COPPER METALLO-BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

这项研究的长期目标是建立一个 详细描述了精华的金属生物化学 酵母中的微量金属铜，酿酒酵母。这个 本研究的意义在于机械论上的局限性。 关于任何必需的细胞生化的信息 任何真核生物中的二价过渡金属离子。这项建议是 旨在测试模型的两个特定方面的吸收和 铜在酿酒酵母中的细胞利用 本提案中所述的前期工作。这两个方面 1)从介质中吸收铜(II)涉及到铜(II) 质膜还原酶催化还原成铜(I) 活性和2)谷胱甘肽是 铜(I)的胞内迁移被带入 手机。描述了针对这些方面的四个具体目标 这也将提供更多的遗传试剂来扩展 这些研究在未来。目标I.克隆并鉴定 突变等位基因CuP3，表现出动力学上更快的铜 积累和相应升高的铜(II)还原酶 活动。建议的模型主要基于我们初步的 对这种突变的研究。目标二.证明减少 中等浓度的铜(II)转化为细胞伴生的铜(I)是铜的一个可能步骤 酿酒酵母的积累，这种活性由 铜(II)还原酶活力及其生化特征 活动。目的三.确定可能的作用(S) 谷胱甘肽(GSH)对铜离子在沙门氏菌胞质中的再分配作用。 Cerevisiae，以及铜硫蛋白作为功能铜库的作用 用于激活载脂蛋白-铜，锌超氧化物歧化酶(SOD-1)。目标 IV.分离出表现出较慢或异常铜的新突变体 在铜(II)还原过程中可能包括突变体的积累， 运输，或细胞内贩运。这些将作为 为模型的未来测试提供基础，特别是测试 铜(II)还原酶和可能的铜转运体的可能性 都是相同的基因产物，并确认其特定的作用 铜处理中的胞内因素。实验设计为 根据酿酒酵母的独特特性 真核生物，包括经典的和分子的 遗传学和体内系统化和可控性的易用性 操纵和分析。大多数试剂都需要从 这项工作已经准备好了。铜的细节 这些研究中产生的矿物生物化学将提供一种 高等植物铜处理可能机制的范式 真核生物。

The long range objective of this research is to establish a detailed description of the metallo-biochemistry of the essential trace metal, copper, in the yeast, Saccharomyces cerevisiae. The significance of this research is that there is limited mechanistic information about the cellular biochemistry of any of the essential divalent transition metal ions in any eukaryote. This proposal is designed to test two specific aspects of a model of the uptake and cellular utilization of Cu in S. cerevisiae based on published and preliminary work described in this proposal. These two aspects are: 1) that uptake of Cu(II) from the medium involves Cu(II) reduction to Cu(I) catalyzed by a plasma membrane reductase activity and 2) that glutathione is a component of the intracellular trafficking of the Cu(I) which is taken into the cell. Four Specific Aims are described which address these aspects and which will also provide additional genetic reagents to extend these studies in the future. Aim I. Clone and characterize the mutant allele, cup3, which exhibits kinetically faster Cu accumulation and a correspondingly elevated Cu(II) reductase activity. The proposed model is largely based on our preliminary studies of this mutation. Aim II. Demonstrate that reduction of medium Cu(II) to cell-associated Cu(I) is a possible step in Cu accumulation by S. cerevisiae, that this activity is represented by the Cu(II) reductase activity, and biochemically characterize this activity. Aim III. Determine the possible function(s) of glutathione (GSH), in the redistribution of Cu in the cytosol of S. cerevisiae, and the role of Cu-thionein as a functional Cu store for the activation of apo-Cu,Zn superoxide dismutase (SOD-1). Aim IV. Isolate new mutants which exhibit slower or aberrant Cu accumulation which may include mutants in Cu(II) reduction, transport, or intracellular trafficking. These will serve as the basis for future tests of the model, in particular, to test the possibility that the Cu(II) reductase and putative Cu-transporter are the same gene product and to confirm the role of specific intracellular factors in Cu-handling. The experimental design is based on the unique characteristics of S. cerevisiae among eukaryotes which include well-established classical and molecular genetics and an ease of systematic and controlled in vivo manipulation and analysis. Most of the reagents needed to begin this work have been prepared. The details of copper metallobiochemistry which emerge from these studies will provide a paradigm for possible mechanisms of Cu-handling in higher eukaryotes.