Assembly of photosystem I in thylakoid membranes

光系统I （PSI）是自然界中最大、最复杂的大分子机制之一。虽然PSI的结构已经被非常详细地解决了，但对于如何将十几种蛋白质亚基和200多种色素和其他辅助因子组合在一起，我们知之甚少。在目前的资助期内，我们已经(i)开发了一种新的生化方法，发现了种子植物PSI组装中的两个中间复合物；（ii）建立了体内标记和亲和纯化方法，为PSI组装机制的新组件产生了许多有趣的候选蛋白质；（iii）设计了一个新的时间分辨率系统来研究绿藻模型衣藻莱茵衣藻PSI组装中的事件序列。我们现在计划在这项工作的基础上：(i)对我们分离出的新的假定PSI组装因子进行功能表征，（ii）确定它们在PSI组装过程中的精确作用，以及（iii）使用衣藻中建立的可诱导PSI敲除和回收系统来生化分解PSI组装中的各个步骤。本研究的最终目标是阐明PSI组装机制的分子结构，确定其组件的功能，建立PSI组装图，提供组装过程中事件的时空序列的完整图像，并阐明与PSI结构和类囊体形态进化相关的组装机制的进化。

Photosystem I (PSI) is one of the largest and most intricate macromolecular machineries in nature. While the structure of PSI has been resolved in great detail, very little is known about how the complex is put together from more than a dozen protein subunits and more than 200 pigments and other co-factors. In the current funding period, we have (i) developed a new biochemical approach that has led to the discovery of two intermediate complexes in PSI assembly of seed plants, (ii) set up in vivo tagging and affinity purification approaches that have yielded a number of interesting candidate proteins for new components of the PSI-assembling machinery, and (iii) designed a new time-resolved system to study the sequence of events in PSI assembly of the green algal model Chlamydomonas reinhardtii. We now plan to build upon this work and (i) functionally characterize the novel putative PSI assembly factors we have isolated, (ii) determine their precise role in the PSI assembly process, and (iii) use the established inducible PSI knock-down and recovery system in Chlamydomonas to biochemically resolve individual steps in PSI assembly. The ultimate goals of the research proposed here are to elucidate the molecular structure of the PSI-assembling machinery and determine the functions of its components, establish an assembly map for PSI that provides a complete picture of the spatial and temporal sequence of events in the assembly process, and elucidate the evolution of the assembly machinery in relation to the evolution of PSI structure and thylakoid morphology.