Membrane protein targeting and assembly in cyanobacteria

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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.