COPPER METALLO BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

酿酒酵母中的铜金属生物化学

• 批准号: 2184279
• 负责人: DANIEL J. KOSMAN
• 金额: $ 18.92万
• 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 1999-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184279
• 关键词: Saccharomyces cerevisiae; X ray crystallography; biological models; biological signal transduction; copper; enzyme activity; fungal genetics; gene complementation; gene expression; membrane transport proteins; metal metabolism; metalloproteins; molecular cloning; oxidative stress; oxidoreductase; protein structure function; recombinant proteins; site directed mutagenesis; superoxide dismutase

The long-term objective of this research is to achieve a detailed understanding of the mechanisms which a eukaryote evolved to accumulate the essential trace nutrient copper, to regulate this accumulation, and to make this redox active metal available for metallating apo-cuproproteins. We intend to continue to exploit the characteristics of the budding yeast, Saccharomyces cerevisiae, which make it unique as a model system to establish possible mechanisms of Cu-handling and its regulation. We have demonstrated biochemically that in the initial step of Cu-accumulation, Cu(II) is reduced by at least two separate reductase activities in the yeast plasma membrane. The expression (synthesis) of these reductases, which support both Cu(II) and Fe(III) accumulation by yeast, requires a protein, Mac1p, whose gene we have cloned. Mac1p also is required for the expression of genes associated with carbon catabolite control, peroxidative stress, and heat shock. Based on its primary sequence, Mac1p may be a metalloprotein. In Specific Aims I and II we will test two hypotheses about Mac1p: 1) that it serves as a primary sensor in a signalling pathway which results in specific gene expression and 2) that a metal ion - either copper or iron - in Mac1p serves as sensor and switch in this signal transduction. Wild type Mac1p will be overexpressed and characterized to determine whether it contains a metal ion, and the spectroscopic and electrochemical properties of the metal-binding site will be evaluated. Whether Mac1p modulates gene expression by binding to (an)other protein and/or to DNA will be determined by in vivo and in vitro approaches. The in vivo function of site-directed Mac1p mutants will be assessed; loss-of-function mutants will be characterized in vitro. We have demonstrated that one of the two reductase activities in the membrane is associated with the product of the FRE1 gene and that this enzyme can use both Fe(III) and Cu(II) as substrate. In Specific Aim III we will test by genetic and biochemical means the hypothesis that the other reductase is a unique Cu(II)-specific enzyme. A mutant in this locus will be generated and the wild type gene cloned by complementation. We have demonstrated that correct intracellular Cu- (and Fe-) trafficking requires the acidification of the yeast vacuole(s). In Specific Aim IV we will test the dual hypothesis that this organelle is the initial site of intracellular Cu-accumulation, and that delivery of copper to apo- cuproproteins occurs from vacuolar stores. The proposed research is based on testable models of copper metabolism and metal-dependent gene regulation in S. cerevisiae. It makes full use of the fact that this organism remains unique among eukaryotes as a cell system in which all of the tools of classical and molecular genetics, cell biology and biochemistry can be used systematically. The details of how S. cerevisiae metabolizes copper may be in part specific to this eukaryote. However, our view is that the aqueous chemistry of copper dictates how this toxic nutrient is dealt with by an aerobic cell, and that the mechanisms suggested by our proposed studies in yeast will serve as paradigms for the design of productive studies in other cell types.

这项研究的长期目标是实现一个详细的 了解真核生物进化积累的机制 必需的微量营养素铜，以调节这种积累， 使这种氧化还原活性金属可用于金属化脱辅基铜蛋白。 我们打算继续开发芽殖酵母的特性， 酿酒酵母，这使得它作为一个独特的模型系统， 建立可能的铜处理机制及其监管。 我们有 生物化学表明，在铜积累的初始步骤， Cu（II）通过至少两种单独的还原酶活性被还原， 酵母质膜 这些还原酶的表达（合成）， 支持Cu（II）和Fe（III）的积累，需要一个 我们克隆了Mac 1 p蛋白的基因。 Mac 1 p也是必需的， 与碳分解代谢物控制相关的基因的表达， 过氧化应激和热休克。 根据其原始序列，Mac 1 p 可以是金属蛋白。 在具体目标I和II中，我们将测试两个 关于Mac 1 p的假设：1）它作为一个主要的传感器， 信号通路，其导致特异性基因表达，和2） Mac 1 p中的金属离子（铜或铁）充当传感器和开关 in this signal信号transduction转导. 野生型Mac 1 p将过表达， 表征以确定其是否含有金属离子，并且 金属结合位点的光谱和电化学性质 将被评估。 Mac 1 p是否通过结合 （a）其他蛋白质和/或DNA将通过体内和体外测定， 接近。 定点Mac 1 p突变体的体内功能将是 评估;将在体外表征功能丧失突变体。 我们 已经证明了细胞膜上的两种还原酶活性之一 与FRE 1基因的产物有关，这种酶可以 同时使用Fe（III）和Cu（II）作为底物。 在第三阶段，我们将 通过遗传和生物化学手段的测试， 还原酶是一种独特的Cu（II）特异性酶。 这个基因座的突变体 产生并通过互补克隆野生型基因。 我们有 表明正确的细胞内Cu-（和Fe-）运输需要 酵母液泡的酸化。 在具体目标4中，我们将 测试双重假设，即这个细胞器是最初的网站 细胞内铜积累和铜向apo- 铜蛋白存在于液泡中。 该研究基于 铜代谢和金属依赖基因的可检验模型 在S.啤酒。 它充分利用了这样一个事实， 生物体作为一种细胞系统在真核生物中仍然是独特的，在这种细胞系统中，所有的 经典和分子遗传学，细胞生物学和 生物化学可以系统地使用。 详细介绍了S.酿酒酵母 铜的代谢可能部分是这种真核生物所特有的。 然而，在这方面， 我们的观点是，铜的水化学性质决定了这种有毒物质是如何 营养物质是由有氧细胞处理的， 我们提出的酵母研究建议将作为范例， 设计其他细胞类型的生产性研究。