Molecular Mechanism of Protein Transport into Chloroplasts

叶绿体是植物细胞中的细胞器，除了负责新陈代谢的许多其他重要方面外，还负责光合作用。然而，叶绿体功能所需的大多数蛋白质是在核基因中编码的，并在细胞质中合成后输入细胞器。虽然已经描述了进口过程的基本轮廓，但许多方面仍然未知。本项目将利用位点特异性交联策略研究前体蛋白导入叶绿体的细节。含有光反应官能团的非天然氨基酸将被纳入前体蛋白的特定位置，然后允许与分离的完整叶绿体的运输装置相互作用。在用紫外光激活交联试剂后，将对产物进行分析，以绘制前体蛋白在叶绿体包膜上运输时发生的一系列相互作用。执行这种专门的交联战略所需的专门知识并不广泛，但在日本名古屋大学可以获得。因此，该项目将与远藤教授的实验室合作完成这些实验。

Chloroplasts are the organelles within plant cells that are responsible for photosynthesis in addition to many other important aspects of metabolism. However, most of the proteins needed for the functions performed by chloroplasts are encoded in nuclear genes and imported into the organelle following their synthesis in the cytosol. While the basic outlines of the import process have been described, many aspects remain unknown. This project will investigate the details of precursor protein import into chloroplasts using a site-specific cross-linking strategy. Unnatural amino acids containing a photoreactive functional group will be incorporated into specific locations within precursor proteins and then allowed to interact with the transport apparatus of isolated intact chloroplasts. Following treatment with UV light to activate the cross-linking reagent, the products will be analyzed in an effort to map the set of interactions that occur as precursor proteins are transported across the chloroplastic envelope membranes. The expertise required to perform this specialized cross-linking strategy is not widely available, but is available at the University of Nagoya in Japan. Thus the project will be done in collaboration with the laboratory of Professor Endo to accomplish these experiments.