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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Carrasco, S. & Meyer, T. STIM proteins and the endoplasmic reticulum-plasma membrane junctions. Annu. Rev. Biochem. 80, 973–1000 (2011).
Elbaz, Y. & Schuldiner, M. Staying in touch: the molecular era of organelle contact sites. Trends Biochem. Sci. 36, 616–623 (2011).
Hogan, P. G., Lewis, R. S. & Rao, A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533 (2010).
Stefan, C. J., Manford, A. G. & Emr, S. D. ER-PM connections: sites of information transfer and inter-organelle communication. Curr. Opin. Cell Biol. 25, 434–442 (2013).
Rhee, H. W. et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science 339, 1328–1331 (2013).
Lam, S. S. et al. Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat. Methods 12, 51–54 (2015).
Kerppola, T. K. Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells. Annu. Rev. Biophys. 37, 465–487 (2008).
Porter, K. R. & Palade, G. E. Studies on the endoplasmic reticulum. III. Its form and distribution in striated muscle cells. J. Biophys. Biochem. Cytol. 3, 269–300 (1957).
Mayer, G. & Bendayan, M. Biotinyl-tyramide: a novel approach for electron microscopic immunocytochemistry. J. Histochem. Cytochem. 45, 1449–1454 (1997).
Bendayan, M. Tech.Sight. Worth its weight in gold. Science 291, 1363–1365 (2001).
Sharma, S. et al. An siRNA screen for NFAT activation identifies septins as coordinators of store-operated Ca2+ entry. Nature 499, 238–242 (2013).
Min, S. W., Chang, W. P. & Sudhof, T. C. E-Syts, a family of membranous Ca2+-sensor proteins with multiple C2 domains. Proc. Natl Acad. Sci. USA 104, 3823–3828 (2007).
Manjarres, I. M., Rodriguez-Garcia, A., Alonso, M. T. & Garcia-Sancho, J. The sarco/endoplasmic reticulum Ca(2 +) ATPase (SERCA) is the third element in capacitative calcium entry. Cell Calcium 47, 412–418 (2010).
Wang, Y. et al. The calcium store sensor, STIM1, reciprocally controls Orai and CaV1.2 channels. Science 330, 105–109 (2010).
Park, C. Y., Shcheglovitov, A. & Dolmetsch, R. The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels. Science 330, 101–105 (2010).
Grigoriev, I. et al. STIM1 is a MT-plus-end-tracking protein involved in remodeling of the ER. Curr. Biol. 18, 177–182 (2008).
Soboloff, J., Rothberg, B. S., Madesh, M. & Gill, D. L. STIM proteins: dynamic calcium signal transducers. Nat. Rev. Mol. Cell Biol. 13, 549–565 (2012).
Giordano, F. et al. PI(4,5)P(2)-dependent and Ca(2 +)-regulated ER-PM interactions mediated by the extended synaptotagmins. Cell 153, 1494–1509 (2013).
Tabb, D. L., McDonald, W. H. & Yates, J. R. 3rd DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26 (2002).
Chang, C. L. et al. Feedback regulation of receptor-induced Ca2+ signaling mediated by E-Syt1 and Nir2 at endoplasmic reticulum-plasma membrane junctions. Cell Rep. 5, 813–825 (2013).
Maleth, J., Choi, S., Muallem, S. & Ahuja, M. Translocation between PI(4,5)P2-poor and PI(4,5)P2-rich microdomains during store depletion determines STIM1 conformation and Orai1 gating. Nat. Commun. 5, 5843 (2014).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Zurek, N., Sparks, L. & Voeltz, G. Reticulon short hairpin transmembrane domains are used to shape ER tubules. Traffic 12, 28–41 (2011).
Russ, W. P. & Engelman, D. M. The GxxxG motif: a framework for transmembrane helix–helix association. J. Mol. Biol. 296, 911–919 (2000).
