INTRACELLULAR PATHWAYS OF COPPER TRAFFICKING

描述：所有生物体都必须调节氧化还原的稳态 活性铜离子是生命所必需的，并且具有潜在的毒性。 这项研究的总体假设是， 铜的分布是由以下因素的作用精确控制的 多个铜"贩运"因素， 另 这里介绍的研究利用了基因定义良好的 面包酵母酿酒酵母研究细胞内途径 铜走私 将探索两个系统。 一个是由 BSD 2，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。 BSD 2的作用 铜通过质膜金属转运蛋白Smflp介导。 第二个铜贩运系统由ATX1表示，编码一个 小胞质铜结合蛋白。 我们的研究表明Atx1p 将铜传递给Ccc2p，Ccc2p是人类铜的酵母同系物 Wilson和Menkes病中受影响的转运蛋白。 以下具体 目的将解决如何积累和细胞内贩运的 酵母中的铜离子由BSD 2-SMF 1途径和 关于ATX1 目的1：了解Smflp参与 铜积累的bsd2p-药物控制。 Smflp的贡献 铜积累将通过测量铜 摄取。 一系列分子和生化研究将探讨 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 Atx1p和Ccc2p之间的相互作用决定了铜的运输。 生物化学和分子遗传学方法将用于测试是否 Atx1p和Ccc2p在物理上相互作用。 通过突变研究，Atx1p 体内活性和体外铜结合所必需的序列将被 鉴定 铜离子从Atx1p到Ccc2p的直接转移将是 在无细胞系统中研究。 其中许多研究还将采用 ATX 1和CCC 2的人类同源物，由HAH 1和Menkes编码， 威尔逊病基因 目的3：确定ATX 1的其他下游靶标： 将利用酵母中的两种遗传筛选来鉴定其他因子， 而不是Ccc2p，其可以用作Atx1p的铜递送的接收者。 总的来说，我们相信这些研究在酵母将提供新的重要 关于贩运铜的复杂途径的资料， 真核生物，用于控制这一重要的氧化还原的稳态 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.