Metal Homeostatic Mechanisms in Yeast

描述（由申请人提供）：本申请的重点是细胞内铜（Cu）和铁（Fe）水平与酵母基因表达的稳态偶联。铜摄取基因的金属调控是由Mac1转录激活子控制的。铁摄取基因的金属调控由两个相关的激活因子Aft1和Aft2控制。这三个因子在缺金属细胞中是转录激活因子，但在富金属细胞中被特异性抑制。主要目的是阐明Mac1感应Cu离子和Aft1和Aft2感应Fe离子的机制。酵母中铁和铜的稳态之间存在着许多相互联系。在过去的资助周期中，我们提供了证据表明Mac1通过直接结合Cu(I)离子在酵母细胞核内感知Cu离子。Cu(I)结合似乎诱导了n端DNA结合域和c端转激活域之间的分子内相互作用。研究提出验证直接感应铜和绘制分子内界面，抑制DNA结合和转激活。一个重要的目标是确定其他酵母蛋白在Cu转运到细胞核或Cu抑制Mac1中起重要作用。酵母体内的铁稳态受Aft1和Aft2调控。铁抑制Aft1和Aft2功能的机制尚不清楚。这些研究的意义在于酿酒葡萄球菌是了解转录调节因子对铁的感知机制的最佳真核系统模型。我们建议确定铁传感是通过直接铁结合、铁介导的翻译后修饰还是通过另一种蛋白质发生的？Aft1和Aft2在酵母中表现出有限的功能冗余。我们建议确定每一种是否表现出不同的生理功能。Mac1和Aftl/Aft2是研究营养控制gone表达的良好模型真核系统。有关Cu和fe调控机制的信息可能适用于其他物种。

DESCRIPTION (provided by applicant): This application focuses on homeostatic coupling of intracellular copper (Cu) and iron (Fe) levels to gene expression in the yeast Saccharomyces cerevisiae. Metalloregulation of Cu uptake genes is controlled by the Mac1 transcriptional activator. Metalloregulation of Fe uptake genes is controlled by two related activators, Aft1 and Aft2. These three factors are transcriptional activators in metal-deficient cells, but specifically inhibited in metal-replete cells. The major objectives are to elucidate the mechanisms of Cu ion sensing by Mac1 and Fe ion sensing by Aft1 and Aft2. Many interconnections exist between Fe and Cu homeostasis in yeast. In the past grant cycle we provided evidence suggestive that Mac1 senses Cu ions within the yeast nucleus through direct binding of Cu(I) ions by Mac1. Cu(I) binding appears to induce an intramolecular interaction between the N-terminal DNA binding domain and C-terminal transactivation domain. Studies are proposed to verify direct sensing of Cu and to map the intramolecular interface that inhibits both DNA binding and transactivation. An important goal is to identify other yeast proteins important in either Cu shuttling to the nucleus or Cu inhibition of Mac1. Fe-homeostasis in yeast is regulated by Aft1 and Aft2. The mechanism of Fe inhibition of Aft1 and Aft2 function is unknown. The significance of these studies is that S. cerevisiae is the best model eukaryotic system to understand the mechanism of Fe sensing by a transcription regulator. We propose to determine whether Fe sensing occurs through direct Fe binding, Fe-mediated post-translational modification or through another protein? Aft1 and Aft2 exhibit limited functional redundancy in yeast. We propose to determine whether each exhibits a distinct physiological function. Mac1 and Aftl/Aft2 are excellent model eukaryotic systems to study nutritional control of gone expression. Information gleaned on the mechanism of Cu- and Fe-regulation may be applicable to other species.