METAL HOMEOSTATIC MECHANISMS IN YEAST

酵母中的金属稳态机制

• 批准号: 2102026
• 负责人: Dennis R. Winge
• 金额: $ 12.29万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-12-15 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2102026
• 关键词: Saccharomyces cerevisiae; alternatives to animals in research; biological signal transduction; copper; genetic library; glutathione; homeostasis; metal metabolism; molecular cloning; transcription factor; yeasts; zinc

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.

该提案重点介绍酿酒酵母的铜稳态作为模型 真核生物。 金属硫硫蛋白生物合成的调节是关键部分 细胞中的金属稳态。 据信该法规紧紧 以高为MT的本构表达控制可能会改变金属离子 细胞分布。 我们对MT的调节的理解 哺乳动物细胞中的生物合成仍然存在描述性。 一种方法 了解该调节的机制是使用生物 例如，酿酒酵母可与遗传操纵相提并论。 表达 MT基因是酿酒酵母，由铜离子特异性调节。 顺式 行动元素和反式因素ACE1对此很重要 CU依赖性调节。 其中存在许多未解决的步骤 这些步骤可能存在途径和调节。 只有很少 信息已知有关质膜传输机制的信息， Cu（I）向细胞核的细胞内转运 cu（i）至ace1。 类似的机制可能与金属诱导的MT有关 哺乳动物细胞中的生物合成。 一个特定目的的重点是 在酿酒酵母中识别Cu信号途径中的其他组件 通过诱变和阳性遗传选择方案。 谷胱甘肽（GSH）在Cu信号转导途径中可能很重要 在Cu（II）还原或Cu（i）呈现中的MT生物合成 ACE1。 我们将评估GSH在该途径中的作用，一般而言 铜和锌代谢通过利用酵母突变细胞 耗尽的GSH水平。 通过过表达γ-谷氨酰胺半胱氨酸 合成酶我们将评估GSH水平升高的结果 金属代谢。 第三个目标是研究的后果 对铜和锌代谢的不受管制的MT合成。 克隆 构成启动子下的酵母MT基因产生高基础水平 公吨。 我们将解决MT的金属隔离是否改变Cu和Zn 细胞内的分布产生局部金属缺乏。 最后 目的是使用我们准备在矢量中的DNA库来筛选 金属反应性启动子序列。 我们将以进餐为特征 - 响应敏感的顺式作用序列，但目的是表征酵母 金属调节并识别跨作用因子的基因 负责金属响应性。 分子遗传学在 解决金属稳态问题的酵母可能会提供有关 金属诱导的癌变的哺乳动物机制和机制。