NUCLEAR GENES THAT MEDIATE CHLOROPLAST ASSEMBLY

所有细胞面临的一个根本问题是需要移位 蛋白质穿过细胞膜并进入细胞膜。现在很明显， 一些远距离相关的膜使用移位机制 它们在进化上是相关的，而其他膜 显然是独一无二的机制。这个项目的目标是 阐明蛋白质靶向于 叶绿体类囊体膜，一种发挥中枢作用的膜 在提供支持陆地生命的固定碳方面的作用。 在理解这一过程方面已经取得了巨大的进展。 最近，很大程度上是由于体外靶向试验的应用。 这些实验提供了几个有力的证据- 蛋白质转运进入或跨过的不同途径 类囊体。其中两条途径与之前的 描述了机制，而第三种似乎是新奇的。充分地 了解靶向路径、自然环境之间的关系 靶向机制和因子在体内的作用 在生化研究中发现，有必要研究 类囊体膜蛋白靶向的遗传破坏的后果。 我们已经在玉米中开发了一种基因系统，我们可以用它 分析这一过程。在此之前，我们分离出破坏 类囊体膜蛋白靶向并克隆了相应的两个 基因。这些实验提出了更充分地利用 本系统要解决以下问题：a)什么是 将蛋白质在细胞内转移的机械部件 类囊体？这个问题将通过克隆两个新的 转座子标记的靶向突变，通过识别 与这些基因的产物相互作用，并通过识别 额外的靶向突变。B)类囊体扮演什么角色 转位机制在类囊体整合中的作用 蛋白质？这个问题将通过分析以下比率来解决 这些蛋白质在易位的突变体中的整合 细胞膜被破坏了。C)不同的类囊体是如何 靶向通路在体内相互作用？要建立冗员和 途径之间的补偿性相互作用，靶向缺陷 双变异体与靶标机制的积累 将对单个突变体的成分进行分析。先验证据 提示新的类囊体靶向途径可能是 涉及一种通用目标机制，该机制先前已 逃脱了侦测。因此，这些实验的结果将 不仅阐明类囊体膜的基本过程 组件，但也可以提供对目标机制的洞察 由所有单元格使用。

A fundamental problem faced by all cells is the need to translocate proteins across and into cellular membranes. It is now clear that some distantly-related membranes employ translocation mechanisms that are evolutionarily related while other membranes employ apparently unique mechanisms. The goal of this project is to elucidate mechanisms by which proteins are targeted to the chloroplast thylakoid membrane, a membrane that plays a central role in providing the fixed carbon that supports terrestrial life. Enormous progress has been made in understanding this process recently, due largely to the application of in vitro targeting assays. These experiments provided evidence for several energetically- distinct pathways by which proteins can translocate into or across the thylakoid. Two of these pathways are related to previously described mechanisms while a third appears novel. To fully appreciate the relationships between targeting pathways, the nature of the targeting machineries, and the roles in vivo of factors identified in biochemical studies, it is essential to study the consequences of genetic disruption of thylakoid protein targeting. We have developed a genetic system in maize with which we can analyze this process. Previously, we isolated mutations that disrupt thylakoid protein targeting and cloned two of the corresponding genes. The experiments proposed more fully exploit the genetics of this system to address the following questions: a) What are the components of the machinery that translocates proteins across the thylakoid? This question will be addressed by cloning two new transposon-tagged targeting mutations, by identifying proteins that interact with the products of these genes, and by identifying additional targeting mutations. b) What roles do the thylakoid translocation machineries play in the integration of thylakoid proteins? This question will be addressed by analyzing the rates of integration of such proteins in mutants in which translocation across the membrane is disrupted. c) How do the different thylakoid targeting pathways interact in vivo? To establish redundancies and compensatory interactions between pathways, targeting defects in double mutants and the accumulation of targeting machinery components in single mutants will be analyzed. Prior evidence suggests that the ~novel" thylakoid targeting pathway may be related to a universal targeting mechanism that has previously escaped detection. Therefore, the results of these experiments will elucidate not only the essential process of thylakoid membrane assembly but may also provide insight into targeting mechanisms employed by all cells.