MATURATION OF CHLOROPLAST C-TYPE CYTOCHROMES

叶绿体 C 型细胞色素的成熟

• 批准号: 3307791
• 负责人: SABEEHA MERCHANT
• 金额: $ 18.12万
• 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 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307791
• 关键词: Chlamydomonas; cell free system; chloroplasts; cytochromes; eukaryote; heme; hemoprotein biosynthesis; intracellular transport; membrane biogenesis; molecular cloning; molecular genetics; mutant; posttranslational modifications; protein transport; site directed mutagenesis; structural genes; ultracentrifugation

In eukaryotic cells, the biosynthesis of the electron transfer complexes of energy transducing membranes requires not only the regulated and coordinate expression of nuclear and organellar genes for the various macromolecular components, but also the correct targeting and processing of the pre-proteins, the association of cofactors with the apoproteins and the stoichiometric assembly of multisubunit structures. These post- translational events are the subject of our long term research program on thylakoid membrane biogenesis. This application focusses on the processing steps - especially heme attachment - involved in the maturation of photosynthetic c-type cytochromes (soluble cyt c6 and membrane-anchored cyt f). The maturation of nuclear-encoded, lumen- targeted cyt c6 includes specific proteolytic processing steps following translocation across the envelope- and thylakoid membranes, and heme attachment following the second translocation. Identifiable intermediates that define the maturation sequence include pre-apocyt c6, intermediate apocyt c6 and apocyt c6. Our previous work suggests that the conversion of apocyt c6 to holocyt c6 is specified by multiple nuclear- and chloroplast-encoded genes whose products function also in the pathway for maturation of organelle-encoded pre-apocyt f. The recognition that there are activities required for the maturation of both proteins simplifies the identification of mutants affected specifically at the post-translational events in c-type cytochrome biosynthesis. The further dissection of the pathway of cyt c6 and cyt f maturation is proposed here, with the goal of identifying the several genes and functions that are necessary at each step of the respective pathways. Parallel biochemical and molecular genetic approaches will be brought to bear on the problem, since the experimental organism, Chlamydomonas reinhardtii, is ideally suited for either strategy. specifically, "tagged" mutants defective in both cyt f and cyt c6 accumulation will be generated to enable the isolation of nuclear genes required for the maturation of chloroplast c-type cytochromes, and chloroplast gene disruption techniques will be exploited to identify the plastid loci. The biochemical functions and sequential action of the gene products at each stage in the pathway will be ascertained by in vivo and in vitro phenotypic characterization of each new tagged or site-directed mutant. The isolation of a cell-free system for heme attachment will permit in vitro analysis, while established techniques will be employed for the in vivo study. The proposed work has as its long term goal, the understanding of the general principles underlying the assembly of the cofactors in the photosynthetic electron transfer membrane. the project has relevance to human health because the same principles apply to respiratory cytochromes, and the anticipated results are expected to enhance our understanding and hence treatment of mitochondrial myopathies, many of which result from deficiencies in respiratory chain components. A unique aspect of this work is the expected identification of biochemical components and genes involves in intracellular heme transport. This is an unexplored area, but one that is ripe for genetic dissection in this organism.

在真核细胞中，电子转移复合物的生物合成 能量转换膜的发展不仅需要调节和 各种核基因和细胞器基因的协调表达 大分子成分，还要正确的靶向和加工 前蛋白、辅因子与脱辅基蛋白的关联 以及多亚基结构的化学计量组装。 这些后 转化事件是我们长期研究计划的主题 类囊体膜生物发生。 该应用程序重点关注 加工步骤 - 特别是血红素附着 - 涉及 光合 c 型细胞色素（可溶性细胞色素 c6 和 膜锚定细胞色素 f)。 核编码、流明的成熟 靶向 cyt c6 包括以下特定的蛋白水解加工步骤 穿过包膜膜和类囊体膜以及血红素的易位 第二次易位后的附着。 可识别 定义成熟序列的中间体包括前 apocyt c6， 中间 apocyt c6 和 apocyt c6。 我们之前的工作表明 apocyt c6 到 holocyt c6 的转换由多个指定 核和叶绿体编码的基因，其产物也在 细胞器编码的前阿波细胞 f 的成熟途径。 这 认识到双方的成熟都需要开展活动 蛋白质简化了受特定影响的突变体的识别 在c型细胞色素生物合成的翻译后事件中。 这 对 cyt c6 和 cyt f 成熟途径的进一步剖析是 在此提出，目的是识别几个基因和 各个路径的每个步骤所必需的功能。 并行生化和分子遗传学方法将被引入 考虑到这个问题，因为实验生物衣藻 莱茵哈特 (reinhardtii) 非常适合这两种策略。 具体来说， 细胞色素 f 和细胞色素 c6 积累均存在缺陷的“标记”突变体将是 生成以能够分离所需的核基因 叶绿体c型细胞色素和叶绿体基因的成熟 将利用破坏技术来鉴定质体基因座。 基因产物的生化功能和顺序作用 该途径的每个阶段都将通过体内和体外确定 每个新标记或定点突变体的表型特征。 用于血红素附着的无细胞系统的分离将允许 体外分析，同时将采用已建立的技术进行体内分析 体内研究。 拟议工作的长期目标是 了解大会的一般原则 光合电子传递膜中的辅助因子。 该项目 与人类健康相关，因为同样的原则适用于 呼吸细胞色素，预期结果 增强我们对线粒体的理解和治疗 肌病，其中许多是由呼吸链缺陷引起的 成分。 这项工作的一个独特之处是预期的识别 细胞内血红素涉及的生化成分和基因 运输。 这是一个尚未探索的领域，但遗传技术已经成熟 在这个有机体中进行解剖。