Molecular Mechanism of Protein Import into Chloroplasts

大多数叶绿体蛋白在细胞核中编码，在细胞质中合成为较高分子量前体，并在翻译后输入到叶绿体中。我们的长期目标是了解这一进口过程是如何发生的。蛋白质进口将在体外重组试验中进行研究。在本实验中，首先通过克隆的前体基因的体外转录和翻译来合成放射性标记的前体蛋白，然后与分离的完整叶绿体孵育。本研究将侧重于进口过程的两个具体方面。第一个是前体蛋白跨两个包膜转运的机制。将努力查明易位中间体，以便更完整地描述进口过程中的各个阶段。还将努力确定参与前体进口的叶绿体被膜的蛋白质。拟议研究的第二个方面将是研究细胞质合成的蛋白质运输到类囊体腔的途径和机制。这个过程特别有趣，因为这些蛋白质在它们的合成位置和最终位置之间穿过三层膜。%从这项工作中获得的见解在两个方面将是重要的。首先，他们将增加有关将细胞质合成的蛋白质定向到真核细胞中适当位置的基础知识。其次，这些结果将在通过基因工程改变叶绿体新陈代谢的努力中得到实际应用。

Most chloroplast proteins are encoded in the nucleus, synthesized in the cytoplasm as higher molecular weight precursors and imported into chloroplasts posttranslationally. Our long term goal is to understand how this import process occurs. Protein import will be studied in an in vitro reconstituted assay. In this assay, radiolabeled precursor proteins are first synthesized by in vitro transcription and translation of cloned precursor genes and then incubated with isolated intact chloroplasts. The present studies will focus on two particular aspects of the import process. The first is the mechanism whereby precursor proteins get translocated across the two envelope membranes. Efforts will be made to identify translocation intermediates in order to gain a more complete description of the stages in the import process. Efforts will also be made to identify proteins of the chloroplast envelope membranes that are involved in precursor import. The second aspect of the proposed studies will be to investigate the pathway and mechanisms whereby cytoplasmically synthesized proteins are transported to the thylakoid lumen. This process is particularly interesting because these proteins cross three membranes between their site of synthesis and their final location.%%% The insights gained from this work will be important in two respects. First, they will add to basic knowledge regarding the targeting of cytoplasmically synthesized proteins to their proper locations in eukaryotic cells. Second, these results will have practical application during efforts to alter the metabolism of chloroplasts by genetic engineering.***//