Junctate is a Ca²⁺-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Abstract

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca²⁺ release-activated Ca²⁺ (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca²⁺ 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 Ca²⁺-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca²⁺ 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 Ca²⁺-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.

Ca²⁺ influx via Ca²⁺ release-activated Ca²⁺ (CRAC) channels is crucial for activation of immune cells (1, 2). Recent studies have established stromal interaction molecule (STIM)1, a Ca²⁺-binding protein localized in the endoplasmic reticulum (ER), as a component of CRAC channels (3). STIM1 contains an EF-hand motif in its N terminus, a single transmembrane (TM) segment, and a long C-terminal cytoplasmic region. Upon ER Ca²⁺ depletion, STIM1 oligomerizes and translocates to plasma membrane (PM)-proximal regions to activate store-operated Ca²⁺ entry (SOCE) (3). Orai1 was identified as a pore subunit of the CRAC channels (1). A cytoplasmic CRAC activation domain/STIM1-Orai1 activating region (CAD/SOAR) fragment of STIM1 directly interacts with and activates Orai1 (1). Orai1 and STIM1 translocate into preexisting junctional areas between the PM and ER membrane upon store depletion (4, 5). Orai1 redistribution into these ER-PM junctions primarily depends on STIM1 (6, 7). Two separate mechanisms of STIM1 translocation are currently known. The C-terminal polybasic residues of STIM1 were shown to interact with PM phosphoinositides (8–10). A mutant of STIM1 lacking these polybasic residues shows a defect in accumulation at the ER-PM junctions (11, 12). This defect of STIM1 mutant can be rescued by coexpression of Orai1, identifying interaction with Orai1 as an alternate mechanism for STIM1 redistribution (11, 13).

Proteins localized to the junctions between the PM and ER/sarcoplasmic reticulum (SR) membranes are known in excitable cells (e.g., neurons and muscle cells) and form a structural foundation for the regulation of the intracellular Ca²⁺ stores and Ca²⁺ entry (3, 14). Proteins at triad/dyad junctions in muscle cells play an important role in coupling the voltage-gated Ca²⁺ channels on the PM to the ryanodine receptors (RyRs) on the SR membrane (3, 14). Various screening approaches have identified junctophilins, mitsugumins, sarcalumenin, junctin, and junctate as important components of these junctions (3, 15, 16). Junctate was identified as an alternative splice isoform of junctin and aspartyl β-hydroxylase (17–20). Whereas junctin is primarily expressed in muscle cells, junctate transcripts are ubiquitously detected (17, 18). Junctate contains several acidic residues in its ER-luminal domain that bind Ca²⁺ and forms a macromolecular complex with the inositol 1,4,5-trisphosphate receptor (InsP₃R) and transient receptor potential canonical (TrpC) channels (17, 21, 22). Furthermore, overexpression of junctate led to a moderate increase in Ca²⁺ stores and SOCE (21). However, the role of junctate in CRAC channel-mediated SOCE, as well as the function of its Ca²⁺-binding domain, remains unknown.

In this study, using affinity protein purification, we have identified junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, using reciprocal affinity purification, we validated Orai1-STIM1 complex as interactors of junctate. Alignment of the C-terminal sequence of junctate with that of EF-hand-containing proteins uncovered a possible EF-hand in junctate. Mutation of this EF-hand facilitated [Ca²⁺]_ER-independent clustering of STIM1 and a corresponding increase in cytoplasmic [Ca²⁺]. Finally, we show that coexpression of the EF-hand mutant of junctate facilitated clustering of a STIM mutant lacking the C-terminal polybasic residues, uncovering a distinct mechanism of STIM1 recruitment into the ER-PM junctions. Based on these results, we propose that junctate is a Ca²⁺-sensing structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact to mediate SOCE.

Results

Identification of Junctate as a Binding Partner of Orai1 and STIM1 Protein Complex.

