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EMBO J. Aug 1, 2012; 31(15): 3282–3296.
Published online Jul 13, 2012. doi:  10.1038/emboj.2012.189
PMCID: PMC3411083

BiP-mediated closing of the Sec61 channel limits Ca2+ leakage from the ER

Abstract

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic protein-conducting channel, the Sec61 complex. Previous work has characterized the Sec61 channel as a potential ER Ca2+ leak channel and identified calmodulin as limiting Ca2+ leakage in a Ca2+-dependent manner by binding to an IQ motif in the cytosolic aminoterminus of Sec61α. Here, we manipulated the concentration of the ER lumenal chaperone BiP in cells in different ways and used live cell Ca2+ imaging to monitor the effects of reduced levels of BiP on ER Ca2+ leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca2+ leakage via the Sec61 complex. When we replaced wild-type Sec61α with mutant Sec61αY344H in the same model cell, however, Ca2+ leakage from the ER increased and was no longer affected by manipulation of the BiP concentration. Thus, BiP limits ER Ca2+ leakage through the Sec61 complex by binding to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344.

Keywords: BiP, calcium homeostasis, diabetes mutation, endoplasmic reticulum, ER calcium leakage, Sec61 complex gating

Introduction

In mammalian cells, the endoplasmic reticulum (ER) represents a major storage compartment for calcium ions (Ca2+) as well as the major site of synthesis of soluble and membrane proteins of various cell organelles (Blobel and Dobberstein, 1975). The latter function involves an aqueous polypeptide-conducting pore in the ER membrane that is large enough to accommodate a polypeptide chain in transit. In contrast, the function as reservoir for receptor-controlled Ca2+ release requires the ER membrane to be more or less impermeable to Ca2+. Otherwise, the Ca2+ gradient between ER lumen and cytosol could not be maintained and Ca2+ could not excert its central role as a second messenger in cellular signalling (Crowley et al, 1994; Berridge, 2002; Rizzuto and Pozzan, 2006). However, the Ca2+ impermeability of the ER membrane is not absolute (Camello et al, 2002). It is one of the functions of the sarcoplasmic ER calcium ATPase (SERCA) to counteract the so-called passive Ca2+ efflux (also termed, Ca2+ leakage) from the ER in order to maintain the Ca2+ gradient (Wuytack et al, 2002).

Many proteins of the ER, ER-Golgi intermediate compartment (ERGIC), Golgi, endosomes, lysosomes, plasma membrane, nucleus, peroxisomes, and the extracellular space are integrated into the ER membrane or transported to the ER lumen by a protein translocase with the heterotrimeric Sec61 complex acting as a dynamic polypeptide-conducting channel (Görlich and Rapoport, 1993; Hartmann et al, 1994). Pore diameters of the Sec61 channel have been found to range from 9–15 Å for the closed to 40–60 Å for the open mammalian Sec61 complex (Hamman et al, 1997; Wirth et al, 2003). After completion of protein translocation, that is, when the Sec61 complex is still ribosome bound but free of a translocating polypeptide chain, or in the ribosome-free state, the Sec61 complex can transiently allow Ca2+ flux (Flourakis et al, 2006; Erdmann et al, 2011; Lang et al, 2011a). Therefore, gating of the Sec61 channel is tightly regulated. Signal sequences of precursor polypeptides destined for the ER contribute to Sec61 channel gating, that is, to opening of the Sec61 complex for partial or full passage of a precursor polypeptide (Kim et al, 2002). Furthermore, ER lumenal BiP and cytosolic Ca2+–calmodulin (CaM) facilitate Sec61 channel gating from the open to the closed state. According to in-vitro experiments, BiP can play a role in opening the Sec61 complex for polypeptide passage together with its nucleotide exchange factor Grp170 (Dierks et al, 1996) and is involved in Sec61 channel closure before and early in translocation together with a hitherto unidentified Hsp40-type co-chaperone (Hamman et al, 1998; Alder et al, 2005). Ca2+–CaM binds to an IQ motif present in the cytosolic aminoterminus of the α-subunit of the heterotrimeric Sec61 complex and limits Ca2+ leakage from the ER by closing the Sec61 channel in a Ca2+-dependent manner. Because the ER is not a major Ca2+ storage organelle in lower eukaryotes, such as yeast, the latter two gating mechanisms may not be relevant to all eukaryotes (Harsman et al, 2011).

