COPPER METALLO-BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的铜金属生物化学

• 批准号: 3306250
• 负责人: DANIEL J. KOSMAN
• 金额: $ 16.99万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306250
• 关键词: Saccharomyces cerevisiae; biological models; copper; enzyme activity; gene mutation; glutathione; intracellular transport; membrane transport proteins; metal metabolism; metallothionein; molecular cloning; mutant; organometallic compounds; oxidoreductase; superoxide dismutase; toxin metabolism

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.

本研究的长期目标是建立一个 必需物质的金属生物化学的详细描述 酿酒酵母中的微量金属铜。 这 这项研究的意义在于，机制有限 有关任何基本细胞生物化学的信息 任何真核生物中的二价过渡金属离子。 这个提议是 旨在测试吸收模型的两个特定方面 酿酒酵母中铜的细胞利用基于已发表的和 本提案中描述的初步工作。 这两个方面 是： 1) 从培养基中摄取 Cu(II) 涉及 Cu(II) 质膜还原酶催化还原为 Cu(I) 活性和 2) 谷胱甘肽是 Cu(I) 的细胞内运输 细胞。 描述了解决这些方面的四个具体目标 并且还将提供额外的遗传试剂来延长 这些研究将在未来进行。 目标 I. 克隆并表征 突变等位基因 cup3，表现出更快的 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 对这种突变的研究。 目标二。 证明减少 介质 Cu(II) 到细胞相关 Cu(I) 是 Cu 的一个可能步骤 酿酒酵母的积累，该活性由 Cu(II) 还原酶活性，并对其进行生化表征 活动。 目标三。 确定可能的功能 谷胱甘肽 (GSH)，参与铜在葡萄球菌细胞质中的重新分配。 酿酒酵母，以及铜硫因作为功能性铜储存的作用 用于激活 apo-Cu,Zn 超氧化物歧化酶 (SOD-1)。 目的 四． 分离表现出较慢或异常 Cu 的新突变体 积累可能包括 Cu(II) 还原突变体， 运输，或细胞内运输。 这些将作为 为模型的未来测试奠定基础，特别是测试 Cu(II) 还原酶和推定的 Cu 转运蛋白的可能性 是相同的基因产物并确认特定的作用 铜处理中的细胞内因子。 实验设计是 基于酿酒酵母的独特特征 真核生物，包括公认的经典和分子 遗传学以及体内系统和控制的容易性 操纵和分析。 开始所需的大部分试剂 这项工作已经准备好了。 铜的详细信息 这些研究中出现的金属生物化学将提供 高等铜处理的可能机制的范式 真核生物。