ER-PM Junctions Research Articles

STIM1-Induced Clustering of Orai1 Channels

Store operated Ca2+ entry is an evolutionarily conserved mechanism in all eukaryotic cells. Decreased ER Ca2+ promotes a transmembrane conformational shift and unfolding of the cytoplasmic STIM1 domain to expose the STIM-Orai Activating Region (SOAR) of STIM1. SOAR1 couples to and activates PM Orai1 channels, mediating Ca2+ entry signals. The F394H mutation in SOAR prevents it coupling with and activating Orai1. SOAR concatemer-dimers containing a single F394H mutated SOAR unit, fully couple to and activate Orai1, suggesting only one functional monomer of the STIM1 dimer is required for coupling to and activating Orai1 channels, consistent with a stoichiometry of one STIM1 dimer binding to each Orai1 monomer in the hexameric Orai1 channel. Using high-resolution fluorescence imaging approaches, we reveal the existence of dense clusters of YFP-tagged concatenated SOAR-dimers dependent on Orai1-His stably expressed in HEK cells. These clusters are absent in cells expressing heterodimers of concatenated-SOAR dimers containing one single F394H-SOAR residue. Concatenated-SOAR dimers with both monomers mutated with F394H, give no visible clustering. Using HEK cells stably expressing Orai1 C-terminally labeled with CFP, wildtype SOAR dimers did not undergo clustering. Thus, the CFP-label appears to sterically block cluster formation mediated by SOAR dimers. 2-APB is able overcome the inhibitory effect of the F394H mutation in SOAR. Interestingly, 2-APB induced recovery of Orai1-dependent clustering of SOAR concatemer dimers heteromeric for the F394H mutation. The results suggest that the second Orai1 binding site within a SOAR dimer can interact with adjacent Orai1 channels to form clusters of Orai1 channels in the PM. This theory is corroborated by recent electron microscopy data showing Orai1 channels spaced approximately the distance of a SOAR1 dimer. Physiologically, Orai1 cluster formation in ER-PM junctions may be important enhancing activation and deactivation kinetics of Orai1 channels and/or amplifying local Ca2+ signals.

Fluorescence Density Mapping: Extending the Possibilities of TIRFM to Study PM-ER Junctions

Total internal reflection microscopy (TIRFM) is an established method to study protein localization, dynamics and interactions of plasmalemmal as well as sub-plasmalemmal cellular structures by fluorescence microscopy. TIRFM features combine high temporal and spatial resolution and thus, in many applications, exceed the possibilities of confocal microscopy. TIRFM has been established as a versatile method to study vesicle traffic, fusion and docking as well as dynamics of proteins within PM-ER junctions. Increasing experimental evidence demonstrates a pivotal role of PM-ER junctional areas in a variety of cellular processes including lipid- and calcium homeostasis which crucially involve close apposition and physical contact between intracellular compartments like the ER and the PM. Microscopic characterization of properties and architecture of PM-ER junctions is currently limited to the use of fluorescent fusion proteins related to ER resident signaling molecules. These markers bear the potential to interfere with native junctional properties. Here we introduce a simple, novel TIRFM approach, which allows to study basolateral junctional areas of cells based on mapping the optical density of small cytosolic fluorophores within the evanescent field. As optical density of small diffusible fluorophores is reduced in junctional membrane appositions, TIRFM can be employed to visualize ER-PM junctions without overexpression of specific ER protein markers and this method enables unprecendeted insight into the dynamics of trans-compartimental signaling. We provide proof of concept by utilizing this new method for in-depth analysis of the role of PM-ER junctions in mast cell calcium signaling. Our results reveal a surprisingly dense, structurally well defined compartment that provides a dynamic platform for key signaling molecules.

Mutant IP3 receptors attenuate store-operated Ca2+ entry by destabilizing STIM-Orai interactions in Drosophila neurons.

