METAL HOMEOSTATIC MECHANISMS IN YEAST

这项建议以酿酒酵母中铜的动态平衡为模型。 真核生物。金属硫蛋白的生物合成调控是一个关键环节 细胞中的金属动态平衡。这一规定被认为是严格的 控制为MTS的高结构性表达可改变金属离子 在细胞中的分布。我们对MT规制的认识 哺乳动物细胞中的生物合成仍然是描述性的。一种方法是 理解这种调节的机制是利用有机体 例如可以进行基因操作的酿酒酵母。的表达 MT基因是酿酒酵母受铜离子特异性调控的基因。独联体- 作用元件和反式作用因子ACE1对此很重要 铜依赖的调控。中存在许多未解决的步骤 在这些步骤中可能存在途径和调节。只有一小部分 关于质膜铜运输机制的信息是已知的， 铜(I)的胞内转运到胞核， Cu(I)至ACE1。类似的机制可能参与了金属诱导的MT 哺乳动物细胞中的生物合成。一个具体目标的重点是 鉴定酿酒酵母铜信号通路中的其他成分 通过突变和积极的基因选择方案。 谷胱甘肽(GSH)可能在铜离子信号转导途径中起重要作用 在铜(II)还原或铜(I)呈递的情况下进行MT生物合成 ACE1.我们将评估GSH在这一途径中的作用以及总体上的作用。 利用酵母突变细胞进行铜和锌的代谢 谷胱甘肽水平耗尽。通过过量表达γ-谷氨酰半胱氨酸 合成酶，我们将评估GSH水平升高的后果 金属代谢。第三个目标是研究 MT合成对铜和锌代谢的影响。克隆基因 在构成启动子下的酵母MT基因产生高基础水平的 Mt.Mt.我们将讨论MT对金属的封存是否会改变铜和锌 细胞内的分布造成局部的金属缺乏。最后 目的是利用我们在载体中制备的DNA文库来筛选 金属反应启动子序列。我们将描述这顿饭的特点- 响应顺式作用序列，但目标是表征酵母菌 受金属调控并识别反式作用因子的基因 负责金属反应。分子遗传学在生物医学中的应用 酵母菌解决金属动态平衡问题可能提供洞察 哺乳动物致癌机制和金属致癌机制。

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.