Localisation of key proteases involved in the assembly and repair of Photosystem II in cyanobacterium Synechocystis sp. PCC 6803.
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All photosynthetic organisms use light as a source of energy, however prolonged excessively high light causes irreversible damage to the main photosynthetic complexes. In particular the D1 polypeptide of Photosystem II is susceptible to damage and must be degraded and replaced. While the concept of PSII repair has attracted intensive research, important details remain to be determined. The sub-cellular localisation of proteases involved in PSII repair and assembly is investigated here in the model cyanobacterium Synechocystis sp. PCC 6803, by employing fluorescent protein tagging and fluorescence imaging in vivo. Results show that all FtsH protease homologues in Synechocystis are localised to distinct regions of the plasma membrane (FtsH1) and thylakoids (FtsH2, FtsH3, FtsH4). Importantly, FtsH2, involved in PSII repair, remains within distinct thylakoid membrane zones when activated by high light, leading to the hypothesis of localised PSII repair centres in the thylakoid membranes. In order to assess composition of the FtsH2-defined membrane zones, a novel technique for isolating membrane sub-fractions by anti-GFP pulldowns was employed. Mass spectrometry identified potentially interacting and neighbouring proteins within the repair centres, whose content changes under different light exposure. Furthermore, observed changes in FtsH2 and FtsH4 distributions under iron and copper deprivation suggest functions in responses to other stress conditions. To find the locations of D1 synthesis during PSII repair and de novo assembly, the D1 C-terminal processing peptidase CtpA was similarly GFP-tagged and observed in vivo. Results suggest that D1 synthesis for PSII repair takes place in the thylakoid membranes, while D1 synthesis for de novo PSII biogenesis takes place in specialised regions at both edges of the thylakoid system, adjacent to the plasma membrane and protruding into the central cytoplasm. By localising crucial cellular enzymes in vivo, this study demonstrates functional compartmentalisation and membrane heterogeneity in a prokaryote.
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