ABSTRACTStore-operated Ca2+ entry (SOCE) occurs when loss of Ca2+ from the endoplasmic reticulum (ER) stimulates the Ca2+ sensor, STIM, to cluster and activate the plasma membrane Ca2+ channel Orai (encoded by Olf186-F in flies). Inositol 1,4,5-trisphosphate receptors (IP3Rs, which are encoded by a single gene in flies) are assumed to regulate SOCE solely by mediating ER Ca2+ release. We show that in Drosophila neurons, mutant IP3R attenuates SOCE evoked by depleting Ca2+ stores with thapsigargin. In normal neurons, store depletion caused STIM and the IP3R to accumulate near the plasma membrane, association of STIM with Orai, clustering of STIM and Orai at ER–plasma-membrane junctions and activation of SOCE. These responses were attenuated in neurons with mutant IP3Rs and were rescued by overexpression of STIM with Orai. We conclude that, after depletion of Ca2+ stores in Drosophila, translocation of the IP3R to ER–plasma-membrane junctions facilitates the coupling of STIM to Orai that leads to activation of SOCE.

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+ signaling1–4. 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 lead 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 signaling. 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.

Fast endocytic recycling determines TRPC1–STIM1 clustering in ER–PM junctions and plasma membrane function of the channel

Stromal interaction molecule 1 (STIM1) senses depletion of ER–Ca2+ store and clusters in ER–PM junctions where it associates with and gates Ca2+ influx channels, Orai1 and TRPC1. Clustering of TRPC1 with STIM1 and Orai1 in these junctions is critical since Orai1-mediated Ca2+ entry triggers surface expression of TRPC1 while STIM1 gates the channel. Thus, plasma membrane function of TRPC1 depends on the delivery of the channel to the sites where STIM1 puncta are formed. This study examines intracellular trafficking mechanism(s) that determine plasma membrane expression and function of TRPC1 in cells where Orai1 and TRPC1 are endogenously expressed and contribute to Ca2+ entry. We report that TRPC1 is internalized by Arf6-dependent pathway, sorted to Rab5-containing early endosomes, and trafficked to ER–PM junctions by Rab4-dependent fast recycling. Overexpression of Arf6, or Rab5, but not the respective dominant negative mutants, induced retention of TRPC1 in early endosomes and suppressed TRPC1 function. Notably, cells expressing Arf6 or Rab5 displayed an inwardly rectifying ICRAC current that is mediated by Orai1 instead of TRPC1-associated ISOC, demonstrating that Orai1 function was not altered. Importantly, expression of Rab4, but not STIM1, with Rab5 rescued surface expression and function of TRPC1, restoring generation of ISOC. Together, these data demonstrate that trafficking via fast recycling endosomes determines TRPC1–STIM1 clustering within ER–PM junctions following ER–Ca2+ store depletion which is critical for the surface expression and function of the channel. Ca2+ influx mediated by TRPC1 modifies Ca2+-dependent physiological response of cells.

Proteomic mapping of ER-PM junctions in living HEK293 cells

Proteomic mapping of ER-PM junctions in living HEK293 cells

Phosphatidylinositol 4,5-Bisphosphate Homeostasis Regulated by Nir2 and Nir3 Proteins at Endoplasmic Reticulum-Plasma Membrane Junctions

Phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) at the plasma membrane (PM) constitutively controls many cellular functions, and its hydrolysis via receptor stimulation governs cell signaling. The PI transfer protein Nir2 is essential for replenishing PM PIP2 following receptor-induced hydrolysis, but key mechanistic aspects of this process remain elusive. Here, we demonstrate that PI at the membrane of the endoplasmic reticulum (ER) is required for the rapid replenishment of PM PIP2 mediated by Nir2. Nir2 detects PIP2 hydrolysis and translocates to ER-PM junctions via binding to phosphatidic acid. With distinct phosphatidic acid binding abilities and PI transfer protein activities, Nir2 and its homolog Nir3 differentially regulate PIP2 homeostasis in cells during intense receptor stimulation and in the resting state, respectively. Our study reveals that Nir2 and Nir3 work in tandem to achieve different levels of feedback based on the consumption of PM PIP2 and function at ER-PM junctions to mediate nonvesicular lipid transport between the ER and the PM.

