Abstract
Ascorbate peroxidase (APEX)-based proximity labeling coupled with mass spectrometry has a great potential for spatiotemporal identification of proteins proximal to a protein complex of interest. Using this approach is feasible to define the proteome neighborhood of important protein complexes in a popular photosynthetic model cyanobacterium Synechocystis sp. PCC6803 (hereafter named as Synechocystis). To this end, we developed a robust workflow for APEX2-based proximity labeling in Synechocystis and used the workflow to identify proteins proximal to the photosystem II (PS II) oxygen evolution complex (OEC) through fusion APEX2 with a luminal OEC subunit, PsbO. In total, 38 integral membrane proteins (IMPs) and 93 luminal proteins were identified as proximal to the OEC. A significant portion of these proteins are involved in PS II assembly, maturation, and repair, while the majority of the rest were not previously implicated with PS II. The IMPs include subunits of PS II and cytochrome b6/f, but not of photosystem I (except for PsaL) and ATP synthases, suggesting that the latter two complexes are spatially separated from the OEC with a distance longer than the APEX2 labeling radius. Besides, the topologies of six IMPs were successfully predicted because their lumen-facing regions exclusively contain potential APEX2 labeling sites. The luminal proteins include 66 proteins with a predicted signal peptide and 57 proteins localized also in periplasm, providing important targets to study the regulation and selectivity of protein translocation. Together, we not only developed a robust workflow for the application of APEX2-based proximity labeling in Synechocystis and showcased the feasibility to define the neighborhood proteome of an important protein complex with a short radius but also discovered a set of the proteins that potentially interact with and regulate PS II structure and function.