COPPER METALLO BIOCHEMISTRY IN SACCHAROMYCES CEREVISIAE

这项研究的长期目标是实现详细的 对真核生物进化积累机制的理解 必需的微量营养铜，以调节这种积累，并 使这种氧化还原活性金属可用于金属化脱脂蛋白铜蛋白。 我们打算继续利用萌芽酵母的特性， 酿酒酵母，这使它成为独一无二的模式系统 建立可能的铜处理及其调控机制。我们有 生物化学证明，在铜积累的初始阶段， 铜(II)被至少两个单独的还原酶活性还原 酵母质膜。这些还原酶的表达(合成)， 同时支持酵母菌积累铜(II)和铁(III)，需要 蛋白质，Mac1p，我们已经克隆了它的基因。Mac1p也是必需的 与碳分解代谢控制相关的基因表达， 过氧化应激和热休克。根据其初级序列，Mac1p 可能是一种金属蛋白。在具体目标一和目标二中，我们将测试两个 关于Mac1p的假设：1)它在 导致特定基因表达的信号通路和2) Mac1p中的金属离子--铜或铁--用作传感器和开关 在这个信号转导中。野生型Mac1p将过度表达和 其特征是确定它是否包含金属离子，以及 金属结合位的光谱和电化学性质 将会被评估。Mac1p是否通过结合来调节基因表达 (An)其他蛋白质和/或DNA将通过体内和体外测定 接近了。Mac1p定点突变体的体内功能将是 评估；功能丧失的突变体将在体外鉴定。我们 已经证明了膜上的两种还原酶中的一种 与FRE1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。在第三个具体目标中，我们将 通过遗传和生物化学手段进行检验的假说 还原酶是一种独特的铜(II)专一性酶。在这个基因座上的突变人会 产生野生型基因，通过互补克隆野生型基因。我们有 证明了正确的细胞内铜(和铁)运输需要 酵母液泡的酸化(S)。在具体目标四中，我们将 检验双重假设，即这个细胞器是 细胞内铜的积累，以及铜向细胞外的输送。 铜原蛋白来自液泡库。拟议的研究是基于 铜代谢与金属依赖基因的可测性模型 对酿酒酵母的监管。它充分利用了这一事实 有机体在真核生物中仍然是独一无二的，它是一个细胞系统，其中所有的 经典和分子遗传学、细胞生物学和 生物化学可以被系统地使用。酿酒酵母是如何 代谢铜可能在一定程度上是这种真核生物所特有的。然而， 我们的观点是，铜的水化学决定了这种毒性 营养物质是由一个好氧细胞处理的，而这种机制 我们在酵母中的拟议研究建议将作为 其他细胞类型的生产性研究的设计。

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.