STIM2 regulates PKA-dependent phosphorylation and trafficking of AMPARs.

STIMs (STIM1 and STIM2 in mammals) are transmembrane proteins that reside in the endoplasmic reticulum (ER) and regulate store-operated Ca(2+) entry (SOCE). The function of STIMs in the brain is only beginning to be explored, and the relevance of SOCE in nerve cells is being debated. Here we identify STIM2 as a central organizer of excitatory synapses. STIM2, but not its paralogue STIM1, influences the formation of dendritic spines and shapes basal synaptic transmission in excitatory neurons. We further demonstrate that STIM2 is essential for cAMP/PKA-dependent phosphorylation of the AMPA receptor (AMPAR) subunit GluA1. cAMP triggers rapid migration of STIM2 to ER-plasma membrane (PM) contact sites, enhances recruitment of GluA1 to these ER-PM junctions, and promotes localization of STIM2 in dendritic spines. Both biochemical and imaging data suggest that STIM2 regulates GluA1 phosphorylation by coupling PKA to the AMPAR in a SOCE-independent manner. Consistent with a central role of STIM2 in regulating AMPAR phosphorylation, STIM2 promotes cAMP-dependent surface delivery of GluA1 through combined effects on exocytosis and endocytosis. Collectively our results point to a unique mechanism of synaptic plasticity driven by dynamic assembly of a STIM2 signaling complex at ER-PM contact sites.

Probing the Functional Coupling Interface between STIM1 and Orai1

Store operated Ca2+ entry (SOCE) plays a crucial physiological roles and involves two key proteins, STIM and Orai. Triggered by store-depletion, ER STIM proteins aggregate at ER-PM junctions and directly activate PM Orai Ca2+ channels. Few SOCE inhibitors have been described that specifically target the STIM/Orai machinery. Recently, we revealed a previously described SOCE modifier, DPB162-AE, potently and specifically blocks STIM1-induced Orai1 channel activation. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not affect SOAR-dimerization nor does it prevent the SOAR-Orai1 interaction. However, it potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores Orai-binding of this SOAR mutant but only slowly restores its activation of Orai1 channel-mediated Ca2+ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. In STIM2, the equivalent residue to Phe-394 is Leu-485 resulting in STIM2's lower efficacy for Orai channel activation. This difference may explain the unusual specificity of DPB162-AE in blocking the action of STIM1 but not STIM2. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.

Testing a Diffusion Trap Model for Store-Operated Calcium Entry by Single Particle Tracking

Depletion of calcium from the endoplasmic reticulum (ER) triggers autonomous assembly of STIM1-Orai1 complexes at ER-plasma membrane (PM) junctions, leading to store-operated calcium entry (SOCE). One hypothesis to explain this process is a diffusion trap in which activated STIM1 diffusing in the ER becomes trapped at junctions by binding to the PM via its C-terminal polybasic domain. STIM1 then traps Orai1 in the PM through binding of STIM1's CRAC activation domain. We tested this diffusion trap model by analyzing STIM1 and Orai1 diffusion using single-particle tracking, photoactivation of protein ensembles, and Monte Carlo simulations. In resting cells, STIM1 diffusion is Brownian with a mean diffusion coefficient (D) of 0.12 µm2/s, while Orai1 is slightly subdiffusive (mean D=0.09 µm2/s). After store depletion both proteins slow to the same speed (mean D≈0.03 µm2/s), consistent with complex formation, and are confined to an area similar in size to ER-PM junctions. At high STIM1:Orai1 expression ratios, the probability that Orai1 escapes the diffusion trap is >1%, but the escape probability is significantly increased by reducing the affinity of STIM1 for Orai1 or by expressing the two proteins at comparable levels. Our results provide direct evidence that STIM1 and Orai1 are trapped by their physical connections across the junctional gap. However, we also find that STIM-Orai interactions are surprisingly dynamic in that readily reversible binding reactions generate free STIM1 and Orai1 which can escape and exchange with extrajunctional pools. These data suggest that STIM1 and Orai1 interact with low affinity, creating the potential for SOCE modulation even when ER calcium stores are mostly depleted, and promoting the reversal of SOCE upon store refilling.

