INTRACELLULAR PATHWAYS OF COPPER TRAFFICKING

描述：所有生物都必须调节氧化还原的动态平衡。 活性铜离子，既是生命所必需的，也是潜在的有毒物质。 这项研究的总体假设是，细胞积累 而铜的分布精确地受到 多个铜“贩运”因素与一个因素沟通和相互作用 又一个。这里提出的研究利用了基因定义良好的 Baker‘s酵母酿酒酵母研究胞内途径 铜走私。将探索两个系统。一种是由 BSD2，编码一种内质网蛋白，有助于防止 不受控制地吸收铜和其他金属。BSD2对BSD2的作用 铜是通过质膜金属转运蛋白Smflp介导的。 第二个铜运输系统由ATX1表示，编码为 胞内小的铜结合蛋白。我们的研究表明，Atx1p 将铜传递给Ccc2p，酵母与人类铜的同系物 肝豆状核变性和门克斯病中受影响的转运蛋白。以下是具体的 AIMS将解决如何积累和细胞内贩运 酵母中铜离子受BSD2-SMF1途径和途径控制 涉及ATX1。目标1：了解SMFLP在 Bsd2p-药物控制铜的积累。Smflp的贡献 将通过测量铜来研究铜的积累 领悟。一系列的分子和生物化学研究将探索 Bsd2突变对Smf1p积累和稳定性的影响 蛋白质及其细胞定位。目标2：了解 控制铜交易的Atx1p和Ccc2p之间的相互作用。 将使用生化和分子遗传学方法来测试 Atx1p和Ccc2p进行物理交互。通过突变研究，Atx1p 体内活性和体外铜结合所需的序列将是 已确认身份。铜离子从Atx1p到Ccc2p的直接转移将是 在无细胞系统中进行研究。这些研究中的许多也将使用 人类与ATX1和CCC2的同源物，由HAH1和Menkes和 威尔逊病基因。目标3：确定ATX1的其他下游靶点： 将利用酵母中的两个基因筛查来确定其他因素 而不是Ccc2p，后者可以作为Atx1p交割铜的接收方。 总体而言，我们相信这些对酵母的研究将提供新的重要的 关于#年铜贩运的复杂途径的信息 控制这种重要氧化还原的动态平衡的真核生物 活性金属。

DESCRIPTION: All living organisms must regulate the homeostasis of redox active copper ions that are both essential for life and potentially toxic. The overall hypothesis of this research is that the cellular accumulation and distribution of copper is precisely controlled by the actions of multiple copper "trafficking" factors that communicate and interact with one another. The research presented here utilizes the genetically well defined baker's yeast Saccharomyces cerevisiae to study intracellular pathways of copper trafficking. Two systems will be explored. One is represented by BSD2, encoding an endoplasmic reticulum protein that helps prevent the uncontrolled uptake of copper and other metals. The action of BSD2 on copper is mediated through the plasma membrane metal transporter, Smflp. The second copper trafficking system is represented by ATX1, encoding a small cytosolic copper binding protein. Our studies indicate that Atx1p delivers copper to Ccc2p, the yeast homologue to the human copper transporters affected in Wilson and Menkes diseases. The following specific aims will address how the accumulation and intracellular trafficking of copper ions in yeast is controlled by the BSD2-SMF1 pathway and by pathways involving ATX1. Aim 1: To understand the involvement of Smflp in the Bsd2p-medicated control of copper accumulation. The contribution of Smflp to copper accumulation will be studied through measurements of copper uptake. A series of molecular and biochemical studies will probe the effects of bsd2 mutations on the accumulation and stability of the Smf1p protein and its cellular localization. Aim 2: To understand the interactions between Atx1p and Ccc2p that govern copper trafficking. Biochemical and molecular genetic approaches will be used to test whether Atx1p and Ccc2p physically interact. Through mutagenesis studies, the Atx1p sequences necessary for activity in vivo and copper binding in vitro will be identified. The direct transfer of copper ions from Atx1p to Ccc2p will be investigated in a cell free system. Many of these studies will also employ the human homologues to ATX1 and CCC2, encoded by the HAH1 and Menkes and Wilson disease genes. Aim 3: To identify other downstream targets of ATX1: Two genetic screens in yeast will be exploited to identify factors other than Ccc2p that may serve as recipients for copper delivery by Atx1p. Overall, we believe that these studies in yeast will provide new important information regarding the complex pathways of copper trafficking in eukaryotes that serve to control the homeostasis of this important redox active metal.