State Key Laboratory of Molecular Developmental Biology, Innovation Academy for Seed Design, CAS, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China; College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China.
State Key Laboratory of Molecular Developmental Biology, Innovation Academy for Seed Design, CAS, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.
Mol Cell Proteomics. 2022 Dec;21(12):100440. doi: 10.1016/j.mcpro.2022.100440. Epub 2022 Nov 8.
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 b/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.
基于抗坏血酸过氧化物酶 (APEX) 的邻近标记结合质谱分析，具有对感兴趣的蛋白质复合物附近的蛋白质进行时空鉴定的巨大潜力。使用这种方法，可以在一种流行的光合模型蓝藻集胞藻 6803 中（以下称为集胞藻），确定重要蛋白质复合物的蛋白质组邻居。为此，我们开发了一种在集胞藻中基于 APEX2 的邻近标记的稳健工作流程，并通过将 APEX2 与腔室 OEC 亚基 PsbO 融合，使用该工作流程来鉴定与光系统 II (PS II) 氧产生复合物 (OEC) 邻近的蛋白质。总共鉴定出 38 种整合膜蛋白 (IMP) 和 93 种腔室蛋白与 OEC 邻近。这些蛋白质中有很大一部分参与 PS II 的组装、成熟和修复，而其余大部分以前与 PS II 没有关联。这些 IMP 包括 PS II 和细胞色素 b/f 的亚基，但不包括 PS I（除了 PsaL）和 ATP 合酶，这表明后两个复合物在空间上与 OEC 分离，距离长于 APEX2 标记半径。此外，由于其腔面向区域仅包含潜在的 APEX2 标记位点，因此成功预测了六个 IMP 的拓扑结构。腔室蛋白包括 66 种具有预测信号肽的蛋白质和 57 种也定位于周质的蛋白质，为研究蛋白质易位的调控和选择性提供了重要的靶标。总之，我们不仅开发了一种在集胞藻中应用基于 APEX2 的邻近标记的稳健工作流程，并展示了使用短半径定义重要蛋白质复合物的邻近蛋白质组的可行性，还发现了一组可能与 PS II 结构和功能相互作用和调节的蛋白质。
