Zhou Maoge, Kong Bingjie, Zhang Xiang, Xiao Ke, Lu Jing, Li Weixing, Li Min, Li Zonghong, Ji Wei, Hou Junjie, Xu Tao
Guangzhou Laboratory, Guangzhou, Guangdong 510005, China.
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
iScience. 2023 Jun 17;26(7):107159. doi: 10.1016/j.isci.2023.107159. eCollection 2023 Jul 21.
Inter-organelle membrane contacts are highly dynamic and act as central hubs for many biological processes, but the protein compositions remain largely unknown due to the lack of efficient tools. Here, we developed BiFCPL to analyze the contact proteome in living cells by a bimolecular fluorescence complementation (BiFC)-based proximity labeling (PL) strategy. BiFCPL was applied to study mitochondria-endoplasmic reticulum contacts (MERCs) and mitochondria-lipid droplet (LD) contacts. We identified 403 highly confident MERC proteins, including many transiently resident proteins and potential tethers. Moreover, we demonstrated that mitochondria-LD contacts are sensitive to nutrient status. A comparative proteomic analysis revealed that 60 proteins are up- or downregulated at contact sites under metabolic challenge. We verified that SQLE, an enzyme for cholesterol synthesis, accumulates at mitochondria-LD contact sites probably to utilize local ATP for cholesterol synthesis. This work provides an efficient method to identify key proteins at inter-organelle membrane contacts in living cells.
细胞器间的膜接触高度动态，是许多生物过程的核心枢纽，但由于缺乏有效的工具，其蛋白质组成在很大程度上仍不清楚。在这里，我们开发了BiFCPL，通过基于双分子荧光互补（BiFC）的邻近标记（PL）策略来分析活细胞中的接触蛋白质组。BiFCPL被应用于研究线粒体-内质网接触（MERC）和线粒体-脂滴（LD）接触。我们鉴定出403种高度可信的MERC蛋白，包括许多瞬时驻留蛋白和潜在的连接蛋白。此外，我们证明线粒体-LD接触对营养状态敏感。一项比较蛋白质组学分析显示，在代谢挑战下，接触位点有60种蛋白质上调或下调。我们证实，胆固醇合成酶SQLE在线粒体-LD接触位点积累，可能是为了利用局部ATP进行胆固醇合成。这项工作提供了一种有效的方法来鉴定活细胞中细胞器间膜接触的关键蛋白。