We asked if the proposed role of BiP in channel closure can be demonstrated at the cellular level and whether the mechanism can be further elucidated. To do so, we manipulated the concentration of available BiP in human cells by different means, such as BIP gene silencing or induction of protein misfolding in the ER, and used live cell Ca2+ imaging to monitor the effects of reduced levels of BiP on ER Ca2+ leakage. Regardless of how the BiP concentration was lowered, the absence of available BiP led to increased Ca2+ leakage from the ER via the Sec61 complex. Recent work on a mouse model for diabetes has indicated that a point mutation in the ER lumenal loop 7 of murine Sec61α leads to a partially deficient Sec61 complex and to β-cell death as well as diabetes (Lloyd et al, 2010). When we replaced wild-type Sec61α with the respective mutant Sec61αY344H in human cells, Ca2+ leakage from the ER was increased and this ER Ca2+ leakage was no longer affected by manipulation of the BiP concentration. The results suggest that BiP limits ER Ca2+ leakage by gating the Sec61 complex via binding to loop 7 of Sec61α. This interpretation is further substantiated by the observations that the Y344H mutation also affects opening of the Sec61 channel for BiP-dependent translocation of certain precursor polypeptides and loop 7 interaction of BiP.

Results

BIP silencing in HeLa cells is tolerable for 96 h

To set the stage for the subsequent experiments, we treated HeLa cells for up to 96 h with one of two different siRNAs that target the coding (BIP siRNA) and the untranslated region (BIP-UTR siRNA) of the BIP mRNA. The maximum silencing effect was seen 72 h after the first transfection (Figure 1A and B). After 48 h, the total number of BIP-silenced cells was similar as that of control cells, and the silencing efficiency was between 60 and 75% (Figure 1A and B). Up to this time point, the BIP-silenced cells also showed no dramatic effect of siRNA on cell division, that is, compared with control siRNA-treated cells (Figure 1C and D). Next, we characterized the morphology of the ER in the silenced cells using 3D-structured illumination microscopy. HeLa cells were treated with control-, BIP-, or BIP-UTR siRNA for up to 96 h and then subjected to immunofluorescence 3D-structured illumination microscopy for an established ER marker protein. Independent of whether the cells were treated with control- or one of the BIP siRNAs, they showed typical ER morphology up to 96 h (Supplementary Figure S1). Thus, these experiments defined an experimental window of around 48 h after first transfection with BIP siRNA that allows functional analysis of the BIP gene product.

Figure 1
Effect of BIP gene silencing on cell proliferation. HeLa cells were cultured in DMEM-medium in 6-cm culture dishes and transfected with BIP siRNA, BIP-UTR siRNA, or control siRNA at a final concentration of 35 nM as indicated. (A, B) Silencing was evaluated ...

BIP silencing stimulates calcium leakage from the ER in intact cells

According to available in-vitro experiments, we expected BiP to contribute to limiting Sec61-mediated Ca2+ efflux from the ER at the cellular level (Haigh and Johnson, 2002). Therefore, we investigated whether silencing the BIP gene in HeLa cells with two different siRNAs enhanced Ca2+ efflux from the ER. Using the Ca2+ indicator Fura-2 in the absence of extracellular Ca2+ aids in visualization of the leakage of Ca2+ from the ER as increased cytosolic calcium concentration in intact cells in response to the irreversible SERCA inhibitor thapsigargin. In Ca2+ imaging experiments, we treated HeLa cells with one of the two BIP siRNAs for 48 h and, subsequently, Ca2+ leakage was unmasked by application of thapsigargin in the presence of EGTA. A third batch of cells was treated with a negative-control siRNA. In contrast to the control siRNA, the two BIP siRNAs had similar enhancing and significant effects on the thapsigargin-induced Ca2+ efflux (Figure 2A and B). Under these conditions, the silencing rate was about 70% (Figure 2C). Thus, BiP contributes to reducing Ca2+ leakage from the ER in human cells. Control experiments demonstrated that the enhanced Ca2+ leakage in BIP siRNA-treated cells was not due to reduced protein synthesis or elevated Ca2+ concentration in these cells (Supplementary Figure S2B and C). Of note, the BIP-silenced cells showed activation of the unfolded protein response at the transcriptional level (Supplementary Figure S2A).

Figure 2
Effect of BIP gene silencing on Ca2+ leakage from the ER. (A) HeLa cells were treated with the indicated siRNAs for 48 h and loaded with the calcium indicator Fura-2 AM as described in Materials and methods. Then live cell Ca2+ imaging ...

