MATURATION OF CHLOROPLAST CYTOCHROMES

描述：该计划广泛的、长期的研究目标是 对叶绿体能量组装过程的理解 传导膜，特别强调辅因子和 多肽被组装在一起以产生特定的功能排列 活着。在本申请中，重点是c型细胞色素， B6/f复合体的可溶性c6和膜锚定的f，以及cyt b6，an B6/f复合体的完整膜亚单位。选择了b6/f综合体 因为它的相对组成简单和明确的辅因子结合 网站。这种复合体对实验中的生长并不是必需的 有机体莱茵衣藻，允许研究计划开发 经典的分子遗传学以及用于研究的生化方法 它的组件。本项目期间的一个主要目标是 对新发现的两种中药成分进行功能研究 类囊体膜相关细胞色素c组装复合体 和CCSL。这些新蛋白质的拓扑结构和方向将是 结合表位暴露分析大肠杆菌的PhoA-融合分析 用纯化的类囊体膜原位检测蛋白水解酶敏感性 膜。这些蛋白质或包含这两种蛋白质的复合体将是 从表达生物素或His6标记的莱茵哈迪氏杆菌菌株中纯化 这两种蛋白质的不同版本，以便确定 并建立了CCS生化活性的测定方法 蛋白质。第二个目标是克隆与之前的 定义了CCS2、3和4基因座，这是组装所有基因所必需的 叶绿体c型细胞色素，其产物被认为相互作用 与CCSL和CCSA。这些基因将从一个有索引的粘粒中识别出来 基于它们拯救非光合作用CCS能力的图书馆 菌株，或通过它们与CCS中的物理关联序列 携带可添加标签的等位基因的菌株。在Cyt b6的情况下，描述 一种独特的装配表型导致了多个CCB基因座的定义 其产品是专门用于将血红素插入到 两个血红素结合部位(bh部位)。第三个目标是通过以下方式克隆这些基因座 应用与CCS基因座相同的方法。增加的 在这个模型系统中关于血红素蛋白组装的知识是直接的 适用于理解含有其他辅因子的组装 电子转移络合物，尤其是结构上更精细的 呼吸性Bc1复合体或吞噬细胞NADPH氧化酶，其功能是 受线粒体肌病和慢性肉芽肿性疾病的影响， 分别进行了分析。

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.