HEME A AND CYTOCHROME OXIDASE BIOSYNTHESIS

真核细胞色素c氧化酶，呼吸道的末端复合物， 链，位于线粒体内膜， 由染色体和细胞核编码的亚基组装而成。 这种异源低聚复合物的生物合成需要表达 大量的核基因，其产物催化 线粒体前体mRNA，mRNA的翻译，以及大量的 翻译后事件。 至少有十几个这样的辅助因素， 在组装过程的后期发挥作用， 在合成、输入和膜插入所有 催化和结构亚基。 我们之前的大部分努力 补助金期间已被用于实现更好的 了解组装缺陷突变体的生化病变。 结果，三种蛋白质被赋予了特定的功能，一种在 血红素A合成（Coxl0p）和两个在线粒体铜稳态中作用 （Coxl7p，Sco1p）。 其他几种线粒体蛋白质参与后， 细胞色素氧化酶组装的翻译阶段也是 表征了 本提案的目标如下。1)重建一个 用于研究铜从Cox17 p转移到Sco1p的体外系统， 随后是细胞色素氧化酶的脱辅基亚基2。 这些研究 将利用纯化的可溶性Cox17 p、脂质体结合的Sco1p和 含有未组装的亚基2的亚线粒体颗粒。2)研究 通过脉冲追踪组装组成亚基的动力学 实验这种技术也将用于探测 这种酶的组装在突变体中被阻断。3)来筛选变种人 在血红素A合成的最后一步4)将联合收割机的基因和 阐明基因产物功能的生物化学方法 以装配缺陷型菌株为代表。 以来 细胞色素氧化酶组装的机制预计是类似的 在所有真核生物中，从这些研究中获得的信息应该是 有助于未来分析细胞色素引起的人类疾病 氧化酶缺乏症

Eucaryotic cytochrome c oxidase, the terminal complex of the respiratory chain, is located in the mitochondrial inner membrane where it is assembled from both mitochondrially and nuclearly encoded subunits. Biosynthesis of this heteroligomeric complex requires the expression of a large number of nuclear genes whose products catalyze processing of the mitochondrial pre-mRNAs, translation of the mRNAs, and a host of post-translational events. At least a dozen such ancillary factors have been implicated to function at late stages of the assembly process, subsequent to synthesis, import and membrane insertion of all the catalytic and structural subunits. Most of our efforts during the prior grant period have been channeled towards attaining a better understanding of the biochemical lesions in assembly-defective mutants. As a result specific functions were ascribed to three proteins, one in heme A synthesis (Coxl0p) and two in mitochondrial copper homeostasis (Coxl7p, Sco1p). Several other mitochondrial proteins involved at post- translational stages of cytochrome oxidase assembly were also characterized. The present proposal has the following goals. 1) To reconstitute an in vitro system for studying copper transfer from Cox17p to Sco1p and subsequently to the apo-subunit 2 of cytochrome oxidase. These studies will make use of purified soluble Cox17p, liposome-bound Sco1p, and of submitochondrial particles containing unassembled subunit 2. 2) To study the kinetics of assembly of the constituent subunits by pulse-chase experiments. This technique will also be used to probe the stages at which enzyme assembly is blocked in mutants. 3) To screen for mutants in the terminal step of heme A synthesis. 4) To combine genetic and biochemical approaches in elucidating the functions of the gene products represented by the class of assembly-defective strains. Since the mechanism of cytochrome oxidase assembly is anticipated to be similar in all eucaryotes, information gained from these studies should be helpful in future analyses of human disorders stemming from cytochrome oxidase deficiencies.