METAL HOMEOSTATIC MECHANISMS IN YEAST

本研究的重点是S.酿酒酵母作为模型 真核生物 金属硫蛋白生物合成的调控是一个关键部分 细胞内金属平衡的关键。 这项规定被认为是严格的 由于MT的高组成性表达可能会改变金属离子， 在细胞中的分布。 我们对MT调节的理解 哺乳动物细胞中的生物合成仍然是描述性的。 一种方法， 理解这种调节的机制是利用生物体 如S.酿酒酵母适合基因操作。 表达 MT基因为S.酿酒酵母是由铜离子特异性调节。 顺- 作用元件和反式作用因子ACE 1对此很重要 Cu依赖性调节。 在这方面存在许多未解决的步骤， 在这些步骤中可能存在通路和调节。 只有很少的 质膜铜转运机制的信息是已知的， Cu（I）向细胞核的细胞内转运， Cu（I）至ACE 1。 类似的机制可能涉及金属诱导的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S中Cu信号通路中的其他组分。酿酒酵母 通过诱变和阳性遗传选择方案。 谷胱甘肽（GSH）可能在Cu ~（2+）信号转导途径中起重要作用 对于MT生物合成，无论是Cu（II）还原还是Cu（I）呈递， ACE 1. 我们将评估GSH在这一途径中的作用， 利用酵母突变细胞进行铜和锌代谢 谷胱甘肽水平下降 通过过度表达γ-谷氨酰半胱氨酸 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合成不受调节对铜和锌代谢的影响。 的克隆 组成型启动子下的酵母MT基因产生高基础水平的 山 我们将讨论金属螯合MT是否改变铜和锌 分布在细胞内，造成局部金属缺乏。 最后一 目的是使用我们在载体中制备的DNA文库来筛选 金属响应启动子序列。 我们将描述膳食- 响应顺式作用序列，但目标是表征酵母 受金属调控并识别反式作用因子的基因 负责金属响应。 分子遗传学在 酵母解决金属稳态问题可能会提供深入了解 哺乳动物的机制和金属诱导的致癌机制。

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.