Lorenz, H., Hailey, D. W., Wunder, C. & Lippincott-Schwartz, J. The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology. Nat. Protoc. 1, 276–279 (2006).
Wu, Y. I. et al. A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104–108 (2009).
Harper, S. M., Neil, L. C. & Gardner, K. H. Structural basis of a phototropin light switch. Science 301, 1541–1544 (2003).
Heo, W. D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006).
Park, C. Y. et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876–890 (2009).
Yuan, J. P. et al. SOAR and the polybasic STIM1 domains gate and regulate Orai channels. Nat. Cell Biol. 11, 337–343 (2009).
Zhou, Y. et al. Initial activation of STIM1, the regulator of store-operated calcium entry. Nat. Struct. Mol. Biol. 20, 973–981 (2013).
Ma, G. et al. Inside-out Ca2+ signaling prompted by STIM1 conformational switch. Nat. Commun. 6, 7826 (2015).
Wu, M. M., Buchanan, J., Luik, R. M. & Lewis, R. S. Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J. Cell Biol. 174, 803–813 (2006).
Lee, O. H. et al. Genome-wide YFP fluorescence complementation screen identifies new regulators for telomere signaling in human cells. Mol. Cell. Proteom. 10, M110001628 (2011).
Zhou, Y. et al. STIM1 gates the store-operated calcium channel ORAI1 in vitro. Nat. Struct. Mol. Biol. 17, 112–116 (2010).
Wang, Y. et al. STIM protein coupling in the activation of Orai channels. Proc. Natl Acad. Sci. USA 106, 7391–7396 (2009).
Zhou, Y., Ramachandran, S., Oh-Hora, M., Rao, A. & Hogan, P. G. Pore architecture of the ORAI1 store-operated calcium channel. Proc. Natl Acad. Sci. USA 107, 4896–4901 (2010).
Wang, X. et al. Distinct Orai-coupling domains in STIM1 and STIM2 define the Orai-activating site. Nat. Commun. 5, 3183 (2014).
Zhang, S. L. et al. Genome-wide RNAi screen of Ca(2 +) influx identifies genes that regulate Ca(2 +) release-activated Ca(2 +) channel activity. Proc. Natl Acad. Sci. USA 103, 9357–9362 (2006).
Zal, T. & Gascoigne, N. R. Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939 (2004).
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.).
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 5 BiFc assay as a secondary assay to validate STIM1 interactors and determination of STIMATE membrane topology (related to Fig. 1).
(a) Scheme for the BiFc assay. The N-terminal portion of YFP (YFPN, residues 1–155) is fused to the STIM1 C-terminus as the bait to prey against a customized library containing candidate genes fused with the C-terminal half of YFP (YFPC, residues 156–238), or vice versa. STIM1 binding to its partners is anticipated to restore the YFP fluorescence. (b) Representative examples of FACS profiles from the BiFc assays. STIM1-YFPN + YFPc was used a negative control, whilst STIMATE-YFPN + ORAI1-YFPC was used as positive control in the assay. In addition to strong YFP signals restored in HEK293 cells coexpressing STIM1-YFPN and a number of YFPC-tagged genes, we also observed efficient recovery of YFP signals in cells coexpressing STIMATE-YFPN and STIMATE-YFPC, implying the tendency of STIMATE to form oligomers. (c) BiFc as a secondary assay to confirm selected STIM1 binding partners in HEK293T cells. YFP-positive cells were counted by FACS ad 20% was set as the threshold for strong positive hits. Store depletion was induced by 1–2 μM thapsigargin (TG), a widely-used inhibitor for SERCA pumps. For hits identified only under resting condition in the proteomic study (Supplementary Table 1), BiFc assay was performed without the addition of TG. Error bars represent s.e.m. from n = 12 wells of cultured cells pooled across 3 independent experiments. Note that this set of data was also used in the scatter plot as X-axis in Fig. 1d. (d) Multiple sequence alignment of the STIMATE (encoded by TEMEM110) proteins in vertebrates and the relative distribution of STIMATE mRNA in human and mouse tissues. The predicted TM segments were