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.

Technical Abstract:
The thylakoids of cyanobacteria are intracytoplasmic membranes that are the sole site of the photosynthetic light reactions and the major site of respiration. Their proteome is strikingly different from that of the plasma membrane. Cyanobacterial membrane targeting systems have never been resolved, and the sites of translation and membrane insertion remain controversial. Using Fluorescent in situ Hybridisation (RNA-FISH), we found that mRNAs encoding core photosynthetic proteins cluster at thylakoid surfaces, suggesting that there are highly localised thylakoid translation zones. We found that thylakoid association persists even when the mRNA is decoupled from the ribosomes, indicating a ribosome-independent mechanism for locating photosynthetic mRNAs to the thylakoid surface. We identified two RNA-binding proteins (RBPs) that are important for locating photosynthetic mRNAs. Now, we will use similar techniques to fully define the role of the thylakoid translation zones. We will test whether different photosynthetic proteins are produced 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 use Atomic Force Microscopy to probe the membrane landscape of the thylakoid translation zones, which we expect to give new insights into reaction centre assembly. We will probe the translation sites of membrane-integral plasma membrane proteins and we will test for the involvement of further RBPs in targeting mRNA molecules to the thylakoid and the plasma membranes, We will look for features of the mRNA molecules that may be recognised by each of the RBPs and we will test these ideas by mutating the mRNA sequences. This will lead towards methods for specific membrane targeting of heterologous proteins and "precision engineering" of cyanobacteria for solar-powered biotechnology.