In our previous studies, using immunoaffinity purification of Orai1 after store depletion, we identified a macromolecular complex containing Orai1, STIM1, and another EF-hand-containing protein, CRAC channel regulator (CRACR)2A (23). CRACR2A was validated as an interactor of Orai1 and STIM1, forming a ternary complex depending on the intracellular Ca²⁺ concentration ([Ca²⁺]_i). From the results of mass spectrometry, in addition to CRACR2A, we identified 21 peptides derived from junctate together with a similar number of peptides of STIM1 (Fig. S1A). To confirm these results, we performed reciprocal large-scale affinity purification to identify interactors of junctate. The results from mass spectrometry showed 5 peptides from Orai1 and 17 peptides from STIM1 (Fig. 1A and Fig. S1A). These results from two independent, unbiased affinity protein purifications strongly suggested junctate as an interacting partner of Orai1-STIM1 complex. The data obtained from mass spectrometry were further validated by immunoprecipitation experiments in HeLa cells expressing FLAG-Orai1, STIM1, and Myc-tagged junctate. FLAG-Orai1 was immunoprecipitated and blotted for detection of STIM1 and junctate. As seen in Fig. 1B, STIM1 and junctate both coimmunoprecipitated with Orai1 upon store depletion. Reciprocal immunoprecipitation of His₆-tagged junctate also resulted in detection of Orai1 and STIM1, and their interactions were enhanced after store depletion (Fig. 1C).

Fig. 1.

Identification of junctate as a binding partner of Orai1 and STIM1. (A) Affinity purification of junctate protein complex. HeLa cells stably expressing Orai1, STIM1 (left lane, HeLa) and FLAG-tagged junctate (right lane, Junct) were treated with thapsigargin and DSP-crosslinked for affinity purification. The eluates were separated on a reducing SDS/PAGE and visualized by silver staining. Asterisk (*) indicates protein bands excised for identity by mass spectrometry. The arrowheads denote protein bands identified as junctate, its multimeric forms, or degradation products. N.S., nonspecific bands.(B) Junctate coimmunoprecipitates with Orai1-STIM1 complex after store depletion. HeLa cells expressing FLAG-tagged Orai1, STIM1, and Myc-His₆-tagged junctate were used as indicated. Anti-FLAG (Orai1) immunoprecipitates were immunoblotted for detection of STIM1, junctate (anti-Myc), and Orai1 (anti-FLAG). (C) Reciprocal immunoprecipitation. HeLa cells expressing Orai1-FLAG, STIM1, and Myc–His₆–junctate were used as indicated. Anti-His₆ (junctate) immunoprecipitates were immunoblotted for detection of junctate, STIM1, and Orai1. In B and C, cells were left untreated or treated with 1 μM thapsigargin (TG). (D) Schematic of junctate. Human junctate contains 299 amino acids with a cytoplasmic N terminus, TM, and negatively charged C terminus in the ER lumen. The truncated mutants used in this study are indicated. (E) C-terminal domain of junctate interacts with STIM1. Pull-down analysis using GST–junctate fragments expressed in bacteria and full-length FLAG-STIM1 expressed in insect cells. (Lower) Ponceau S staining of the same blot. (F) Mutation in the EF-hand motif of junctate decreases Ca²⁺ binding. ⁴⁵Ca²⁺ overlay experiments were performed with indicated amounts (in micrograms) of purified GST-fused WT junctate and junctate^EFmut. (Upper) Ponceau S staining of the same blot. (G) Schematic showing the functional domains of junctate including its STIM1-interaction region.

Identification of a Ca²⁺-Binding EF-Hand Domain in the ER Luminal Side of Junctate.

To examine interaction between junctate and Orai/STIM proteins, we first validated the intracellular topology of junctate using fluorescence protease protection assays (24). As reported previously, our data showed that junctate contains a short cytoplasmic N terminus, single TM segment, and a long C-terminal ER-luminal region enriched in negatively charged residues (Fig. S1 B and C) (21). Next, we checked protein interaction of junctate with Orai1 and STIM1. In GST pull-down analysis, we did not observe a significant interaction between junctate and Orai1 (Fig. S2A). However, full-length STIM1 bound to the recombinant junctate protein (Fig. 1 D and E). Further binding experiments with truncation mutants of junctate showed that STIM1 interacted with the ER-luminal region of junctate between residues 71–236 (Fig. 1E and Fig. S2B). Reciprocal binding experiments using recombinant full-length junctate and STIM1 fragments showed binding of junctate to the N terminus of STIM1 (Fig. S2 C and D). This direct interaction of junctate with STIM1 was further validated by immunoprecipitation experiments without coexpression of Orai1 (Fig. S2E). Together, these studies indicate that a direct interaction between the ER-luminal regions of junctate and STIM1 contributes to coimmunoprecipitation of junctate with the Orai1-STIM1 complex.