highlighted in yellow. Regions enriched with positively- or negatively residues in the STIMATE C-tail were boxed in blue or red, respectively. The secondary structure elements were displayed on the top of aligned primary sequences. Cylinder, α-helix; Arrow, β-strand. Sequence accession numbers in the Uniprot database are: human, Q86TL2; mouse, Q3UF25; fish, Q7SY08; frog, Q28EE1. (e–h) Determination of STIMATE topology by the fluorescence protease protection (FPP) assay. (e) Schematic representation of the FPP assay. Digitonin is added to HEK293 cells to permeabilize the plasma membrane and causes diffusion of cytosolic proteins outside the cell. The amount of digitonin is optimized to maintain the membrane integrity of ER. Subsequent addition of trypsin would cause the degradation of fluorescent protein tags exposed toward the cytosol (e.g., STIM1-YFP, panel f), thus resulting in significant quenching of fluorescence. In contrast, the fluorescence tags residing within the subcellular organelles (e.g., EGFP-STIM, panel f) remain intact and emit fluorescence. (f) Confocal images of HEK293 cells expressing either N- or C-terminally FP tagged STIM1 or STIMATE or mCherry alone that were sequentially treated with digitonin and trypsin. Both fluorescence protein tagged constructs showed a pronounced reduction of fluorescence upon addition of trypsin, indicating that N and C-termini of STIMATE face the cytosolic side of ER. Note that STIMATE-YFP and mCherry-STIMATE were co-transfected into the same cells. (g) Time course of fluorescence imaging following sequential addition of digitonin and trypsin. (h) Cartoon depicting the tentative membrane topology of STIMATE. Computational prediction of membrane topology yielded two models. Most programs including TOPCONS, TMHMM and TMPred predicted five transmembrane segments in STIMATE. TM4 is predicted to contain ∼30 amino acids (Supplementary Fig. 1d), a length that far exceeds the average length of typical integral membrane proteins of the ER (approximately 20 amino acids). It is likely that TM4 adopts a hairpin structure to penetrate but not span across the ER membrane, thus allowing both the N- and C-termini of STIMATE facing toward the same side. In model II, only four TM segments were predicted by the programs SCAMPI and SOSUI. The second TM segment in Model I was absent in Model II.
Supplementary Figure 6 Expression profiles of STIMATE and confirmation of STIMATE knockdown or knockout (related to Figs 1 and 3).
(a) mRNA expression profiles of STIMATE in mouse (left) and human (right) tissues (n = 3 independent experiments). Data were plotted as average ± s.d. (b) Relative STIMATE mRNA levels in HEK293 cells transfected with indicated siRNA oligos. Error bars denote s.e.m. from n = 3 independent experiments. (c) Design of the guide sequence (sgRNA) to target the human STIMATE exon 4 for gene disruption by the Cas9 targeting vector pX459. Seven stable cell lines after puromycin selection were maintained and sequenced. Surveyor nuclease assay was further used to confirm gene disruption in stable clones after puromycin selection. (d) Confirmation of gene disruption at STIMATE exon 4 with Sanger’s sequencing. (e) Ionomycin-induced Ca2+ flux in normal and STIMATE-KO HEK293 cells. Error bars represent s.e.m. from assays performed with n = 30 (control), 36 (STIMATE KO#3), or 40 cells (STIMATE KO#7), respectively, pooled across three independent experiments.
(a) Confocal images of HEK293, HeLa or COS-7 cells expressing STIMATE with FP tagged to its N- or C-terminus. Regardless of the position of FP tags, STIMATE displayed ER-like distribution. Blue, nuclear staining with Hoechst 33342. Scale bar, 10 μm. (b) Colocalization of EGFP-STIMATE with ER marker proteins DsRed-ER or mCherry-Sec61β. Nuclei were stained in blue by Hoechst 33342. The insets show at higher magnification the regions outlined by dotted line. Scale bar, 10 μm.
