HEME A AND CYTOCHROME OXIDASE BIOSYNTHESIS

血红素 A 和细胞色素氧化酶生物合成

• 批准号: 6329750
• 负责人: ALEXANDER A TZAGOLOFF
• 金额: $ 18.73万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-01-15 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329750
• 关键词: Saccharomyces cerevisiae; apoenzymes; complementary DNA; copper; cytochrome oxidase; enzyme biosynthesis; enzyme deficiency; enzyme mechanism; enzyme reconstitution; fungal genetics; gene complementation; heme; human genetic material tag; liposomes; microorganism metabolism; mitochondria; mutant; porphyrin biosynthesis; posttranslational modifications; protein structure function

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.

真核细胞色素C氧化酶，呼吸道的末端复合体 链，位于线粒体内膜上 由线粒体和核编码的亚基组装而成。 这种异寡聚体复合体的生物合成需要表达 大量的核基因，其产物催化加工 线粒体前mRNAs，mRNAs的翻译，以及一系列 翻译后事件。至少有十几个这样的辅助因素 被牵连到组装过程的后期阶段， 在合成、进口和膜植入之后，所有的 催化亚基和结构亚基。我们在前一年的大部分努力 授权期已经被用来实现更好的 了解装配缺陷突变体的生化损伤。 因此，特定的功能被归因于三种蛋白质，其中一种是 血红素A合成(Coxl0p)和两个线粒体铜稳态 (Cox17p，Sco1p)。其他几种线粒体蛋白参与后- 细胞色素氧化酶组装的翻译阶段也是 特色化的。 本提案有以下目标。1)重组IN 研究铜从Cox17p到Sco1p转移的体外体系和 随后是细胞色素氧化酶的脱辅基2亚单位。这些研究 将利用纯化的可溶性Cox17p、脂质体结合的Sco1p和 含有未组装亚单位的亚线粒体颗粒2.2)进行研究 脉冲追踪法组装组成亚基的动力学 实验。这项技术也将被用来探测 哪种酶的装配在突变体中被阻止。3)筛选突变体 在血红素A合成的最后一步。4)结合遗传和 生物化学方法在阐明基因产物功能中的应用 以装配缺陷菌株类为代表。自.以来 细胞色素氧化酶的组装机制预计是相似的 在所有真核生物中，从这些研究中获得的信息应该是 有助于未来对细胞色素引起的人类疾病的分析 氧化酶缺乏症。