The metal Copper (Cu) serves as an essential biochemical co-factor for a wide variety of enzymes such as Cu, Zn superoxide dismutase, involved in oxidative stress protection, cytochrome oxidase, which plays a key role in respiration, lysyl oxidase, which crosslinks collagen molecules for proper connective tissue formation, dopamine beta-hydroxylase, involved in neurotransmitter biosynthesis and other critical enzymatic activities. Cu is highly toxic, however, due to its ability to engage in redox reactions generating oxygen-derived free radicals which damage proteins, membranes and genetic material. The essential yet highly toxic nature of Cu demands that all cells establish and maintain Cu homeostatic mechanisms to allow sufficient levels of Cu to accumulate for essential biochemical reactions, but prevent the accumulation of Cu to cytotoxic concentrations. This absolute necessity for Cu homeostatic control is underscored by the existence of two human genetic disorders of Cu homeostasis, Menkes syndrome and Wilson's disease. In this proposal experiments are outlined to investigate, using yeast cells as model eukaryotic systems, fundamental mechanisms of Cu homeostasis. First, the AMT1 Cu Metalloregulatory Factor (CuMRTF) from the opportunistic pathogenic yeast Candida glabrata will be studied as a model for Cu sensing transcription factors. DNA binding studies and gene expression studies in vivo will be used to investigate the molecular interactions of CuMRTF proteins with the Metal Response Element (MRE) in the AMT1 gene promoter. Furthermore, the mechanism by which the C. glabrata AMT1 gene is rapidly transcriptionally autoactivated, via other promoter elements, will be investigated. Secondly, a new member of the CuMRTF protein family in the yeast Saccharomyces cerevisiae, denoted MRTF-X, will be characterized. The subcellular location of the MRTF-X protein will be ascertained by indirect immunofluorescence microscopy (IIM), and the expression of the MRTF-X gene and the physiological consequences of inactivation of this gene will be studied. In vitro DNA binding studies will be conducted to study interactions between MRTF-X and known Cu homeostasis gene promoters and to identify other sequences bound by MRTF-X. Third, a new gene involved in high affinity Cu transport in S. cerevisiae, denoted CTR2, will be investigated to determine the role of the encoded protein in Cu transport. The cellular location of the CTR2 protein will be investigated by IIM and the importance of CTR2 Cysteine residues in high affinity Cu transport will be investigated by mutagenesis, phenotypic and Cu transport studies. Fourth, the cis- and trans-acting elements responsible for the Cu repression and Cu-starvation-induction of CTR2 mRNA levels will be investigated by promoter mutagenesis and fusion experiments, in vivo gene expression studies, in vitro DNA binding experiments and through the isolation of trans-acting regulatory mutations and the corresponding regulatory genes. The experiments proposed in this application are aimed at elucidating the fundamental processes by which yeast, and all organisms, establish and maintain normal Cu homeostasis.
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