Proteomic mapping of ER-PM junctions identifies STIMATE as a regulator of Ca2+ influx

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signalling(1-4). Store-operated Ca2+ entry mediated by dynamic STIM1-ORAI1 coupling represents a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously unrecognized protein regulators facilitate this process remain ill-defined. Using a combination of spatially restricted biotin labelling in situ coupled with mass spectrometry(5,6) and a secondary screen based on bimolecular fluorescence complementation(7), we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches led to the discovery of an ER-resident multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of Ca2+ influx in vertebrates. STIMATE physically interacts with STIM1 to promote STIM1 conformational switch. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the Ca2+-NFAT signalling. Our findings enable further genetic studies to elucidate the function of STIMATE in normal physiology and disease, and set the stage to uncover more uncharted functions of hitherto underexplored ER-PM junctions.National Institutes of Health [R01 GM112003, R01 AI084167, R01 CA143811, R01 GM110397]; Leukemia & Lymphoma Society [LLS 3013-12]; Robert A. Welch Endowed Chair in Chemistry [BE-0023]; China Scholarship Council; National Natural Science Foundation of China [NSFC31471279, NSFC-81222020]; Recruitment Program for Young Professionals of China; Program for New Century Excellent Talents in University [NCET-13-0061]; American Heart Association SDG [13SDG17200006]; Cancer Prevention Research Institute of Texas grant; Texas A&M University Health Science Center Startup FundSCI(E)PubMedARTICLEwyoujun@bnu.edu.cn; yzhou@ibt.tamhsc.edu101339-+1