TARGETING AND ASSEMBLY OF THYLAKOID MEMBRANE PROTEINS

真核细胞区室化需要靶向各种靶向蛋白。 组分，例如蛋白质，脂质等，进入特定的亚细胞 结构. 在人类中，错误的估计往往会导致严重的疾病。 蛋白质的靶向总是涉及它们的运输穿过或进入 膜。 目前，实现这一目标的机制是 大部分未知。 我们的长期目标是了解生物化学 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 植物叶绿体已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一个 三个阶段的过程，包括：运输通过两个信封 膜，蛋白质介导的基质穿越，并插入到 类囊体双层。 每个步骤的体外复溶测定 这个过程已经发展起来了。 该提案描述了生物化学 研究旨在最终确定每种疾病的潜在机制， 步 光介导的细胞凋亡过程中包膜蛋白的变化分析 激活蛋白质输入装置将识别蛋白质 易位机制的组成部分。 基质蛋白 维持膜蛋白溶解性和插入所需的因子 能力将被净化，其作用方式将被确定。 纯化将通过常规以及亲和层析来完成。 技术. 最后，类囊体蛋白插入和 大会将确定。 这将通过扣留或 降低了对工艺的要求， 中间体的 阻止中间体的能力应该提供一个 识别插入装置的蛋白质成分的装置。 本项目的成功完成将提供基本的见解 蛋白质与两种不同的蛋白质相互作用的方式 蛋白质易位系统，并将阐明的方式， 细胞器内的可溶性蛋白可以稳定膜蛋白 在它们插入双层之前。

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.