Dynamic regulation of ER‐PM junctions facilitates PI(4,5)P2 replenishment during receptor‐induced Ca2+ signaling (599.3)

ER‐PM junctions are membrane interfaces between the endoplasmic reticulum (ER) and the plasma membrane (PM), permitting direct molecular interactions and exchange of signals between the two subcellular compartments. ER‐PM junctions have been implicated as important subcellular loci for lipid metabolism and calcium signaling. However, the molecular mechanisms underlying the regulation and functions of ER‐PM junctions are not well understood. Using a genetically‐encoded marker and live‐cell imaging, we found that ER‐PM junctions are dynamically regulated during calcium signaling. Elevation of cytosolic calcium induces the translocation of the ER membrane protein extended synaptotagmin‐1 (E‐Syt1) to ER‐PM junctions and enhances ER‐to‐PM connection. E‐Syt1‐mediated enhanced ER‐to‐PM connection facilitates the recruitment of Nir2, a phosphatidylinositol (PI) transfer protein, to ER‐PM junctions during receptor‐induced calcium signaling. Nir2 promotes replenishment of PM PI(4,5)P2 following receptor‐induced hydrolysis. Disruption of the enhanced ER‐to‐PM connection or Nir2 expression resulted in reduced PM PI(4,5)P2 replenishment and defective calcium signaling. These results demonstrate that calcium‐mediated dynamic regulation of ER‐PM junctions is crucial for maintaining PI(4,5)P2 levels at the PM and sustaining calcium signaling.

Distinct Orai-Coupling Domains in Stim1 and Stim2 Define the Orai-Activating Site

The ER membrane-spanning STIM1 protein is a finely-tuned sensor of ER luminal Ca2+. Small changes in ER Ca2+ induce STIM1 to undergo an intricate self-triggering process, causing it to translocate into ER-PM junctions where it couples with and activates the highly Ca2+-selective family of Orai channels in the PM. The entering Ca2+ sustains Ca2+ oscillations, maintains Ca2+ homeostasis, and provides crucial long-term Ca2+ signals in many cell types which control gene expression and cellular growth. Similar in structure and also widely expressed among cells, the little-studied STIM2 protein is reported to differ subtly from STIM1 in its N-terminal domain, affecting luminal Ca2+-sensitivity and the rate of unfolding and self-activation. The STIM1 cytoplasmic C-terminus contains the STIM-Orai activating region (SOAR) which has been structurally resolved. While the corresponding SOAR sequence in STIM2 is highly conserved, we reveal it has a profoundly diminished interaction with and ability to gate Orai1 channels. We narrowed this distinction in Orai1 activation to a small sequence in SOAR, within which substitution of a single phenylalanine in STIM1 with leucine in STIM2 confers a severe decrease in Orai1 channel-gating efficacy. This residue is strategically positioned at the structural apex of the SOAR domain. Modification of this single residue within the intact STIM1 protein reveals its pivotal role in both interaction with and gating of the Orai1 channel. The results not only pinpoint a crucial locus of STIM-Orai coupling but also reveal a physiologically profound distinction between STIM1 and STIM2.

Feedback Regulation of Receptor-Induced Ca2+ Signaling Mediated by E-Syt1 and Nir2 at Endoplasmic Reticulum-Plasma Membrane Junctions

