Membrane protein targeting and assembly in cyanobacteria

蓝藻是一种光合作用细菌，在地球生态中发挥着至关重要的作用，也是太阳能生物技术的有前途的载体。蓝藻具有比大多数其他细菌更复杂的细胞结构：它们有一个复杂的内部膜系统，称为类囊体膜。类囊体充满了参与光合作用和呼吸作用的蛋白质复合体。相比之下，细胞周围的质膜专用于其他功能，在两个膜系统中发现了截然不同的蛋白质组。虽然我们知道哪些蛋白质存在于哪个膜中，但我们不知道它们整合到每个膜中的位置，也不知道是什么把它们送到那里的。我们已经从一种使用荧光显微镜来寻找编码类囊体膜蛋白的特定mRNA分子的位置的方法中获得了这两个问题的线索。我们使用了一种名为RNA-FISH的技术，在这种技术中，我们对细胞进行化学固定，然后用连接到荧光分子的短合成DNA序列来探测它们。DNA探针与细胞中的目标mRNA结合，然后我们可以观察它们在细胞内的位置。使用这项技术，我们已经证明了编码光合作用装置核心组件的mRNA分子聚集在类囊体膜表面最接近发现DNA的细胞中央部分的非常特定的位置。我们有证据表明，这些位置对应于光合作用蛋白首先产生并整合到类囊体膜的“翻译区”，我们也有证据表明，mRNA分子通过与特定的mRNA结合蛋白相互作用而被定向到类囊体膜上。这种相互作用对于将膜蛋白定向到正确的膜上可能是至关重要的。我们鉴定的mRNA结合蛋白在不同物种的蓝藻中高度保守，甚至在植物叶绿体中也有同源物。这表明，在植物细胞内从自由生活的蓝藻到叶绿体的十多亿年的进化过程中，信使核糖核酸靶向系统的元件已经被保守。在这个建议中，我们将进行进一步的RNA-FISH研究，以找到蓝藻中更广泛的信使核糖核酸物种的位置。我们将测试不同的类囊体膜蛋白是否在相同的区域翻译，或者每种蛋白质是否有自己的特定翻译区域。我们将测试新产生的光合作用复合体是停留在转换区，还是迁移到膜上的其他地方进行进一步组装。我们还将检查编码质膜蛋白的mRNAs的位置，看看质膜上是否有类似的翻译区。我们将使用一种名为原子力显微镜的技术来检查类囊体膜转换区的组织，这种技术可以告诉我们单个蛋白质复合体是如何在膜中排列的。我们希望这将使我们对光合作用复合体组装的精细和协调过程有新的见解。我们将测试进一步的RNA结合蛋白是否参与将mRNA分子靶向类囊体膜和质膜，我们将寻找可能被每个RNA结合蛋白识别的mRNA分子的特征。然后，我们将通过生产具有突变的mRNA序列的细胞来测试我们的想法，我们预计这些序列将改变与蛋白质的关联和mRNA在细胞中的位置。我们希望该项目的这一部分能够告诉我们蓝藻细胞如何能够将特定的蛋白质靶向特定的膜，为蓝藻用于太阳能生物技术的“精确工程”方法奠定基础。

Cyanobacteria are photosynthetic bacteria that play a crucial role in the ecology of the planet, and are also promising vehicles for solar-powered biotechnology. Cyanobacteria have a more complex cell structure than most other bacteria: they have an intricate internal membrane system called the thylakoid membranes. The thylakoids are packed with protein complexes involved in photosynthesis and respiration. By contrast, the plasma membrane surrounding the cell is dedicated to other functions and very different sets of proteins are found in the two membrane systems. Although we know which proteins are found in which membrane, we don't know where they are integrated into each membrane, and we don't know what sends them there. We have gained clues to both questions from an approach using fluorescence microscopy to find the locations of specific mRNA molecules that encode thylakoid membrane proteins. We used a technique called RNA-FISH in which we chemically fix the cells and then probe them with short synthetic sequences of DNA linked to fluorescent molecules. The DNA probes bind to the target mRNA in the cell, and we can then observe their location within the cell. Using this technique we have shown that the mRNA molecules that code for core components of the photosynthetic apparatus cluster at very specific locations at the parts of the thylakoid membrane surface that are nearest to the central part of the cell where the DNA is found. We have evidence that these locations correspond to "translation zones" where the photosynthetic proteins are first produced and integrated into the thylakoid membrane, and we also have evidence that the mRNA molecules are directed to the thylakoid membrane by interaction with specific mRNA-binding proteins. Interactions of this sort may be crucial for targeting membrane proteins to the correct membrane. The mRNA-binding proteins that we identified are strongly conserved in different species of cyanobacteria, and they even have homologs in plant chloroplasts. This suggests that elements of an mRNA targeting system have been conserved over more than a billion years of evolution from a free-living cyanobacterium to a chloroplast within a plant cell.In this proposal, we will carry out further RNA-FISH to studies to find the locations of a wider range of mRNA species in cyanobacteria. We will test whether different thylakoid membrane proteins are translated at the same zones, or whether each protein has its own specific translation zone. We will test whether the newly-produced photosynthetic complexes stay at the translation zones, or whether they migrate elsewhere in the membrane for further assembly. We will also check the location of mRNAs encoding plasma membrane proteins to see if there are comparable translation zones at the plasma membrane. We will examine the organisation of the thylakoid membrane translation zones using a technique called atomic force microscopy, which can tell us how individual protein complexes are arranged in the membrane. We expect this to give us new insights into the elaborate and co-ordinated process by which photosynthetic complexes are assembled. We will test for the involvement of further RNA-binding proteins in targeting mRNA molecules to the thylakoid and the plasma membranes, and we will look for features of the mRNA molecules that may be recognised by each of the RNA-binding proteins. We will then test our ideas by producing cells with mutated mRNA sequences that we expect to change the association with the protein and the location of the mRNA in the cell. We expect this part of the project to tell us how cyanobacterial cells are able to target specific proteins to a specific membrane, laying the foundations for methods for "precision engineering" of cyanobacteria for solar-powered biotechnology.