Molecular Mechanism of Protein Transport into Chloroplasts

细胞生物学中一个重要的未解决的问题是理解 细胞质合成的蛋白质是如何靶向 真核细胞的不同区室。 在植物细胞中， 叶绿体为研究这个问题提供了一个很好的系统。 大多数叶绿体蛋白质在细胞核中编码， 在细胞质中作为较高分子量的前体合成。 这些前体在翻译后被输入叶绿体 穿过两个包膜膜，并随后被引导至 在细胞器中的正确位置。 的长期目标 这个项目是为了了解如何进口过程发生。 在 目前，获得更详细的 对蛋白质转运到叶绿体的理解是缺乏的， 关于运输中间体的身份的信息。 的 该项目的基本策略将是阻止蛋白质运输， 特定阶段，从而产生易位中间体 在运输过程中的离散点含有前体。 将使用交联试剂来识别运输 在物理上与被困物非常接近的组件 中间体的 随着推定成分的确定，进一步的工作 将被要求确认他们参与运输， 在运输过程中发挥作用。 到 为了实现这些目标，针对 将制备每种推定的组分，并将编码 每种成分都将被分离和测序。 这些抗体 cDNA克隆将是研究 假定组件的功能。 %%% 这项工作的重要性至少有两个原因。 首先，它补充说， 对基本细胞过程的一般理解，即 细胞内蛋白质运输 二是具有实用性 的重要性，努力改变代谢途径， 叶绿体利用基因工程技术，其中外来 蛋白质需要被引导到叶绿体中。

A significant unresolved problem in cell biology is understanding how cytoplasmically synthesized proteins are targeted to the various compartments of eukaryotic cells. In plant cells, chloroplasts offer an excellent system for studying this problem. Most chloroplastic proteins are encoded in the nucleus and synthesized in the cytoplasm as higher molecular weight precursors. These precursors are imported postranslationally into chloroplasts across the two envelope membranes and are subsequently directed to the proper location within the organelle. The long term goal of this project is to understand how the import process occurs. At the present time, the major barrier to gaining a more detailed understanding of protein transport into chloroplasts is a lack of information regarding the identity of transport intermediates. The basic strategy of the project will be to block protein transport at specific stages, thereby generating translocation intermediates containing precursors at discrete points in the transport process. Cross-linking reagents will be used to identify transport components that are in close physical proximity to the trapped intermediates. As putative components are identified, further work will be required to confirm their involvement in transport and to determine their function during the transport process. To accomplish these objectives, specific antibodies directed against each putative component will be prepared and cDNA clones encoding each component will be isolated and sequenced. These antibodies and CDNA clones will be valuable tools for investigating the functions of the putative components. %%% This work is significant for at least two reasons. First, it adds to the general understanding of a basic cellular process, i.e. intracellular protein trafficking. Second, it has practical significance for efforts to alter the metabolic pathways of chloroplasts using genetic engineering techniques, in which foreign proteins will need to be directed into chloroplasts.