MATURATION OF CHLOROPLAST CYTOCHROMES

叶绿体细胞色素的成熟

• 批准号: 2693246
• 负责人: SABEEHA MERCHANT
• 金额: $ 19.75万
• 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/2693246
• 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 了解叶绿体能量的组装过程 转导膜，特别强调辅因子和 多肽被组装以产生特定的功能排列 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 和膜锚定的 f，以及细胞色素 b6， b6/f 复合物的完整膜亚基。 选择b6/f复合物 由于其相对组成简单和明确的辅因子结合 网站。 该复合物对于实验中的生长不是必需的 莱茵衣藻生物体允许研究计划利用 经典的分子遗传学以及生化研究方法 其组装。 当前项目期间的首要目标是 对两个新发现的组件进行功能研究 假定的类囊体膜相关细胞色素 c 组装复合物，CcsA 和抄送。 这些新蛋白质的拓扑结构和方向将是 通过大肠杆菌中的 phoA 融合分析结合表位暴露进行分析 和用纯化的类囊体进行原位蛋白酶敏感性测定 膜。 蛋白质或含有两种蛋白质的复合物将是 从表达生物素或 his6 标签的莱茵衣藻菌株中纯化 这两种蛋白质的版本，以便识别该蛋白质的其他成分 复杂并开发 Ccs 生化活性的检测方法 蛋白质。 第二个目标是克隆与先前的基因相对应的基因 定义了CCS2、3和4基因座，这些基因座是组装所有基因座所必需的 叶绿体 c 型细胞色素，其产物被认为可以相互作用 与 Ccsl 和 CcsA。 将从索引粘粒中鉴定基因 图书馆基于其拯救非光合作用CCS的能力 菌株，或凭借它们与 ccs 中的 BLE 序列的物理关联 携带 ble 标记等位基因的菌株。 对于 cyt b6，描述 独特的装配表型导致了多个 CCB 位点的定义 其产品专门需要将血红素插入其中之一 两个血红素结合位点（bH 位点）。 第三个目标是通过以下方式克隆这些基因座 应用针对 CCS 基因座描述的相同方法。 增加的 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因子的组装 电子转移配合物，尤其是结构更复杂的 呼吸性 bc1 复合物或吞噬细胞 NADPH 氧化酶，其功能是 受线粒体肌病和慢性肉芽肿病的影响， 分别。