We not only analysed Ca2+ leakage from the ER indirectly, that is, as increased Ca2+ concentration in the cytosol, but also did so directly by monitoring ER lumenal Ca2+ concentration. BIP was silenced as described above in a stable HeLa cell line, termed HeLa-CES2, containing ER lumenal carboxylesterase, which allows improved loading of AM-dyes into the ER (Rehberg et al, 2008). Using this targeting-esterase loading method, we monitored the ER lumenal Ca2+ concentration with Fluo5N. Under these conditions, BIP silencing also led to increased Ca2+ leakage from the ER after addition of thapsigargin (Figure 2D and E). Of note, BiP depletion did not lead to reduced levels of ER lumenal carboxylesterase (Figure 2F).

To confirm these conclusions, we attempted expression of the BIP cDNA lacking the BIP-UTR in the presence of the BIP-UTR siRNA and observed rescue of the BIP silencing phenotype in form of restoration of the basal Ca2+ leakage seen in control cells (Figure 3A and C). Transfection with a negative-control plasmid, however, had no effect. According to western blot analysis, the complementation efficiency of the BIP expression plasmid was around 250% (Figure 3D). We also carried out plasmid transfections with vector control and the BIP expression plasmid for cells that had been pretreated with control siRNA and observed that three-fold BIP overexpression did not result in significant changes in the thapsigargin responses (Figure 3A and C).

Figure 3
Effect of BIP and KAR2 expression on Ca2+ leakage from the ER. HeLa cells were treated with the indicated siRNAs and plasmids for about 48 h and loaded with the calcium indicator Fura-2 AM as described in Materials and methods. Then live cell ...

The question was if the observed effect of BiP on cellular Ca2+ homeostasis is specific or a general phenomenon of the major chaperones of the ER. Therefore, we investigated whether silencing the PDIA1 (PDI)-, GRP94, or Calreticulin (CALR)-gene in HeLa cells with validated siRNAs enhanced Ca2+ efflux from the ER. In contrast to the BIP siRNA, the PDI, GRP94 and CALR siRNAs did not have a stimulatory effect on the thapsigargin-induced Ca2+ efflux (Figure 3E). On average, the silencing rate was 66±4% under these conditions (n was between 10 and 13). Thus, the observed effect is specific to BiP.

Based on the available in-vitro experimental results (Alder et al, 2005), we expected that the yeast orthologue of BiP, Kar2p, would be unable to limit Sec61-mediated Ca2+ efflux in HeLa cells. Therefore, we also attempted expression of the KAR2 cDNA in the presence of the BIP-UTR siRNA and observed that it was unable to rescue the BIP silencing phenotype; it did not restore the normal level of Ca2+ leakage (Figure 3B and C). Western blot analysis showed that the KAR2 expression plasmid restored an ER lumenal Hsp70 concentration comparable to control cells (Figure 3D). Thus, the observed effect of BiP on cellular Ca2+ homeostasis is highly specific.

As a means of further validation of the BIP silencing approach, mutant BiPR197E was employed that is inhibited in its ability to cooperate with Hsp40-type co-chaperones (Awad et al, 2008) and had to be expected to be unable to facilitate Sec61 gating based on the in-vitro experiments (Alder et al, 2005). Therefore, we expressed the BIPR197E cDNA in the presence of the BIP-UTR siRNA and observed no rescue of the BIP silencing phenotype; BiPR197E did not restore the normal level of Ca2+ leakage (Figure 3F). According to western blot analysis, the complementation efficiency of the BIPR197E expression plasmid was 250±24% (n=3). Thus, the observed effect of BiP on cellular Ca2+ homeostasis involves an ER resident Hsp40 and, in addition, bears the hallmarks of a substrate interaction of the chaperone.

Next, we asked whether the effect of BiP on ER Ca2+ leakage could be linked to the Ca2+ permeable Sec61 complex. To address this question, we treated HeLa cells for 48 h with siRNA directed against BIP plus either SEC61A1 siRNA or a negative-control siRNA. The results of Ca2+ imaging experiments for cells with BIP siRNA plus the control siRNA were indistinguishable from those of the BIP siRNA-treated cells (Figure 4A and B). Additional silencing of SEC61A1 by siRNA, however, had an inhibitory effect on the BIP silencing-induced Ca2+ efflux (Figure 4A and B). According to western blot analysis, the silencing efficiency of both siRNAs was >70% (Figure 4C). Thus, BiP contributes to reducing Ca2+ leakage from the ER at the level of the Sec61 complex. We also carried out double-siRNA transfections with a two-fold concentration of control siRNA and observed no significant changes in the thapsigargin responses (Figure 4B). Of note, HeLa cells treated with the combination of SEC61A1 and BIP siRNA for 48 h were indistinguishable from control siRNA-treated cells in cell growth (Figure 4D) and showed a typical ER morphology in 3D-structured illumination microscopy (Supplementary Figure S3).

