Membrane protein targeting and assembly in cyanobacteria

蓝藻是一种光合细菌，在地球生态中起着至关重要的作用，也是太阳能生物技术的有希望的载体。蓝藻的细胞结构比大多数其他细菌更复杂：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物。相比之下，细胞周围的质膜致力于其他功能，并且在两个膜系统中发现了非常不同的蛋白质组。虽然我们知道哪些蛋白质存在于哪个膜上，但我们不知道它们在哪里被整合到每个膜上，也不知道是什么把它们送到那里的。我们利用荧光显微镜找到编码类囊体膜蛋白的特定mRNA分子的位置，从而获得了这两个问题的线索。我们使用了一种叫做RNA-FISH的技术，我们用化学方法固定细胞，然后用与荧光分子相连的短合成DNA序列探测它们。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。利用这项技术，我们已经证明，编码光合装置核心成分的mRNA分子聚集在类囊体膜表面非常特定的位置，这些位置最靠近发现DNA的细胞中心部分。我们有证据表明，这些位置对应于“翻译区”，在那里光合作用蛋白首次产生并整合到类囊体膜中，我们也有证据表明mRNA分子通过与特定mRNA结合蛋白的相互作用被引导到类囊体膜。这种相互作用对于将膜蛋白靶向到正确的膜上可能是至关重要的。我们发现的mrna结合蛋白在不同种类的蓝藻中都是高度保守的，它们甚至在植物叶绿体中也有同源物。这表明mRNA靶向系统的元素已经保守了超过10亿年的进化，从一个自由生活的蓝藻到植物细胞内的叶绿体。在本提案中，我们将开展进一步的RNA-FISH研究，以寻找蓝藻中更广泛的mRNA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，或者每个蛋白质是否有自己特定的翻译区域。我们将测试新产生的光合复合体是留在翻译区，还是迁移到膜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mrna的位置，看看在质膜上是否有类似的翻译区。我们将使用一种叫做原子力显微镜的技术来检查类囊体膜翻译区的组织，这种技术可以告诉我们单个蛋白质复合物是如何在膜中排列的。我们希望这能给我们提供新的见解，了解光合复合体组装的复杂而协调的过程。我们将测试进一步的rna结合蛋白参与将mRNA分子靶向到类囊体和质膜，我们将寻找可能被每种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mRNA序列的细胞来测试我们的想法，我们希望改变与蛋白质的关联以及mRNA在细胞中的位置。我们希望这个项目的这一部分能告诉我们蓝藻细胞是如何将特定的蛋白质靶向到特定的膜上的，为蓝藻的太阳能生物技术“精密工程”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.