Proteomic Mapping and Optogenetic Control of ER-PM Junctions in Living Cells

Abstract

Specialized junctional sites that connect the plasma membrane (PM) and endoplasmic reticulum (ER) play critical roles in controlling lipid metabolism and calcium signaling. Store-operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes a classical molecular event occurring at ER-PM junctions, but the protein composition and how previously-unrecognized protein regulators facilitate calcium influx at this particular subcellular compartment remain poorly defined. By taking advantage of a spatially-restricted biotin-labeling technique coupled with mass spectrometry, we systematically mapped the proteome of intact ER-PM junctions in living cells while maintaining their architectural integrity. Using a secondary screen based on bimolecular fluorescence complementation, we further discovered a novel multi-transmembrane protein that we call STIMATE (STIM-activating enhancer, encoded by TMEM110) as a positive regulator of calcium influx in vertebrates. STIMATE colocalizes with STIM1 to the ER and promotes STIM1 conformational switch through physical contact with the STIM1 juxtamembrane coiled coil domain. This represents a novel regulatory mechanism to modulate STIM1-dependent calcium flux in mammalian cells. Genetic depletion of STIMATE substantially reduces STIM1 puncta formation at ER-PM junctions and suppresses the calcium-NFAT signaling. To noninvasively manipulate membrane contact sites, we also created a novel tool to reversibly control the formation of ER-PM junctions by harnessing the power of light. This optogenetic tool makes it possible for future screening of protein/chemical regulators that are involved in maintaining ER-PM junctions. Our findings not only afford an initial view on the protein composition of intact ER-PM junctions under physiological conditions, but also provide a solid framework to uncover more uncharted functions of hitherto underexplored ER-PM junctions. Increasing our comprehension of knowledge on this elusive and intriguing subcellular structure will lead to better design of therapeutic strategies to treat human disorders associated with aberrant lipid metabolism and calcium signaling.