A proximity labeling strategy enables proteomic analysis of inter-organelle membrane contacts
Maoge Zhou,
Bingjie Kong,
Xiang Zhang,
Ke Xiao,
Jing Lu,
Weixing Li,
Min Li,
Zonghong Li,
Wei Ji,
Junjie Hou,
Tao Xu
Affiliations
Maoge Zhou
Guangzhou Laboratory, Guangzhou, Guangdong 510005, China; Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Bingjie Kong
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Xiang Zhang
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Ke Xiao
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Jing Lu
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Weixing Li
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Min Li
Guangzhou Laboratory, Guangzhou, Guangdong 510005, China
Zonghong Li
Guangzhou Laboratory, Guangzhou, Guangdong 510005, China
Wei Ji
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China; Corresponding author
Junjie Hou
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; Corresponding author
Tao Xu
Guangzhou Laboratory, Guangzhou, Guangdong 510005, China; Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China; School of Biomedical Engineering, Guangzhou Medical University, Guangzhou, Guangdong 511436, China; Corresponding author
Summary: 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.
