NUCLEAR GENES THAT MEDIATE CHLOROPLAST ASSEMBLY

所有细胞都面临的一个基本问题是 蛋白质穿过并进入细胞膜。现在已经清楚 一些远缘相关膜采用移位机制 在进化上是相关的，而其他的膜 显然是独特的机制。该项目的目标是 阐明蛋白质靶向靶向 叶绿体类囊体膜，一种起着中心作用的膜， 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，主要是由于体外靶向测定的应用。 这些实验为几个能量- 不同的途径，通过这些途径，蛋白质可以转移到 类囊体其中两条途径与以前的 描述的机制，而第三个似乎新颖。充分 理解靶向途径之间的关系， 的靶向机制，以及在体内的作用的因素 在生物化学研究中发现，研究 类囊体蛋白靶向的遗传破坏的后果。 我们已经开发了一个玉米遗传系统， 分析这个过程。之前，我们分离出了 靶向类囊体蛋白，并克隆了两个相应的 基因.提出的实验更充分地利用了 该系统解决以下问题：a）什么是 蛋白质在细胞中的转运机制的组成部分 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，通过识别蛋白质， 与这些基因的产物相互作用， 额外的靶向突变。B）类囊体在植物生长过程中 转运机制在类囊体整合中的作用 蛋白质？这个问题将通过分析 这些蛋白质在突变体中的整合， 膜被破坏。c）不同的类囊体 靶向通路在体内相互作用？建立冗余， 途径之间的补偿性相互作用， 双突变体和靶向机制的积累 将分析单个突变体中的组分。先前的证据 这表明，新的”类囊体靶向途径可能是 与一种通用的靶向机制有关， 逃脱了侦查因此，这些实验的结果将 不仅阐明了类囊体膜的基本过程， 组装，但也可以提供对靶向机制的深入了解 所有细胞都在使用。

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.