Cyanobacteria use the structural features of their messenger RNA molecules to guide newly-made proteins to the correct membrane inside the cell, and researchers want to crack that code. This matters because cyanobacteria are photosynthetic bacteria that could serve as solar-powered factories, producing fuels, chemicals, or medicines from sunlight and CO₂. But engineering them for that purpose requires placing synthetic proteins precisely where they are needed—inside the thylakoid membrane (where photosynthesis happens) or at the plasma membrane (where products could be exported). Currently, the protein-sorting machinery in these cells is poorly understood, making such precision impossible. The project will systematically test which parts of mRNA molecules determine where they are translated, identify the chaperone proteins that escort plasma-membrane mRNAs past the thylakoid layers, and then apply those rules to target a synthetic fluorescent protein to a chosen membrane as a proof of concept. If successful, this fundamental science will provide a toolkit for precision engineering of cyanobacterial cell architecture. That could eventually enable more efficient production of biofuels, bioplastics, or other high-value compounds—reducing reliance on fossil feedstocks and making industrial biotechnology more sustainable.
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Context Cyanobacteria are photosynthetic prokaryotes that play a huge role in the global ecosystem and show outstanding potential for exploitation as solar-powered microbial cell factories. Their photosynthetic physiology is enabled by an unusually complex prokaryotic cell architecture, with sub-cellular compartments that include carboxysomes for CO2 fixation, and the thylakoid membranes, which are the site of the photosynthetic light reactions. The thylakoid membranes form an extensive set of flattened membrane sacs that reside in the cytoplasm and do not appear contiguous with the plasma membrane. Maintenance of the complex cell organisation of cyanobacteria demands a precise protein sorting machinery, which remains poorly understood. In a current BBSRC-funded project (BB/W001012/1 “Membrane protein targeting and assembly in cyanobacteria”: 2022-2025), we have been following the sorting and translation of cyanobacterial proteins. We have done this primarily by looking at the locations and interactions of mRNA molecules, but also by tracking the locations of newly-assembled proteins and the ribosomes that translate them. Our results suggest that the sub-cellular sorting of some key proteins is determined by the structural features of mRNA molecules. In the case of thylakoid membrane proteins, we have identified a small family of RNA binding proteins that bind some thylakoid mRNAs and help to locate them at a specific thylakoid membrane surface, where they encounter a dedicated pool of thylakoid-associated ribosomes. This ensures that thylakoid membrane proteins are inserted into the correct membrane. Recognition of some thylakoid mRNAs appears to be mediated by a stem-loop structure at the 3’ end of the coding sequence. We can see that mRNAs encoding plasma membrane proteins are directed to the plasma membrane for translation. We have less information about the factors controlling this process, but it is clear that plasma membrane mRNAs have to bypass multiple thylakoid membrane layers on their way to the plasma membrane. Therefore, we can infer a requirement for mRNA chaperones to prevent premature translation and degradation of plasma membrane mRNAs during their journey from the nucleoid to the plasma membrane. Challenge Taking our understanding of protein targeting and the maintenance of cell organisation in cyanobacteria to the next level requires identification of the specific features of mRNA molecules that target them to different destinations in the cell. We can do this by combining the methods that we and our collaborators have developed for probing the locations and interactions of cyanobacterial mRNAs and proteins with a systematic approach to manipulating the sequences of test mRNA species. On the principle that “what I cannot create I do not understand” we can apply what we learn about the rules for mRNA targeting to locate a synthetic membrane protein in the plasma membrane or the thylakoid membrane. Objectives Identify the features of mRNA molecules that target them to different cell structures (thylakoid membrane, plasma membrane and carboxysomes), by fusing different regions of mRNAs to a test mRNA species. Test candidate mRNA chaperones that may be involved in locating plasma membrane mRNAs at the plasma membrane. Apply the rules for mRNA targeting to locate a synthetic fluorescent membrane protein in either the thylakoid membrane or the plasma membrane, as a proof-of-concept for precision engineering of the cell. Potential applications Targeting heterologous proteins to specific cell locations will enable more precise modification of cyanobacterial cells for solar-powered generation and export of products.
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