Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and Ca2+ signalling1,2,3,4. 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 spectrometry5,6 and a secondary screen based on bimolecular fluorescence complementation7, 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.
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We are grateful to R. Lewis at Stanford University for the HRP–STIM1, mCherry–STIM1, and mCherry–CAD constructs. We thank J. Liou at University of Texas Southwestern Medical Center for sharing with us the MAPPERs construct, and Z. Songyang at Baylor College of Medicine for the BiFc-related constructs. We thank M. Höök at Texas A&M University for access to the Biacore 3000, D. Liu at Baylor College of Medicine for access to the Cell Based Assay Screening Facility and advice on BiFc, and R. Payne at Texas A&M University for technical support in electron microscopy studies. This work was supported by National Institutes of Health grants (R01 GM112003 to Y.Z., R01 AI084167, R01 CA143811 to C.L.W., and R01 GM110397 to P.G.H.), a Special Fellow Award from the Leukemia & Lymphoma Society (LLS 3013-12 to Y.Z.), a Robert A. Welch Endowed Chair in Chemistry (BE-0023) to C.L.W., the China Scholarship Council (to J.J.), the National Natural Science Foundation of China (NSFC31471279 to Y.W. and NSFC-81222020 to L.C.), the Recruitment Program for Young Professionals of China (to Y.W.), the Program for New Century Excellent Talents in University (NCET-13-0061 to Y.W.), the American Heart Association SDG (13SDG17200006 to S.L.Z.), a Cancer Prevention Research Institute of Texas grant (to Y.H.), and by an allocation from the Texas A&M University Health Science Center Startup Fund (to Y.Z.).
Integrated supplementary information
Side-by-side comparison of the time course of GFP-STIM1 puncta formation in normal HEK293 (WT, left) and STIMATE knockout HEK293 cells (STIMATE-KO, right) under TIRF microscope.
The total fluorescence intensities of these two cells were comparable under epifluorescence microscope. Appended in the movie includes the time points after TG (1 μM)-induced store depletion.
Light-inducible accumulation of LiMETER at cortical ER in normal HEK293 (WT, left) and STIMATE knockout HEK293 cells (STIMATE-KO, right) under TIRFM.
Appended in the movie includes the time points after light stimulation at 488 nm.