Zinc is essential yet too much zinc can be toxic. Therefore, regulatory mechanisms are present that control the intracellular concentration of this metal. The long-range goal of this research is to determine the molecular basis of zinc homeostasis in eukaryotic cells. This proposal describes a combined genetic, molecular, biochemical, and biophysical approaches to the study of these processes in the yeast Saccharomyces cerevisiae. Yeast has proven to be an ideal model system for recent studies of iron and copper homeostasis and will no doubt prove to be equally valuable for the study of zinc metabolism. Zinc uptake in yeast is mediated by two separate systems. One system has a high affinity for zinc and the ZRT1 gene encodes the transporter protein of this system. The second system has a lower affinity for substrate and the ZRT2 gene encodes its transporter. An important facet of zinc homeostasis in yeast is the regulation of zinc uptake by transcriptional control of these two genes. ZRT1 and ZRT2 are both expressed at high levels in zinc-limited cells but at low levels in zinc-replete cells. Preliminary studies have identified a new gene, ZAP1 (for Zinc-responsive Activator Protein), that plays a critical role in this regulation. As the name implies, the current hypothesis is that ZAP1 encodes a transcriptional activator that binds to sequences (ZREs) in the promoters of ZRT1 and ZRT2. DNA binding to the ZREs is conferred by zinc finger domains in Zap1p and the rates transcription of these genes are increased through the action of acidic activation domains in the protein. It is also proposed that Zap1p acts as the zinc """"""""sensor"""""""" and its transcriptional activation domains are repressed by binding of zinc ions directly to the protein. To test the various aspects of this hypothesis, the following specific aims are proposed: 1) Precisely identify the ZREs and determine if Zap1p binds to these sequences. 2) Perform a functional dissection of the Zap1p protein. 3) Determine the in vivo effects of zinc on Zap1p function. 4) Identify potential intermolecular repressors of Zap1p activity. 5) Characterize zinc-binding by Zap1p in vitro. These experiments will lead to a greater understanding of zinc homeostasis in human cells.
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