Molecular basis of FtsH function in the cyanobacterium Synechocystis PCC 6803

蓝藻是一种多样化的细菌群，它们有能力进行类似植物的光合作用，利用太阳能将二氧化碳固定在有机物中，并从水中释放氧气。它们存在于水生环境中，对全球的含氧光合作用做出了重要贡献。因此，它们的光合作用活动对于确定大气中二氧化碳（一种温室气体）的含量至关重要。蓝藻也与植物中的叶绿体密切相关，许多生化过程是保守的。因此，蓝藻被广泛用作研究叶绿体相关过程的模型，例如参与光合作用的蛋白质成分是如何工作的。特别是蓝藻胞囊藻6803是一种广泛使用的实验系统，因为在这种生物中进行基因工程实验很容易。最近，Peter Nixon和他的同事们发现了一种被称为FtsH2的蓝藻蛋白酶，它参与了光损伤后进化氧气的光系统II复合体的修复。在叶绿体中，FtsH2的同源物也参与了相同的过程。随后，FtsH2被证明参与了Synechocystis 6803对许多其他环境胁迫（包括高温、高盐和无机碳可用性降低）的成功驯化。因此，FtsH2是一个重要的因素，控制许多不同的生理过程对蓝藻的生存至关重要。在本提案的背景工作中，我们开发了实验工具，对这类FtsH蛋白酶进行重要的基础研究。我们已经开发了纯化FtsH2蛋白酶的技术，并表明它与相关的FtsH3蛋白酶形成复合物。通过电子显微镜，我们首次发现FtsH2/FtsH3复合物由6个亚基组成。在这个应用程序中，我们描述了一系列的实验来评估FtsH2和其他三个FtsH蛋白酶家族成员的结构和功能，这些成员在胞囊藻6803中发现。我们建议确定每个FtsH亚基在细胞中的位置，确定细胞中不同类型FtsH复合物的数量和组成，确定每种FtsH蛋白酶的潜在靶标，并确定FtsH2突变体所显示的生理效应是由于FtsH复合物的蛋白水解活性还是FtsH复合物帮助拉或折叠细胞靶标的能力。除了提高我们对蓝藻如何应对各种环境胁迫的理解外，我们的结果对试图了解FtsH在叶绿体中的作用的植物科学家也具有特殊意义。最终，对FtsH蛋白酶的研究可能会产生光合生物，如农作物，它们能够在包括强光胁迫在内的一系列环境条件下更有效地生长。

Cyanobacteria are a diverse group of bacteria that have the ability to carry out plant-like photosynthesis, using solar energy to drive the fixation of carbon dioxide into organic matter and the liberation of oxygen from water. They are found in aquatic environments and make an important contribution to global oxygenic photosynthesis. Their photosynthetic activity is therefore crucial to determining levels of carbon dioxide, a greenhouse gas, in the atmosphere. Cyanobacteria are also closely related to the chloroplasts found in plants and many of the biochemical processes are conserved. Consequently cyanobacteria have been widely used as models to study related processes in chloroplasts, such as how the protein components involved in photosynthesis work. In particular the cyanobacterium Synechocystis 6803 is a widely used experimental system because of the ease of doing genetic engineering experiments in this organism. Recently Peter Nixon and colleagues identified a cyanobacterial protease, termed FtsH2, which was involved in the repair of the oxygen-evolving Photosystem II complex following light damage. Homologues of FtsH2 are also involved in the same process in chloroplasts. Subsequently FtsH2 has been shown to be involved in the successful acclimation of Synechocystis 6803 to a number of other environmental stresses including heat, high salt and reduced availability of inorganic carbon. FtsH2 is therefore an important factor controlling a number of different physiological processes vital for survival of the cyanobacterium. In background work to this proposal we have developed the experimental tools to conduct important fundamental studies on this class of FtsH protease. We have developed techniques to purify the FtsH2 protease and have shown that it forms a complex with the related FtsH3 protease. By using electron microscopy we could show for the first time that this FtsH2/FtsH3 complex is made of six subunits. In this application we describe a series of experiments to assess the structure and function of FtsH2 and the other three members of the FtsH protease family found in Synechocystis 6803. We propose to identify the location of each of the FtsH subunits in the cell, determine the number and composition of the different types of FtsH complex in the cell, identify potential targets for each of the FtsH proteases and determine if the physiological effects displayed by the FtsH2 mutants is as a result of the proteolytic activity of the FtsH complex or the ability of FtsH complexes to help pull or refold cellular targets. Beside improving our understanding of how cyanobacteria respond to various environmental stresses, our results will also be of special significance to plant scientists who are trying to understand the role of FtsH in the chloroplast. Ultimately work on the FtsH proteases might allow the generation of photosynthetic organisms, such as crop plants, that are able to grow more productively under a range of environmental conditions including high light stress.