MATURATION OF CHLOROPLAST C-TYPE CYTOCHROMES

在真核细胞中，电子传递复合物的生物合成 能量转换膜不仅需要调节， 协调表达核和细胞器基因的各种 大分子成分，而且正确的靶向和处理 辅因子与脱辅基蛋白的结合 以及多亚基结构的化学计量组装。 这些后- 翻译事件是我们长期研究计划的主题 对类囊体膜生物发生的影响 此应用程序侧重于 涉及的加工步骤--尤其是血红素附着-- 光合C型细胞色素（可溶性CytC 6和 膜锚定细胞色素F）。 成熟的核编码，腔- 靶向的CytC 6包括特定的蛋白水解加工步骤， 穿过被膜和类囊体膜的转运，以及血红素 在第二次易位之后附着。 可识别 确定成熟序列的中间体包括前脱细胞C6， 中间体脱细胞C6和脱细胞C6。 我们之前的研究表明， 脱细胞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apocyt f成熟的途径。 的 认识到需要开展活动，使两者都成熟起来， 蛋白质简化了突变体的鉴定， 在C型细胞色素生物合成的翻译后事件。 的 对细胞色素C6和细胞色素F成熟途径的进一步剖析是 在这里提出，目标是确定几个基因， 各自途径的每个步骤所需的功能。 平行的生物化学和分子遗传学方法将被带到 与这个问题有关，因为实验生物衣原体 reinhardtii，理想地适合于任一策略。 具体地说， 在细胞色素F和细胞色素C6积累中有缺陷的“标记”突变体将是 产生，以使分离所需的核基因， 叶绿体c型细胞色素成熟，叶绿体基因 将利用破坏技术来鉴定质体基因座。 基因产物的生物化学功能和序列作用 将通过体内和体外方法确定途径中的每个阶段 每个新的标记的或定点突变体的表型表征。 用于血红素附着的无细胞系统的分离将允许 体外分析，而建立的技术将用于在 体内研究 拟议工作的长期目标是， 对大会所依据的一般原则的理解 光合电子传递膜中的辅因子。 项目 与人类健康有关，因为同样的原则适用于 呼吸细胞色素，预期的结果预计将 增强我们对线粒体疾病的理解， 肌病，其中许多是由呼吸链缺陷引起的 件. 这项工作的一个独特方面是预期的识别 与细胞内血红素有关的生化成分和基因 运输 这是一个尚未探索的领域，但一个成熟的遗传学领域。 在这个有机体中进行解剖。

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.