COPPER METALLO-BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

这项研究的长期目标是建立一个 详细描述的金属生物化学的基本 微量金属铜存在于酿酒酵母中 的 这项研究的意义在于， 关于任何重要的细胞生物化学的信息 二价过渡金属离子。 这项建议是 旨在测试吸收模型的两个具体方面， S中铜的细胞利用。酿酒厂根据已发表的和 本提案中所述的初步工作。 这两方面 是：1）从介质中吸收Cu（II）涉及Cu（II） 通过质膜还原酶催化还原为Cu（I） 活性和2）谷胱甘肽是一种成分， Cu（I）的细胞内运输，其被带入 cell. 四个具体的目标是针对这些方面的描述 它还将提供额外的基因试剂， 这些研究在未来。 艾姆岛 克隆并表征 突变等位基因cup 3，其表现出动力学更快的Cu 积累和相应升高的Cu（II）还原酶 活动 所提出的模型主要基于我们的初步 研究这种突变。 Aim II. 证明减少 中Cu（II）到细胞缔合Cu（I）是Cu（II）中的可能步骤。 积累S。酿酒酵母，这种活性是由 Cu（II）还原酶活性，并对其进行生物化学表征 活动 Aim III. 确定以下各项的可能功能 谷胱甘肽（GSH）参与了Cu在S. 啤酒，铜硫蛋白作为功能铜库的作用 激活apo-Cu，Zn超氧化物歧化酶（SOD-1）。 目的 四. 分离出表现出较慢或异常Cu的新突变体 可能包括Cu（II）还原突变体的积累， 运输或细胞内运输。 这些将作为 该模型未来测试的基础，特别是测试 铜（II）还原酶和推定的铜转运蛋白 是相同的基因产物，为了证实特异性 细胞内因素在铜处理。 实验设计 根据S.酿酒厂之间 真核生物，包括公认的经典和分子生物学 遗传学和易于系统和控制的体内 操作和分析。 开始所需的大部分试剂 这项工作已经准备好了。 铜的细节 从这些研究中产生的金属生物化学将提供一个 范例的可能机制铜处理在较高的 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.