The ER-luminal C terminus of junctate is enriched in negatively charged residues and binds Ca²⁺ with a K_D of 217 ± 20 μM (17). To delineate the boundaries of the Ca²⁺-binding domain of junctate, we measured Ca²⁺ binding of the truncated forms of junctate. As reported previously (17), full-length junctate showed strong Ca²⁺ binding (Fig. S3A). Whereas deletion of the N terminus did not influence Ca²⁺ binding, deletion of the C-terminal 223 amino acids completely abolished Ca²⁺ binding, suggesting that the critical domain exists between amino acid 77–176. Careful sequence analysis of this region accompanied by structural modeling uncovered a putative EF-hand motif between residues 77 and 88 (N-⁷⁷DADGDGDFDVDD⁸⁸-C) that is highly conserved with the known signature of the EF-hand motifs (N-Dx[D/N]x[D/N]GxIxx[E/D][E/D]-C; http://structbio.vanderbilt.edu/cabp_database, http://pfam.sanger.ac.uk/family/PF00036) and is also similar to the EF-hand sequence of STIM1 (Fig. S3B). To determine the role of this putative EF-hand, we generated a junctate mutant (junctate^EFmut) with substitutions of ⁷⁷DAD⁷⁹ to ⁷⁷AAA⁷⁹. These two aspartic acids were predicted to be important for Ca²⁺ binding based on a model generated from the structure of parvalbumin, a classic EF-hand motif-containing protein (Fig. S3C) (25). In ⁴⁵Ca²⁺ overlay experiments, junctate^EFmut showed a dramatic decrease in Ca²⁺ binding, suggesting an important role of the EF-hand motif in Ca²⁺ binding (Fig. 1F). Previously, it was shown that 21 Ca²⁺ ions bind to a single molecule of junctate (17). Considering that only a single Ca²⁺ ion binds to an EF-hand motif, it is possible that binding of Ca²⁺ to the EF-hand domain influences Ca²⁺ binding of other residues. In summary, domain studies revealed that junctate contains a Ca²⁺-sensing EF-hand motif and a STIM1-interacting region in its ER-luminal C terminus (Fig. 1G).

Expression of Junctate^EFmut Increases the Cytoplasmic Ca²⁺ Concentration in T cells Independently of Store Depletion.

The role of junctate in SOCE in HEK293 cells has been determined using overexpression and siRNA-mediated depletion (21). However, its function in regulation of CRAC channel activity has not been tested, particularly with siRNA that specifically depletes junctate and not other alternatively spliced isoforms including junctin and aspartyl β-hydroxylase. To specifically deplete junctate, we designed siRNA targeting the unique 3′-untranslated region (UTR) sequence of junctate mRNA. Junctate depletion resulted in a significant decrease in SOCE in Jurkat T cells (Fig. S4 A and B). The reduction in SOCE was specific to reduced junctate levels, because this defect was recovered by ectopic expression of junctate (Fig. S4A). Furthermore, decreased SOCE attributable to depletion of junctate also reduced the transcriptional activity of nuclear factor of activated T cells (NFAT), using a NFAT-dependent luciferase reporter in Jurkat T cells (Fig. S4C).

To examine whether junctate affects CRAC channel activity in T cells and to investigate a functional role of its EF-hand, we measured SOCE in primary CD4⁺ T cells stably expressing WT junctate or junctate^EFmut. Expression of WT junctate or junctate^EFmut in T cells resulted in a marginal, but significant increase in SOCE compared with empty vector-transduced cells (Fig. 2A). Interestingly, the resting [Ca²⁺]_i levels were high in T cells expressing junctate^EFmut (Fig. 2A, initial 150 s). We further examined this observation by measuring store-independent Ca²⁺ entry. These experiments were performed by sequentially exposing the cells to external solution with or without 2 mM Ca²⁺ in the absence of store depletion (Fig. 2B). Again, we observed significantly increased [Ca²⁺]_i levels in cells expressing junctate^EFmut. This increased store-independent Ca²⁺ entry could be blocked by 1 μM Gd³⁺ and coexpression of a dominant-negative mutant of Orai1, Orai1^E106Q, suggesting that Ca²⁺ entry induced by junctate^EFmut was mediated by Orai proteins (26, 27) (Fig. 2B). A STIM1 mutant (STIM1^EFmut) harboring mutations in its EF-hand domain (Fig. S3B) was used as a positive control for store-independent CRAC channel activity (28, 29). Expression of STIM1^EFmut in T cells induced store-independent Ca²⁺ entry that was suppressed by 1 μM Gd³⁺ and coexpression of Orai1^E106Q (Fig. 2C). These data suggested that junctate^EFmut induces an increase in cytoplasmic [Ca²⁺] via activation of endogenous CRAC channels, albeit to a lesser extent than STIM1^EFmut (summarized in Fig. 2C, bar graph). Together with results obtained from knockdown experiments, these data demonstrated that junctate plays an important role in CRAC channel activity in T cells and its EF-hand domain is important for sensing [Ca²⁺]_ER.

