Molecular Analysis of a Chloroplast Protein Import Intermediate

蛋白质跨细胞膜转运的机制不是 很好理解。 这项建议将侧重于 蛋白质跨越两个膜的叶绿体包膜期间， 蛋白质导入豌豆叶绿体的过程。 这是我们的目标 定义和表征蛋白酶抗性片段的建议， 当细胞器暴露在低温下或 解偶联剂尼日利亚菌素可阻断β-磷酸化。 三 已经提出了替代方案来解释形成 抗蛋白酶进口中间体。 A.两个步骤 前体首先连续穿过外表面， 包膜，然后是内包膜; B）和 替代的两步法，其中前体跨越 膜间隙，同时穿过内外包膜 C）包膜处的一步易位机制 膜接触部位。 我们建议在体外测试这些替代的输入途径。 叶绿体导入测定。 对于该测定法，使用了编码抗肿瘤基因的特异性mRNA。 核酮糖1，5-二磷酸羧化酶前体小亚基 将通过体外转录从质粒表达。 的 前体蛋白将在无细胞小麦胚芽中翻译 翻译系统 通过调节进口温度，我们将 定义蛋白酶保护的片段 积累以及进口的允许温度范围。 的 将分析捕获的中间体以确定：a）哪个结构域或 保护前体的区域免受蛋白水解; B）其中 c）如果其它的叶绿体区室，则前体片段被保护; 叶绿体前体遵循类似的输入途径;以及; d） 低温和尼日利亚菌素抑制输入的生化基础。 预计这项研究的结果不仅将作为一项 细胞中其他蛋白质转运途径的模型系统，但 也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.