Key result
Spatially-restricted biotin-labeling with mass spectrometry identifies ~70 candidate proteins at ER-PM junctions in living cells.
The study successfully mapped the proteome of intact ER-PM junctions in living HEK293 cells, identifying over 70 candidate proteins.
Advances molecular mapping of ER-PM junctions; leaves open validation in cardiomyocytes and relevance to cardiac calcium signaling.
Specialized junctional sites that connect the plasma membrane \(PM) and endoplasmic reticulum \(ER) are intimately involved in controlling lipid metabolism and calcium signaling in mammalian cells.Store operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes one of the most wellestablished molecular events occurring at ER-PM junctions, but the protein composition at this particular subcellular compartment remain poorly de ned.Using an in situ spatially-restricted biotin-labeling coupled with mass spectrometry, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity.Our approaches lead to the discovery of >70 candidate proteins at ER-PM junctions, with the majority falling into the categories of ER/PM-resident proteins, cytoskeletal components, and proteins functioning in intracellular membrane tra cking or posttranslational modi cations.Although the current protocol is limited to ER-PM junctions, the methods described herein can be readily extended to study other types of inter-membrane appositions in various types of cells.
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Zhou et al. (2015) studied this question. APEX2-catalyzed in situ biotin-labeling vs. Control samples without H2O2 was evaluated on Proteomic mapping of intact ER-PM junctions. In situ spatially-restricted biotin-labeling coupled with mass spectrometry identified over 70 candidate proteins at ER-PM junctions in living HEK293 cells.
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