Fig. 2.

Mutation in the EF-hand domain of junctate increases intracellular Ca²⁺ concentrations in T cells. (A) SOCE measurements in primary CD4⁺ T cells retrovirally expressing WT junctate or junctate^EFmut. Intracellular stores were depleted with thapsigargin (TG) (1 μM), and SOCE was measured by perfusion with 2 mM Ca²⁺-containing solution. Traces show averaged (± SEM) responses from T cells: vector (n = 55 cells), junctate (n = 59), or junctate^EFmut (n = 60). Bar graph shows peak SOCE ± SEM from three independent experiments with 50–70 cells in each experiment. P values are indicated. (B) Effect of junctate EF-hand mutant on store-independent Ca²⁺ entry. The levels of [Ca²⁺]_i were measured in primary T cells by exchanging Ca²⁺-free extracellular solution with 2 mM Ca²⁺-containing solution. Each trace shows averaged (± SEM) responses from 55 (vector), 60 (junctate), 67 (junctate^EFmut), 58 (junctate^EFmut with Gd³⁺) and 52 (junctate^EFmut + Orai1^E106Q) cells. When indicated, 1 μM Gd³⁺ was added. (C) Effect of STIM1 EF-hand mutant on store-independent Ca²⁺ entry. Each trace shows averaged (± SEM) responses from 46 (STIM1^EFmut), 51 (STIM1^EFmut with Gd³⁺) and 52 (STIM1^EFmut + Orai1^E106Q) cells. Bar graphs show averaged Ca²⁺ ± SEM without store depletion from three independent experiments with 40–70 cells in each experiment. *P < 0.005; **P < 0.05.

Junctate^EFmut Induces Localization of STIM1 in Clusters Independently of Store Depletion.

It is possible that expression of junctate^EFmut induces activation of STIM1 and Orai1 indirectly by altering [Ca²⁺]_ER. To examine this possibility, we measured [Ca²⁺]_ER after treating the cells with ionomycin. Consistent with previous observations (21), [Ca²⁺]_ER was increased in cells overexpressing junctate (Fig. S5). However, we did not detect any difference in [Ca²⁺]_ER between cells expressing junctate and junctate^EFmut, suggesting that activation of CRAC channels by junctate^EFmut was not mediated by a change in [Ca²⁺]_ER. We then examined whether the Ca²⁺-unbound form of junctate (attributable to mutations in the EF-hand) can induce local clustering of STIM1 at the ER-PM junctions by visualizing its subcellular localization in Jurkat T cells. In cells coexpressing WT junctate and STIM1, STIM1 showed an uniform distribution in the ER membrane (Fig. 3A) and clustered into the ER-PM junctions upon store depletion, as seen in epifluorescence and total internal reflection fluorescence (TIRF) images (Fig. 3A, arrows). WT junctate showed a predominant ER membrane localization in epifluorescence images, as well as accumulation at the ER-PM junctions in TIRF images (Fig. 3A). Upon store depletion, junctate showed a mild change in its localization, whereas STIM1 showed a pronounced accumulation at the ER-PM junctions, where it colocalized with junctate. Interestingly, in cells coexpressing junctate^EFmut, STIM1 showed clustering at the ER-PM junctions even without any store depletion (Fig. 3B, arrows). Similar observations were made with STIM1^EFmut, which showed constitutive localization at the ER-PM junctions (Fig. 3B, graph) (28, 29).

Fig. 3.

