A Proximity Labeling Strategy Enables Spatial Proteomic Analysis of Inter-organelle Membrane Contacts in Living Cells

Inter-organelle membrane contacts especially mitochondria-endoplasmic reticulum contacts (MERC) conduct important biological functions including exchange of lipids and ions, and modulation of membrane dynamics. Alterations of inter-organelle membrane contacts have been implicated with the pathogenesis of diseases, such as neurodegenerative diseases, cancers and type 2 diabetes. However, the protein compositions of inter-organelle contacts remain largely unknown as no biomarker has been discovered and it’s difficult to purify intact inter-organelle contact proteins. Here, we applied a systematic approach to probe the spatial proteome of MERC in living cells by combining the bimolecular fluorescence complementation assay and a proximity labeling strategy based on APEX2. As a result, we discovered 403 highly confident MERC proteins including many well-known MERC proteins and a variety of novel protein species as well. We further validated that WFS1, BAG2, SPTLC1 and GLUD1 are enriched at MERC with high resolution fluorescent imaging. Our study provides a powerful tool to characterize the spatial proteomes of inter-organelle membrane contacts in living cells.