TARGETING AND ASSEMBLY OF THYLAKOID MEMBRANE PROTEINS

真核细胞区隔需要各种不同的靶向 成分，如蛋白质、脂类等，进入特定的亚细胞 结构。在人类中，错误的目标往往会导致严重的疾病。 靶向蛋白质总是涉及到它们通过或进入 膜。目前，实现这一目标的机制是 很大程度上是未知的。我们的长期目标是了解生化 参与细胞膜组装的机制，特别是 这些膜的蛋白质成分。体内的类囊体膜 植物叶绿体被选为实现这一目标的模型。 核编码的类囊体膜蛋白定位于 包括以下三个阶段的流程：跨越两个信封的运输 膜，蛋白质介导的间质穿透和插入 类囊体双层。每一步的体外重组检测 这种工艺已经被开发出来了。这份提案描述了生化 研究的最终目的是确定每种疾病的潜在机制 一步。光诱导过程中包膜蛋白变化的分析 激活蛋白质输入装置将识别蛋白质 移位机械的部件。基质蛋白 维持膜蛋白溶解和插入所需的因子(S) 能力将被净化，其行动模式将被确定。 纯化将通过常规和亲和来完成。 技巧。最后，类囊体膜蛋白的插入步骤和 集合将被确定。这将通过扣留或 降低对工艺的要求并进行表征 中间体。逮捕中间人的能力应该提供一个 识别插入装置的蛋白质组分的装置。 该项目的成功完成将提供基本的见解 蛋白质可以通过不同方式与两个 蛋白质转位系统，并将阐明 细胞器内的可溶性蛋白(S)可以稳定膜蛋白 在它们插入到双层中之前。

Eukaryotic cell compartmentalization requires the targeting of various components, e.g. proteins, lipids, etc, into specific subcellular structures. In humans, mistargeting often results in serious disease. Targeting of proteins invariably involves their transport across or into membranes. At present, the mechanisms by which this is accomplished are largely unknown. Our long term goal is to understand biochemical mechanisms involved in the assembly of cell membranes, especially the protein components of those membranes. The thylakoid membrane within plant chloroplasts has been chosen as a model to achieve this goal. Nuclear-encoded thylakoid membrane proteins are localized in a three-stage process that involves: transport across the two envelope membranes, protein-mediated traversal of the stroma, and insertion into the thylakoid bilayer. In vitro reconstituted assays for each step of this process have been developed. This proposal describes biochemical studies designed ultimately to determine underlying mechanisms of each step. Analysis of changes in envelope proteins during a light-mediated activation of the protein import apparatus will identify protein components of the translocation machinery. The stromal protein factor(s) required to maintain membrane protein solubility and insertion competence will be purified and its mode of action determined. Purification will be accomplished by conventional as well as affinity techniques. Finally, the steps of thylakoid protein insertion and assembly will be determined. This will be accomplished by withholding or lowering the requirements for the process and characterizing intermediates. The ability to arrest intermediates should provide a means of identifying protein components of the insertion apparatus. Successful completion of this project will provide fundamental insight into the manner by which proteins can differentially interact with two protein translocation systems and will elucidate the manner by which soluble protein(s) within organelles can stabilize membrane proteins prior to their insertion into the bilayer.