Copper is an essential, but potentially toxic nutrient. Homeostatic mechanisms exist to regulate the cellular concentration of copper ions, thus maintaining Cu balance and minimizing the deleterious effects of Cu ions. Metal sensory systems are one facet of metal ion homeostasis. Signals from these systems often result in specific metal-regulation of biosynthesis of proteins involved in homeostasis. In yeast, copper regulation consists of both Cu-mediated induction and Cu-mediated inhibition of transcription. This grant focuses on mechanisms by which yeast cells sense the intracellular level of Cu ions and regulate gene expression in a Cu-dependent manner. The expression of a number of genes encoding products involved in Cu ion uptake in yeast is specifically inhibited by Cu ions. We demonstrated that Cu-attenuation of gene expression occurs through Cu-mediated repression of the transactivation activity of the Mac1 transcription factor. Elucidation of the mechanism of Cu-mediated repression of Mac1 is a major focus of this proposal. Activation domain activity in Mac1 maps to a region containing two Cys-rich sequence motifs. One hypothesis to be tested is that Cu(I) binding to the Cys-rich motifs in the Mac1 activation domain is an initial event in Cu-repression of Mac1 function. Experiments are outlined to discern whether repression occurs through an intramolecular or intermolecular process. A significant feature of the Mac1 studies is that there are limited examples of regulation of transcriptional activation function. Conditions of excess copper ions in the environment result in the induced expression of a different subset of genes whose products have protective roles. Cu-induction of gene expression in yeast is mediated by the Ace1 transcription factor. Cu-activation of Ace1 requires Cu(I) ions to be present within the nucleus. We postulate that a specific nuclear Cu(I) transporter exists analogous to known Cu transporters specific for the mitochondrion and the secretory pathway. Our objective is to identify gene products that are required for Cu-activation of Ace1. If a candidate nuclear Cu transporter is identified, we want to determine whether it also participates in nuclear transport of Cu(I) for regulation of Mac1 function.
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