Supplementary Figure 8 TIRF imaging of STIMATE in indicated cells with or without coexpression of STIM1 or STIM1 + ORAI1 (related to Figs 2 and 3). 1 μM TG was added to induce store depletion. Scale bar, 10 μm.
(a) Representative TIRF images of HEK293, HeLa or COS-7 cells expressing STIMATE with mCherry tagged to its N-terminus or EYFP at its C-terminus. (b) TIRF images of COS-7 or HeLa cells cotransfected with mCherry-STIMATE (red) and EGFP-STIM1 (green). (c) TIRF images of EGFP-STIM1/ORAI1 stable cell lines transfected with mCherry-STIMATE. (d) Quantification of STIM1-STIMATE colocalization by the Pearson correlation coefficient. mCherry-STIMATE was transfected into HEK293 or HeLa cells coexpressing STIM1 (blue) or STIM1 + ORAI1 (red) as illustrated in panels c-d. Error bars denote s.e.m. from n = 6 (STIM1) or 10 (STIM1 + ORAI1) cells pooled across two independent experiments.
Supplementary Figure 9 Accumulation of STIM1 mutants or MAPPER at ER-PM junctions and representative examples of electron micrographs for HRP-STIM1 in normal or STIMATE-KO HEK293 cells (related to Figs 2 and 3).
(a–d) Effects of STIMATE depletion or overexpression on the action of STIM1 gain-of-function mutants. TIRF images (a) of EGFP-STIM1-D76A or EGFP-STIM1-L258G were acquired 0 or 300 seconds after store depletion induced by TG in normal or STIMATE-KO HEK293 cells. Scale bar, 10 μm. (b) Quantification of puncta size of WT and mutant STIM1 in normal or STIMATE-KO HEK293 cells. Note that the data for WT STIM1 was also used in Fig. 3e. Error bars denote s.e.m. for n = 6 cells pooled across two independent experiments. ∗∗∗ P < 0.001 (compared to control, paired student’s t-test). (c,d) Constitutive Ca2+ influx elicited by STIM1 gain-of-function mutant D76A (c) or L258G (d) in HE293 cells co-transfected with mCherry (control, black) or mCherry-STIMATE (red). Constitutive Ca2+ influx was monitored by Fura-2 when switching the external medium from 0 mM Ca2+ to 1 mM Ca2+. Error bars denote s.e.m. from n = 25 (control) and 28 (+ STIMATE) cells for D76A, or n = 30 (control) and 40 cells (+ STIMATE) for L258G pooled across three independent experiments. (e) Representative examples of EM images of HRP-STIM1 in normal or STIMATE-KO HEK293 cells. Compared to normal HEK293 cells, HRP-STIM1 exhibited less efficient translocation toward the ER-PM junction upon store depletion in STIMATE-KO cells. Arrowhead, HRP-STIM1 located at ER-PM junction; arrow, representative HRP-STIM1 staining in the cytosol (non-cortical ER regions). N stands for nucleus. Scale bar, 4000 nm. (f–h) cER labeling by MAPPER in normal or STIMAT-KO HEK293 cells. (f) Domain architecture of the genetically-encoded cER marker MAPPER. (g–h) TIRF images (g) and quantification (h) of MAPPER accumulation at cER in control (empty vector) or STIMATE-KO HEK293 stable cells. Error bars denote s.e.m. for n = 20 cells pooled across three independent experiments. Scale bar, 10 μm.
Supplementary Information (PDF 1817 kb)
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. (AVI 1781 kb)
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. (AVI 1232 kb)
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Jing, J., He, L., Sun, A. et al. Proteomic mapping of ER–PM junctions identifies STIMATE as a regulator of Ca2+ influx. Nat Cell Biol 17, 1339–1347 (2015). https://doi.org/10.1038/ncb3234
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