Molecular Analysis of a Chloroplast Protein Import Intermediate

蛋白质跨细胞膜转运的机制还不是很清楚。这项建议将集中在蛋白质输入豌豆叶绿体的过程中，蛋白质跨叶绿体被膜的两个膜的转运。这项建议的目标是定义和表征当细胞器暴露在低温下或当解偶联剂黑素阻止光磷酸化时捕获的蛋白酶抗性片段。已经提出了三种替代方案来解释抗蛋白酶进口中间体的形成。它们是：A)两步过程，其中前体首先穿过外膜，然后是内膜；B)交替的两步过程，其中前体跨越膜间空间，同时跨越外膜和内膜；以及C)在被膜接触点的一步转位机制。我们建议在体外叶绿体导入实验中测试这些替代的导入途径。在本实验中，编码核酮糖1，5-二磷酸羧基酶前体(PRSS)小亚基的特定mRNAs将通过体外转录从质粒中获得表达。前体蛋白将在无细胞的小麦胚芽翻译系统中进行翻译。通过调节进口温度，我们将确定蛋白酶保护片段的积累温度以及允许进口的温度范围。捕获的中间体将被分析以确定：a)保护前体的哪个结构域或区域不被蛋白质降解；b)在哪个叶绿体隔间保护前体片段；c)其他叶绿体前体是否遵循类似的输入途径；以及d)低温和黑素抑制进口的生化基础。希望这项研究的结果不仅可以作为细胞内其他蛋白质转运途径的模型系统，而且还将扩大我们对叶绿体生物发生的理解。

The mechanism of protein translocation across cellular membranes is not well understood. This proposal will focus on the translocation of proteins across the two membranes of the chloroplast envelope during the course of protein import into pea chloroplasts. It is the goal of this proposal to define and characterize a protease resistant fragment that is trapped when the organelles are exposed to low temperature or when photophosphorylation is blocked by the uncoupler nigericin. Three alternatives have been proposed to account for the formation of a protease resistant import intermediate. These are: A) a two step process in which the precursor successively crosses first the outer envelope membrane and then the inner envelope membrane; B) and alternative two step process in which the precursor spans the intermembrane space while crossing both the outer and inner envelope membranes; and C) a one step translocation mechanism at envelope membrane contact sites. We propose to test these alternative import pathways in an in vitro chloroplast import assay. For this assay, specific mRNAs encoding the small subunit of ribulose 1,5-bisphosphate carboxylase precursor (prSS) will be expressed by in vitro transcription from plasmids. The precursor proteins will be translated in a cell free wheat germ translation system. By modulating the temperature of import, we will define the temperatures at which the protease protected fragment accumulates as well as the permissive temperature range of import. The trapped intermediate will be analyzed to determine: a) which domain or region of the precursor is protected from proteolysis; b) in which chloroplast compartment the precursor fragment is protected; c) if other chloroplast precursors follow a similar import pathway; and; d) the biochemical basis of import inhibition by low temperature and nigericin. It is expected that the results of this study will not only serve as a model system for other protein translocation pathways in the cell, but will also expand our understanding of chloroplast biogenesis.