Junctate EF-hand mutant induces clustering of STIM1 at the ER-PM junctions in T cells. (A) Epifluorescence and TIRF images of Jurkat T cells coexpressing STIM1–mCherry and junctate–GFP. The graph on the right represents time course of averaged normalized fluorescence intensity ± SEM for STIM1–mCherry (blue trace) and junctate–GFP (black trace) from 23 cells. (Scale bar: 5 μm.) (B) Epifluorescence and TIRF images of Jurkat T cells expressing STIM1–mCherry and junctate^EFmut–GFP. The graph on the right represents time course of averaged normalized fluorescence intensity ± SEM for cells expressing STIM1–mCherry with WT junctate (black trace; same as that in A), STIM1–mCherry and junctate^EFmut–GFP (blue trace, 32 cells), and STIM1^EFmut (green trace, 23 cells). (Scale bar: 5 μm.) In A and B, ER Ca²⁺ was depleted with 1 μM thapsigargin at the initial time point (t = 20 s). Arrows depict sites of clustering where STIM1 and Junctate^EFmut accumulate. (C) Truncations of the N or C terminus of junctate^EFmut abolishes constitutive STIM1 clustering. Epifluorescence and TIRF images of Jurkat T cells expressing STIM1–mCherry with ΔC123-junctate^EFmut–GFP or ΔN-junctate^EFmut–GFP. The bar graph on the right summarizes the number of cells showing STIM1 clusters without store depletion. Numbers at the top of each bar represent the number of cells showing STIM1 in clusters/total number of cells examined. (Scale bar: 5 μm.)

To examine whether the C-terminal ER-luminal region of junctate was important for constitutive clustering of STIM, we generated truncation mutants of junctate^EFmut. Truncation of the C-terminal 123 amino acids of junctate^EFmut (ΔC123^EFmut) did not influence its localization in the ER membrane (Fig. 3C, Upper, epifluorescence images) and at the ER-PM junctions (Fig. 3C, TIRF images; and Fig. S6A). However, this truncation abolished store-independent STIM1 clustering at the ER-PM junctions (Fig. 3C, TIRF images and graph). These results suggested that the STIM1 interaction domain between amino acid 71–236 is important for store-independent STIM1 recruitment by junctate^EFmut. We also examined the localization of the N-terminally truncated junctate^EFmut (ΔN^EFmut). The truncation of the cytoplasmic, N-terminal 26 amino acids did not change the localization of junctate on the ER membrane (Fig. 3C, Lower, epifluorescence images). However, ΔN^EFmut did not properly localize at the ER-PM junctions, and, therefore, it did not induce store-independent STIM1 clustering (Fig. 3C and Fig. S6A). The cytoplasmic N terminus of junctate was identified as a protein interaction domain for the InsP₃R and has been shown to play an important role in SOCE (21, 22). It is possible that junctate localization at the ER-PM junctions depends on interaction with other proteins via its N terminus in T cells. To examine the effect of N- or C-terminally truncated mutants of junctate^EFmut on store-independent Ca²⁺ entry, we performed Ca²⁺ measurements in T cells overexpressing either the ΔN^EFmut or the ΔC123^EFmut. In agreement with the TIRF data, expression of either of these mutants had a very mild effect on the levels of store-independent or store-dependent Ca²⁺ entry (Fig. S6 B–E). Immunoprecipitation experiments showed a strong interaction of junctate^EFmut with STIM1 in the absence of store depletion (Fig. S7A). Furthermore, area analysis of TIRF images did not show any significant difference between the junctional areas in cells expressing WT junctate and junctate^EFmut (Fig. S7 B and C). These data suggest that store-independent STIM1 recruitment and Ca²⁺ entry by junctate^EFmut are directly mediated by protein interactions. In summary, our results suggest that the EF-hand of junctate acts as an ER-luminal Ca²⁺ sensor that facilitates STIM1 clustering at the ER-PM junctions upon store depletion. In addition, the protein interaction domains of junctate play an important role in its localization at the ER-PM junctions (N terminus) and interaction with STIM1 (C terminus).

Junctate Recruits STIM1 into the ER-PM Junctions in a Manner Independent of Phosphoinositide and Orai1 Binding.

