MATURATION OF CHLOROPLAST CYTOCHROMES

该计划的广泛，长期的研究目标是 了解叶绿体能量的组装过程 转导膜，特别强调如何辅因子和 多肽被组装以产生特定的功能排列， vivo. 在本申请中，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复合物的完整膜亚单位。 选择b6/f复合物 由于其相对简单的组成和明确的辅因子结合， 网站. 这种复合物在实验中对生长不是必需的。 这使得研究计划能够利用 经典的分子遗传学和生物化学方法 的集会。 本项目期间的主要目标是 对新确定的两个组成部分进行功能研究， 类囊体膜结合细胞色素c组装复合物 和Ccsl。 这些新蛋白质的拓扑结构和方向将是 通过在E.大肠杆菌结合表位暴露 用纯化的类囊体原位进行蛋白酶敏感性测定 膜。 这些蛋白质或含有这两种蛋白质的复合物将被 从C.表达生物素-或His 6-标记的莱茵哈德氏菌菌株 这两种蛋白质的版本，以确定其他成分的 复杂的，并开发测定的生化活动的Ccs proteins. 第二个目标是克隆与先前的基因相对应的基因。 定义的CCS 2，3和4位点，这是所有组装所需的， 叶绿体c型细胞色素，其产物被认为是相互作用的 使用Ccsl和CcsA。 这些基因将从索引粘粒中鉴定出来 基于它们拯救非光合CCS的能力， 菌株，或凭借它们与CCS中的序列的物理关联 携带ble-tagged等位基因的菌株。 在细胞色素b6的情况下，描述 一个独特的组装表型导致多个CCB基因座的定义 其产品是专门用于血红素插入到其中一个 两个血红素结合位点（bH位点）。 第三个目标是克隆这些基因座， 应用针对CCS基因座描述的相同方法。 增加的 在该模型系统中血红素蛋白组装知识直接 适用于理解其他含辅因子的组装 电子转移复合物，特别是结构上更复杂的 呼吸道bc 1复合物或吞噬细胞NADPH氧化酶，其功能是 影响线粒体肌病和慢性肉芽肿病， 分别

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.