ASSEMBLY OF CYTOCHROME C OXIDASE

我们建议研究细胞色素c氧化酶，它有亚基编码 在线粒体和细胞核中，都在内部组装 薄膜。这一过程可能对人类疾病很重要。 因为最近很明显，衰老和许多OXPHO 疾病表现为细胞色素C氧化酶缺乏症。这些是 特定于退行性组织的疾病，包括：致命和良性 婴儿肌病，利综合征，缺血性心脏病， 阿尔茨海默氏症和帕金森氏症。其确切原因是 这些疾病是未知的。尽管许多OXPHOS疾病已经被 与线粒体基因相关联的是，最近已经清楚地表明，它们的 遗传学是复杂的，核基因和线粒体基因都是 牵涉其中。为了开始解决这个问题，我们将首先研究 酵母细胞色素C氧化酶的组装。该蛋白的生物发生 需要3个线粒体基因和至少38个核基因。几个 最近的研究表明，这种多聚体的组装 蛋白质是由许多蛋白质促进的，包括--热 休克蛋白和细胞色素c氧化酶特异性的“组装促进剂” 蛋白质。我们的总体目标是阐明 亚基组装，~识别作为中间体的部分复合体 这一过程，并决定了细胞色素c氧化酶如何特异的“组装” “促进者”蛋白质在这一途径中发挥作用。我们的实验 策略将结合使用新奇的集合《集结 缺陷“宠物突变体，这些基因的条件性突变体，条件性 和COX结构基因的零突变，体内动力学分析， 生化和免疫化学分级研究，同步进行 体内组装系统和一种新开发的体外线粒体 支持导入、导出和组装的系统。一旦我们有了 在酵母中确立了“组装促进器”蛋白质的角色 问相似的蛋白质是否在人类细胞色素c的组装中起作用 氧化物酶。这将通过克隆人类基因来实现 在功能上补充众所周知的“组装缺陷”酵母 变种人。这些人类基因的可获得性应该提供一个 开始讨论细胞色素c的分子基础的机会 OXPHOS病中的氧化酶缺陷。

We propose to study how cytochrome c oxidase, which has subunits encoded in both the mitochondrion and nucleus, is assembled in the inner membrane. This process is likely to be important to human disease because it has become clear recently that both aging and many OXPHOS diseases exhibit cytochrome c oxidase deficiencies. These are degenerative tissue specific diseases that include: fatal and benign infantile myopathies, Leigh's syndrome, ischemic heart disease, Alzheimer's disease, and Parkinson's disease. The precise cause of these diseases is unknown. Although many OXPHOS diseases have been linked to mitochondrial genes it has become clear recently that their genetics is complex and that both nuclear and mitochondrial genes are involved. To begin to address this problem we will first study the assembly of yeast cytochrome c oxidase. The biogenesis of this protein requires 3 mitochondrial genes and at least 38 nuclear genes. Several recent studies have suggested that the assembly of this multimeric protein is facilitated by a number of proteins, including -both heat shock proteins and cytochrome c oxidase specific "assembly facilitator" proteins. Our overall objectives are to elucidate the sequence in which subunits assemble,~ identify partial complexes that are intermediates in the process, and determine how cytochrome c oxidase specific "assembly facilitator" proteins function in this pathway. Our experimental strategy will combine the use of a collection of novel "assembly defective" pet mutants, conditional mutants in these genes, conditional and null mutants in COX structural genes, in vivo kinetic analyses, biochemical and immunochemical fractionation studies, a synchronized in vivo assembly system, and a newly developed mitochondrial in vitro system that supports import, export, and assembly. Once we have established a role for "assembly facilitator" proteins in yeast we will ask if similar proteins function in the assembly of human cytochrome c oxidase. This will be approached by cloning human genes that functionally complement well-understood "assembly defective" yeast mutants. The availability of these human genes should provide an opportunity to begin to address the molecular basis for cytochrome c oxidase defects in OXPHOS diseases.