Previous studies have identified two independent mechanisms important for STIM1 accumulation at the ER-PM junctions, including binding of STIM1 to PM phosphoinositides and interaction with Orai1 (8–11, 13). We hypothesized that junctate binding may provide an alternative mechanism of STIM1 accumulation. As reported previously (8–10), STIM1-ΔK failed to form clusters at ER-PM junctions in Jurkat T cells and HEK293 cells because of a loss of phosphoinositide binding (Fig. 4A and Fig. S8A). Coexpression of Orai1 induced clustering of STIM1-ΔK after store depletion, which was consistent with the previous observations (Fig. 4A and Fig. S8A) (11). Interestingly, coexpression of junctate^EFmut induced clustering of STIM1-ΔK at ER-PM junctions even without store depletion, in both Jurkat and HEK293 cells (Fig. 4B and Fig. S8B). We further validated that recruitment of STIM1-ΔK into the junctions by junctate^EFmut was independent of Orai1 because it was not influenced by depletion of endogenous Orai1 (Fig. S9). These results identify a third mechanism of STIM1 recruitment into ER-PM junctions, by interaction with Junctate (summarized in Fig. 4C).

Fig. 4.

Junctate mediates STIM1 recruitment into ER-PM junctions in a manner independent of Orai1 and phosphoinositide interaction. (A) Representative TIRF images of Jurkat T cells expressing STIM1-YFP and STIM1-ΔK-YFP (Left). ER Ca²⁺ was depleted with 1 μM of thapsigargin where indicated. Images at right show TIRF images of Jurkat T cells expressing STIM1-ΔK and Orai1–mCherry under resting conditions (Upper) and after store depletion (Lower). (Scale bar: 5 μm.) (B) Representative TIRF images of Jurkat T cells expressing STIM1-ΔK-YFP and junctate^EFmut–mCherry under resting conditions. (Scale bar: 5 μm.) (C) Schematic depicting the domains of STIM1 involved in its recruitment into the ER-PM junctions.

Improper regulation of CRAC channel activity by expression of the STIM1 EF-hand mutant induces cell death (28). Because overexpression of the junctate EF-hand mutant in Jurkat T cells induced high resting [Ca²⁺]_i, we determined its physiological consequences in primary CD4⁺ T cells. Consistent with the increased [Ca²⁺]_i, expression of junctate^EFmut induced higher levels of cell death at 48 and 96 h poststimulation compared with control cells or those expressing WT junctate (Fig. S10). Therefore, high resting [Ca²⁺]_i levels correlated with a proportional increase in cell death in primary CD4⁺ T cells overexpressing junctate^EFmut.

Discussion

Our study has identified junctate as a structural component of junctional regions between the PM and ER membrane in T cells, where Orai1 and STIM1 cluster and interact. The interaction between junctate and Orai1-STIM1 complex was detected from two unbiased affinity protein purifications using Orai1 and junctate as bait, respectively (Fig. 1). Their functional interactions were further validated by checking the levels of store-independent Ca²⁺ entry and their colocalization at the ER-PM junctions (Figs. 2 and 3). Whereas Orai1 and STIM1 show major rearrangement and accumulation into the junctions upon store depletion, localization of junctate did not change dramatically, suggesting that junctate acts as a structural platform to recruit Orai1 and STIM1 (Fig. 3). In physiological conditions where the amounts of Orai1 and STIM1 proteins are limited, the recruitment of STIM1 into specific loci at the ER-PM junctions can be important for local concentration of Orai1 and STIM1 or for polarized Ca²⁺ entry observed in various cell types including T cells (e.g., the immunological synapse) (30–32).

In the current studies, a Ca²⁺-sensing role of junctate was demonstrated after uncovering an EF-hand domain in its C terminus. Mutations of the EF-hand of junctate increased the probability of STIM1 clustering into the PM-proximal area, activation of CRAC channels without store depletion, and cell death (Figs. 2 and 3 and Fig. S10). Although the junctate EF-hand mutant induced store-independent Ca²⁺ entry, it is doubtful that junctate directly activates Orai1 gating because junctate did not show a significant interaction with Orai1 (Fig. S2A), and the level of store-independent Ca²⁺ entry induced by the junctate mutant was significantly less than that of STIM1 mutant (Fig. 2). Therefore, currently, the most probable hypothesis is that junctate EF-hand mutant facilitates STIM1 clustering at the ER-PM junctions and increases the chance of Orai1-STIM1 interaction.

The N terminus of junctate comprises of ∼26 amino acids. A truncation of these residues prevents junctional accumulation of junctate (Fig. 3C). Electron microscopy (EM) and chemical-bridging studies have shown a gap of 10–25 nm with variations at the contact sites of ER-PM junctions (4, 5). One of the recent EM studies in HeLa cells suggested that the minimum distance between the ER and PM at junctions varies between 1.3 and 14.7 nm (average, 8.3 nm) (33). Thus, it is still possible that junctate itself may interact with the PM, assuming it extends at 0.29 nm per residue, similar to that of a collagen triplex, to generate a 7.54-nm-long N-terminal fragment that can encompass the junctional gap (34). However, junctate N terminus lacks obvious predicted lipid-interacting motifs, suggesting its interaction with other lipid-binding proteins at the ER-PM junctions.

