Department of Biochemistry, University of Colorado, Boulder, CO, 80309, USA.
Renewable and Sustainable Energy Institute, University of Colorado, Boulder, CO, 80309, USA.
Photosynth Res. 2021 Feb;147(2):177-195. doi: 10.1007/s11120-020-00806-y. Epub 2020 Dec 6.
Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.
蓝细菌具有独特的细胞内组织。许多蛋白质组学研究已经研究了蓝细菌的不同特征，以了解细胞内结构及其各自的功能。虽然这些研究在理解蓝细菌生理学方面取得了很大进展，但用于纯化细胞结构的常规分级分离方法存在局限性；具体来说，某些细胞区域无法用现有的分级分离方法进行纯化。基于邻近的蛋白质组学技术是为了克服生化分级分离在蛋白质组学方面的局限性而开发的。基于邻近的蛋白质组学依赖于时空蛋白质标记，然后对标记的蛋白质进行质谱分析，以确定感兴趣区域的蛋白质组。我们使用过表达的 APEX2 酶（一种工程化的抗坏血酸过氧化物酶）在蓝细菌集胞藻 PCC 7002 中进行了基于邻近的蛋白质组学研究。我们使用 APEX2 与 PsbU 之间的翻译融合，确定了类囊体腔的蛋白质组，类囊体腔是一个用现有方法仍然难以研究的细胞区域，PsbU 是光系统 II 的腔室亚基。我们的结果证明了 APEX2 作为一种工具的强大功能，可用于研究包括蓝细菌中光系统 II 组装在内的细胞内特征和过程的细胞生物学，具有增强的时空分辨率。
