MATURATION OF CHLOROPLAST CYTOCHROMES

叶绿体细胞色素的成熟

• 批准号: 6018927
• 负责人: SABEEHA MERCHANT
• 金额: $ 19.74万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018927
• 关键词: Chlamydomonas; Escherichia coli; bioenergetics; chloroplasts; cofactor; cytochrome b; cytochrome c; electron transport; genetic library; heme; hemoprotein biosynthesis; intracellular transport; membrane biogenesis; membrane proteins; molecular cloning; molecular genetics; mutant; posttranslational modifications; protein purification; protein transport; site directed mutagenesis

DESCRIPTION: The broad, long term research objective of this program is the understanding of the process of assembly of the chloroplast energy transducing membrane, with particular emphasis on how the cofactors and polypeptides are assembled to yield a specific functional arrangement in vivo. In this application, the emphasis is on the c-type cytochromes, soluble c6 and membrane-anchored f of the b6/f complex, and cyt b6, an integral membrane subunit of the b6/f complex. The b6/f complex is chosen for its relative compositional simplicity and well-defined cofactor binding sites. This complex is not essential for growth in the experimental organism Chlamydomonas reinhardtii which allows the research plan to exploit classical molecular genetics as well as biochemical approaches for the study of its assembly. A prime objective during the present project period is to undertake functional studies of two newly identified components of a putative thylakoid membrane-associated cytochrome c assembly complex, CcsA and Ccsl. The topology and orientation of these novel proteins will be analyzed by phoA-fusion analysis in E. coli combined with epitope exposure and protease susceptibility assays in situ with purified thylakoid membranes. The proteins, or a complex containing the two proteins, will be purified from C. reinhardtii strains that express biotin- or his6-tagged versions of the two proteins, in order to identify other components of the complex and to develop assays for the biochemical activities of the Ccs proteins. A second aim is to clone genes corresponding to the previously defined CCS2, 3 and 4 loci, which are required for the assembly of all chloroplast c-type cytochromes, and whose products are proposed to interact with Ccsl and CcsA. The genes will be identified from an indexed cosmid library on the basis of their ability to rescue non-photosynthetic ccs strains, or by virtue of their physical association wit ble sequences in ccs strains carrying ble-tagged alleles. In the case of cyt b6, the description of a unique assembly phenotype led to the definition of multiple CCB loci whose products are required specifically for heme insertion into one of the two heme binding sites (bH site). A third aim is to clone thes loci by applying the same methods described for the CCS loci. The increased knowledge of heme protein assembly in this model system is directly applicable to the understanding of the assembly of other cofactor-containing electron transfer complexes, especially the structurally more elaborate respiratory bc1 complex or phagocyte NADPH oxidase whose functions are affected in mitochondrial myopathies and chronic granulomatous disease, respectively.

描述：该计划的广泛长期研究目标是 了解叶绿体能量组装过程 转导膜，特别强调辅助因子和 组装多肽以产生特定的功能布置 体内。 在此应用中，重点是C型细胞色素， B6/F复合物的可溶性C6和膜锚定F，以及Cyt B6 B6/F复合物的整体膜亚基。 选择B6/F复合物 因为它的相对组成简单性和定义明确的辅因子结合 站点。 该复合物对于实验的增长不是必不可少的 有机体衣原体Reinhardtii，允许研究计划利用 经典分子遗传学以及研究的生化方法 它的组装。 在本项目期间的一个主要目标是 对两个新鉴定的组件进行功能研究 推定的类囊体膜相关的细胞色素C组合络合物CCSA 和CCSL。 这些新蛋白的拓扑和方向将是 通过大肠杆菌与表位暴露结合的Phoa融合分析分析 和蛋白酶的敏感性测定原位用纯化的类囊体 膜。 蛋白质或包含两种蛋白质的复合物将是 从表达生物素或His6标签的C. renhardtii菌株中纯化 这两种蛋白质的版本，以识别 复杂并开发CCS生化活性的测定法 蛋白质。 第二个目的是克隆基因对应于先前 定义的CCS2、3和4位基因座，所有这些基因座是所有人组装所必需的 叶绿体C型细胞色素，并提议其产物相互作用 与CCSL和CCSA。 这些基因将从索引的宇宙中识别 图书馆根据其挽救非光合合成CC的能力 菌株，或者凭借其在CCS中的身体关联序列 带有BLE标签等位基因的菌株。 对于Cyt B6，描述 独特的组装表型导致了多个CCB基因座的定义 需要专门将其产品的产品插入其中一个 两个血红素结合位点（BH位点）。 第三个目标是克隆该基因座 应用针对CCS基因座描述的相同方法。 增加 该模型系统中血红素蛋白组装的知识直接 适用于理解其他含辅助因子的组装 电子转移复合物，尤其是结构上更精致的 呼吸道BC1复合物或吞噬细胞NADPH氧化酶的功能是 受到线粒体肌病和慢性肉芽肿性疾病的影响， 分别。