This proposal focuses on copper homeostasis in S. cerevisiae as a model eukaryote. Regulation of metallothionein biosynthesis is a critical part of metal homeostasis in cells. This regulation is believed to be tightly controlled as high constitutive expression of MTs may alter the metal ion distribution in cells. Our understanding of the regulation of MT biosynthesis in mammalian cells remains descriptive. One approach toward understanding the mechanism of this regulation is the use of organism such as S. cerevisiae amenable to genetic manipulation. Expression of MT genes is S. cerevisiae is specifically regulated by copper ions. Cis- acting elements and a trans-acting factor, ACE1, are important for this Cu-dependent regulation. A number of unresolved steps exist in this pathway and regulation may exist at these steps. Only a paucity of information is known of mechanisms for plasma membrane copper transport, intracellular transport of Cu(I) to the nucleus, the presentation of Cu(I) to ACE1. Similar mechanisms may be involved in metal-induced MT biosynthesis in mammalian cells. The focus of one specific aim is to identify other components in the Cu-signal pathway in S. cerevisiae through mutagenesis and a positive genetic selection protocol. Glutathione (GSH) may be important in the Cu-signal transduction pathway for MT biosynthesis in either Cu(II) reduction or Cu(I) presentation to ACE1. We will evaluate the role of GSH in this pathway and in general copper and zinc metabolism by taking advantage of yeast mutant cells with depleted GSH levels. By over-expressing the gamma-glutamyl cysteine synthetase we will evaluate the consequence of elevated levels of GSH in metal metabolism. A third objective is to study the ramifications of unregulated MT synthesis on copper and zinc metabolism. Cloning of the yeast MT gene under a constitutive promoter yields high basal levels of MT. We will address whether metal sequestration by MT alters Cu and Zn distribution within cells creating localized metal deficiency. The last objective is the use of a DNA library we prepared in vector to screen for metal-responsive promoter sequences. We will characterize meal- responsive cis-acting sequences but the goal is to characterize yeast genes that are metal-regulated and identify the trans-acting factors responsible for metal-responsiveness. The use of molecular genetics in yeast to address issues of metal homeostasis may provide insight into mammalian mechanisms and mechanism of metal-induced carcinogenesis.
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