STIM1 contains a polybasic stretch of amino acids in its C terminus that bind phosphoinositides, and is important for its clustering at ER-PM junctions (9, 10, 12). Truncation of this polybasic domain abolished STIM1 accumulation at ER-PM junctions, and overexpression of Orai1 recovered accumulation of STIM1-ΔK mutant into the ER-PM junctions (11, 12). Our data showed that overexpression of the junctate EF-hand mutant can also induce accumulation of STIM1-ΔK at the ER-PM junctions (Fig. 4 and Fig. S8). These results suggest that there are multiple pathways of STIM1 accumulation; (i) a pathway mediated by direct phosphoinositide binding of STIM1; (ii) an Orai1-dependent mechanism mediated by direct interaction, and (iii) a junctate-dependent mechanism mediated by protein interaction between STIM1 and junctate (Fig. 4C). In an overexpression system of Orai1, STIM1, and junctate, these mechanisms are likely to be redundant. In a physiological condition when the concentration of Orai1 and STIM1 is low, multiple mechanisms of STIM1 accumulation can be important for efficient and timely assembly of active CRAC channel protein complex. Because phosphoinositide binding is an intrinsic property of STIM1, it should play a predominant role in its accumulation. However, depending on composition of the PM phosphoinositides, or abundance of Orai1 and junctate in specific cell types, it is possible that binding to Orai or junctate could be important. This can account for the moderate decrease in SOCE observed after depletion of junctate in Jurkat T cells (Fig. S4).

Based on these observations, we propose a multiple Ca²⁺ sensor model to account for the role of junctate in CRAC channel regulation (Fig. S11). In this model, when ER Ca²⁺ store is filled, junctate exists at the ER-PM junctions in a Ca²⁺-bound form, possibly in a protein complex with other lipid-binding proteins. Ca²⁺-bound STIM1 is widely distributed in the ER membrane. Upon store depletion, Ca²⁺-unbound STIM1 clusters, translocates into the junctions, and recruits Orai1. For accumulation of STIM1 at the ER-PM junctions, direct binding to PM phosphoinositides plays an important role (8–10, 12). Protein interaction between STIM1 and Orai1 ensure proper, reciprocal accumulation of both the proteins at the ER-PM junctions. Junctate plays an additional role in STIM1 recruitment by defining the ER-PM junctions for clustering of Orai1 and STIM in a Ca²⁺-dependent manner. In summary, we propose that CRAC channel activity in T cells is regulated by multiple Ca²⁺-sensing proteins to detect the ER Ca²⁺ levels (e.g., STIM1 and junctate) and the cytoplasmic Ca²⁺ levels (e.g., calmodulin and CRACR2A) (23, 35).

Materials and Methods

Plasmids and Cells.

Full-length cDNAs of Orai1, junctate, and STIM1 were subcloned into pMSCV-CITE-eGFP-PGK-Puro, pFastBac1, pN1–mCherry and pEYFPN1 vectors using primers described in Table S1. Cell lines were obtained from American Type Culture Collection. See SI Materials and Methods for details.

Immunoprecipitation and GST Pull-Down Analysis.

Transfected HEK293 or HeLa cells were harvested, dithiobis succinimidyl propionate (DSP)-crosslinked, and lysed, and the lysates were immunoprecipitated with anti-FLAG or anti-His₆ antibody. For pull-down analysis, full-length junctate or its fragments, and lysates from Sf9 cells expressing STIM1 were incubated with glutathione Sepharose 4B beads in binding buffer. After extensive washing, proteins bound to the beads were analyzed by immunoblotting. See SI Materials and Methods for a detailed description.

Single-Cell Ca²⁺ Imaging and TIRF Microscopy.

T Cells were loaded with 1 μM Fura 2-AM and attached to poly-d-lysine-coated coverslips. For each experiment, 50–80 individual T cells were analyzed. TIRF microscopy (TIRFM) was performed using an Olympus IX2 illumination system as described previously (23). Acquisition and image analysis were performed using Slidebook (Intelligent Imaging Innovations). See SI Materials and Methods for details.