will be investigated using genetic approaches. The second specific aim is to identify a network of genes that regulate Zn2+- homeostasis. The research design and methods include conducting genetic screens for mutations that affect C. elegans Zn2+ metabolism. Genetic methods will be used to determine the specific role of newly identified genes in Zn2+ metabolism. The role of these genes in cell fate specification will be determined to characterize the relationship between Zn2+ metabolism and Ras-mediated signaling. Molecular approaches will be used to clone the affected genes and reveal the mechanisms used by these proteins to regulate Zn2+ metabolism. These studies are likely to provide significant new insights into Zn2+ metabolism by establishing the role of CDF-1 in an intact animal, elucidating the biochemical mechanism of CDF-1, and identifying and characterizing new proteins that regulate Zn2+.
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