MATURATION OF CHLOROPLAST C-TYPE CYTOCHROMES

在真核细胞中，电子转移复合体的生物合成 能量传导膜的发展不仅需要被调节的和 细胞核和细胞器基因在不同物种中的协调表达 大分子成分，还要正确的靶向和加工 在前蛋白中，辅因子与脱辅基蛋白的联系 以及多亚基结构的化学计量组装。这些帖子- 翻译活动是我们长期研究计划的主题 类囊体膜生物发生的研究。此应用程序侧重于 加工步骤--尤其是血红素附着--涉及 光合作用C型细胞色素(可溶性细胞色素C6和 膜锚定Cyt f)。核编码的管腔的成熟- 靶向cyt c6包括以下特定的蛋白降解处理步骤 跨被膜和类囊体膜的转运，以及血红素 第二次移位后的附着。可识别的 定义成熟序列的中间产物包括细胞前C6， 中间细胞C6和细胞C6。我们之前的工作表明 细胞c6向全细胞c6的转化由多个 核和叶绿体编码的基因，其产物也在 细胞器编码的前体细胞成熟的途径。 认识到两者的成熟都需要开展一些活动 蛋白质简化了受特定影响的突变体的鉴定 在C型细胞色素生物合成的翻译后事件中。这个 进一步剖析了细胞色素C6和细胞成熟的途径 在这里提出的，目的是识别几个基因和 在各个路径的每一步所必需的功能。 并行的生化和分子遗传学方法将被引入到 关于这个问题，因为实验有机体衣藻 Renehardtii非常适合这两种策略。具体地说， 在cyt f和cyt c6积累方面都有缺陷的“标记”突变体将被 生成以实现分离所需的核基因 叶绿体C型细胞色素和叶绿体基因的成熟 中断技术将被用来识别叶绿体基因座。 基因产物的生化功能和序列作用 途径的每个阶段都将通过体内和体外来确定 每个新标记突变体或定点突变体的表型特征。 隔离无细胞的血红素附着系统将允许在 体外分析，而成熟的技术将被用于 活体研究。拟议的工作有一个长期目标，即 理解大会的一般原则 光合作用电子传递膜中的辅因子。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，预期结果为 提高我们对线粒体的认识和治疗 肌病，其中许多是由于呼吸链缺陷引起的 组件。这项工作的一个独特方面是预期的身份识别 与细胞内血红素有关的生化成分和基因 运输。这是一个尚未开发的地区，但也是一个适合遗传的地区。 在这种有机体中进行解剖。

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.