Endoplasmic reticulum (ER)-plasma membrane (PM) junctions are highly conserved subcellular structures. Despite their importance in Ca(2+) signaling and lipid trafficking, the molecular mechanisms underlying the regulation and functions of ER-PM junctions remain unclear. By developing a genetically encoded marker that selectively monitors ER-PM junctions, we found that the connection between ER and PM was dynamically regulated by Ca(2+) signaling. Elevation of cytosolic Ca(2+) triggered translocation of E-Syt1 to ER-PM junctions to enhance ER-to-PM connection. This subsequently facilitated the recruitment of Nir2, a phosphatidylinositol transfer protein (PITP), to ER-PM junctions following receptor stimulation. Nir2 promoted the replenishment of PM phosphatidylinositol 4,5-bisphosphate (PIP2) after receptor-induced hydrolysis via its PITP activity. Disruption of the enhanced ER-to-PM connection resulted in reduced PM PIP2 replenishment anddefective Ca(2+) signaling. Altogether, our results suggest a feedback mechanism that replenishes PM PIP2 during receptor-induced Ca(2+) signaling viathe Ca(2+) effector E-Syt1 and the PITP Nir2 at ER-PM junctions.

Interorganellar Membrane Microdomains: Dynamic Platforms in the Control of Calcium Signaling and Apoptosis

The dynamic interplay among intracellular organelles occurs at specific membrane tethering sites, where two organellar membranes come in close apposition but do not fuse. Such membrane microdomains allow for rapid and efficient interorganelle communication that contributes to the maintenance of cell physiology. Pathological conditions that interfere with the proper composition, number, and physical vicinity of the apposing membranes initiate a cascade of events resulting in cell death. Membrane contact sites have now been identified that tether the extensive network of the endoplasmic reticulum (ER) membranes with the mitochondria, the plasma membrane (PM), the Golgi and the endosomes/lysosomes. Thus far, the most extensively studied are the MAMs, or mitochondria associated ER membranes, and the ER-PM junctions that share functional properties and crosstalk to one another. Specific molecular components that define these microdomains have been shown to promote the interaction in trans between these intracellular compartments and the transfer or exchange of Ca2+ ions, lipids, and metabolic signaling molecules that determine the fate of the cell.

Septins Set the Stage for Orai1 to Bind STIM1 at ER-PM Junctions

The endoplasmic reticulum (ER) Ca(2+) sensor STIM1 recruits and activates the plasma membrane (PM) Ca(2+) channel Orai1 at ER-PM junctions for store-operated Ca(2+) entry (SOCE). Reporting in Nature, Sharma et al. (2013) showed that septins are necessary for Orai1 recruitment and SOCE, implicating these scaffolding proteins in signaling at ER-PM junctions.

The C‐ and N‐terminal STIM1 binding sites on Orai1 are required for both trapping and gating CRAC channels

Ca(2+) release-activated Ca(2+) (CRAC) channels are activated through a mechanism wherein depletion of intracellular calcium stores results in the aggregation of stromal interaction molecule 1 (STIM1), the endoplasmic reticulum (ER) Ca(2+) sensor, and Orai1, the CRAC channel protein, at overlapping sites in the ER and plasma membranes (PMs). The redistribution of CRAC channels is driven through direct STIM1-Orai1 binding, an important event that not only controls gating, but also regulates Orai1 ion selectivity. Orai1 harbours two STIM1 binding sites, one each on the intracellular C- and N-termini. Previous studies have proposed modular functions for these sites, with the C-terminal site thought to regulate STIM1-Orai1 binding and trapping of Orai1 at the ER-PM junctions, and the N-terminal site mediating gating. However, here we find that a variety of mutations in the N-terminal site impair the binding of Orai1 to STIM1 and to the soluble CRAC activation domain (CAD). Gating could be restored in several N- and C-terminal point mutants by directly tethering the minimal STIM1 activation domain (S) to Orai1 (Orai1-SS channels), indicating that loss of gating in these mutants by full-length STIM1 results from insufficient ligand binding. By contrast, gating could not be restored in mutant Orai1-SS channels carrying more drastic deletions that removed the STIM1 binding sites (1-85, 73-85, or 272-279 Orai1), suggesting that STIM1 binding to both sites is essential for channel activation. Moreover, analysis of ion selectivity indicated that the molecular requirements for gating and modulation of ion selectivity are similar, yet substantively different from those for Orai1 puncta formation, suggesting that ion selectivity and gating are mechanistically coupled in CRAC channels. Our results indicate that the C- and N-terminal STIM1 binding sites are both essential for multiple aspects of Orai1 function including STIM1-Orai1 association, Orai1 trapping, and channel activation.

