Biogenesis of Chloroplasts: Import and Assembly of Nuclear Encoded Chloroplast Proteins

克莱恩博士希望确定在细胞质中合成的多肽被运输到叶绿体内的类囊体膜的机制。类囊体是叶绿体内部的膜。大约50%的类囊体多肽是在叶绿体内合成的；剩下的在细胞质中。最近的证据表明，细胞内合成的蛋白质通过“可溶性中间体”途径实现其功能定位，其中前体蛋白通过两个包膜，以可溶性形式穿过水基质，并通过第二次运输（组装）过程插入类囊体。克莱恩博士的实验室已经用叶绿体亚组分重建了组装过程，但还没有严格证明它参与了一个完整的系统。拟议的研究将通过在阻止组装的条件下将类囊体前体蛋白导入叶绿体，直接研究可溶性中间途径和参与体外组装过程。选择条件将通过对装配过程的能量学进行彻底的分析来确定。第二个目标是研究叶绿体合成蛋白质的可能性。输入的蛋白质通过同样的装配装置插入到类囊体中。叶绿体合成蛋白的组装将在体外进行重组，并与组装进口蛋白的组装要求进行比较。这项工作将增加我们对叶绿体膜蛋白生物发生的认识，并将增加对蛋白质如何跨膜的更一般的理解。

Dr. Cline wishes to determine the mechanism by which polypeptides synthesized in the cytosol are transported into the thylakoid membranes within the chloroplasts. Thylakoids are internal membranes within cholroplasts. Approximately 50% of thylakoid polypeptides are synthesized within the chloroplast; the remainder in the cytosol. Recent evidence suggests that the cytosolically synthesized proteins achieve their functional location by a "soluble intermediate' pathway, in which precursor proteins pass through both envelope membranes, traverse the aqueous stroma in soluble form, and insert into thylakoids by a second transport (assembly) process. Dr. Cline's laboratory has reconstituted the assembly process with chloroplast subfractions, but has not yet rigorously demonstrated its involvement in an intact system. The proposed studies will directly investigate the soluble intermediate pathway and involvement of the in vitro assembly process by importing a thylakoid precursor protein into chloroplasts under conditions that prevent assembly. Selective conditions will be identified by thorough analysis of the energetics of the assembly process. A second objective will examine the possibility that chloroplast synthesized proteins. and imported proteins are inserted into thylakoids by the same assembly apparatus. Assembly of chloroplast synthesized proteins will be reconstituted in vitro and the assembly requirements compared with those for assembling imported proteins. This work will increase our knowledge of the biogenesis of chloroplast membrane proteins and will add to a more general understanding of how proteins cross membranes.