Orai Channel Pore Properties and Gating by STIM: Implications from the Orai Crystal Structure

The Orai channels are unusual, yet prominent, calcium (Ca2+) signal mediators in most cell types. Orai proteins are structurally unique, having little sequence homology with other ion channels. They are also functionally unique with exceedingly high selectivity for Ca2+, mediating both short-term Ca2+ homeostasis and long-term Ca2+ signals important for transcriptional control. Operating in the plasma membrane (PM), Orai channel regulation is unprecedented among ion channels; channel gating occurs through an elaborate intermembrane coupling with stromal interaction molecule (STIM) proteins in the endoplasmic reticulum (ER). STIM proteins function as sensors of Ca2+ stored in the ER lumen and translocate into ER-PM junctions to tether and activate Orai channels when ER Ca2+ concentration decreases. Crystallization studies reveal an unexpected hexameric structure for the Orai channel and provide important insights into the pore architecture, the structural basis of its unusual cation selectivity, and how channel gating occurs through its coupling with STIM proteins.

Visualization of STIM1 and Orai1 by Electron Microscopy

STIM1 and Orai1 constitute the core machinery of store-operated calcium entry. We used transmission and freeze-fracture electron microscopy to visualize these proteins at endoplasmic reticulum (ER)-plasma membrane (PM) junctions. In thin sections, ER formed frequent extensive cortical junctions in HEK-293 cells overexpressing STIM1 and Orai1 and exposed to thapsigargin (TG), but small and rare junctions in control cells. Extended molecules, most likely STIM1, decorated the ER and bridged the ER-PM gap. Freeze-fracture replicas of cells treated in the same manner showed elevated PM domains (“puncta”) packed with particles at a higher density than on the surface of control cells (2564±443/µm2 versus 1863±331/µm2). Puncta varied in size (∼0.01 to 0.45 µm2) and frequency. Clusters of particles in puncta typically exhibited a clear sharp border, but particle density sometimes declined more gradually to background. Since the puncta most likely correspond to sites of STIM1-Orai1 co-clustering at ER-PM junctions, targeting to puncta was used to guide the classification of particles potentially representing Orai1 channels based upon structural parameters. A high percentage of particles in puncta exhibited a common signature; “large and tall” particles were present at a significantly higher density within puncta than in untransfected cells (average 1300±309 versus 225±84/µm2). Three characteristics of these particles are similar to the tetrameric CaV1.1 channels of skeletal muscle: 1) diameter, only slightly smaller than that of CaV1.1; 2) unusual height indicated by the platinum free “shadow”; 3) square, slightly scalloped outline of the same shadow in some images. It is reasonable to postulate that the “large and tall” particles represent tetrameric Orai1 channels clustered in correspondence with cortical Stim-bearing ER junctions. These observations introduce a new approach to visualize individual unlabeled Stim and Orai molecules in situ. Funded by NIH RO1 HL-48093 (CFA) and NS-14609 (MDC).

Junctate is a Ca 2 + -sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

SARAF Inactivates the Store Operated Calcium Entry Machinery to Prevent Excess Calcium Refilling

Store operated calcium entry (SOCE) is a principal cellular process by which cells regulate basal calcium, refill intracellular Ca(2+) stores, and execute a wide range of specialized activities. STIM and Orai proteins have been identified as the essential components enabling the reconstitution of Ca(2+) release-activated Ca(2+) (CRAC) channels that mediate SOCE. Here, we report the molecular identification of SARAF as a negative regulator of SOCE. Usingheterologous expression, RNAi-mediated silencing and site directed mutagenesis combined with electrophysiological, biochemical and imaging techniques we show that SARAF is an endoplasmic reticulum membrane resident protein that associates with STIM to facilitate slow Ca(2+)-dependent inactivation of SOCE. SARAF plays a key role in shaping cytosolic Ca(2+) signals and determining the content of the major intracellular Ca(2+) stores, a role that is likely to be important in protecting cells from Ca(2+) overfilling.