Figure 4
Effect of combined BIP and SEC61A1 silencing on Ca2+ leakage from the ER. HeLa cells were treated with the indicated siRNAs for 48 h and loaded with the calcium indicator Fura-2 AM as described in Materials and methods. Then live cell Ca2+ ...

Protein misfolding stimulates calcium leakage from the ER in intact cells by reducing the pool of available BiP

We reasoned that reproducing the effect of BIP silencing should be possible by reducing the ER lumenal concentration of available BiP using an entirely unrelated approach. Protein misfolding in the ER leads to sequestration of BiP by misfolded polypeptides, induction of the unfolded protein response, and ultimately apoptosis or necrosis. Typical reagents for the induction of protein misfolding in the ER and, thus for ER stress in living cells are dithiothreitol (DTT) and tunicamycin, which interfere with disulphide bridge formation and N-glycosylation, respectively (Helenius et al, 1992; Ellgaard et al, 1999). Therefore, we investigated whether reducing the level of available BiP in the ER of HeLa cells using the two different folding antagonists enhances Ca2+ efflux from the ER. In Ca2+ imaging experiments with Fura-2, we treated HeLa cells with either DTT or tunicamycin for 3 min; subsequently, Ca2+ leakage was visualized by application of thapsigargin in the presence of external EGTA. The two folding antagonists had similar enhancing and significant effects on the thapsigargin-induced Ca2+ efflux (Figure 5A–C), that is, phenocopied the effect of BIP gene silencing by siRNA. Thus, BiP contributes to reducing Ca2+ leakage from the ER based on these results, as well. Of note, treatment of HeLa cells with tunicamycin or DTT for the duration of these experiments did not elicit UPR (Supplementary Figure S2A) and did not affect total cellular Ca2+ content and ER morphology (Supplementary Figures S2C and S4). Furthermore, this short treatment did not result in phosphorylation of eIF2α (data not shown).

Figure 5
Effect of ER stress inducers on Ca2+ leakage from the ER. HeLa cells were loaded with the calcium indicator Fura-2 AM as described in Materials and methods. Then live cell Ca2+ imaging was carried out. (A, B) As indicated with arrow, the ...

We wondered if treatment of HeLa cells for 3 min was sufficient to affect protein N-glycosylation and, therefore, causes protein misfolding. Thus, HeLa cells were treated with tunicamycin for 3 min and converted to semi-permeabilized cells. Subsequently, the glycosylation capacity of the ER of these semi-permeabilized cells was analysed in an established assay that employs in-vitro synthesis of different model precursor proteins, SDS–PAGE and phosphorimaging (Wilson et al, 1995). Indeed, tunicamycin treatment of HeLa cells for 3 min was sufficient to specifically inhibit N-gylcosylation of newly synthesized polypeptides (Figure 5D), suggesting that the dolichol-glycan pool is rather small in HeLa cells under the used conditions.

To further substantiate above conclusions, we silenced the BIP gene prior to treatment with the stress inducers and observed that there is no additive effect of either drug with BIP-UTR siRNA (Figure 6A–C). According to western blot analysis, the silencing efficiency of BIP siRNA was >75% (Figure 6D). The negative-control siRNA had no influence on the drug effect (Figure 6A and C). Thus, the folding antagonists and BIP silencing indeed have the same stimulatory effect on ER Ca2+ leakage.

Figure 6
Effect of ER stress inducers and BIP silencing on Ca2+ leakage from the ER. HeLa cells were treated with the indicated siRNAs for 48 h and loaded with the calcium indicator Fura-2 AM as described in Materials and methods. Then live cell Ca2+ ...

Next, we asked if the effect of the two folding antagonists on ER Ca2+ leakage could also be attributed to the Sec61 channel. We treated HeLa cells with DTT or tunicamycin after incubation with either SEC61A1-UTR siRNA or a negative-control siRNA for 96 h. The results of Ca2+ imaging experiments for cells with DTT or tunicamycin in combination with the control siRNA were indistinguishable from those of the drug-treated but untransfected cells (Figure 7A and E). Silencing of SEC61A1 by siRNA, however, inhibited the drug-induced Ca2+ efflux (Figure 7B and E). Western blot analysis demonstrated that the silencing efficiency of the SEC61A1 siRNA was around 85% (Figure 7F). To confirm this result, we attempted expression of the SEC61A1 cDNA, lacking the UTR of the SEC61A1 gene, in the presence of the SEC61A1-UTR siRNA and observed rescue of the SEC61A1 silencing phenotype as partial restoration of the drug effects on Ca2+ leakage (Figure 7C and E). According to western blot analysis, the complementation efficiency of the SEC61A1 expression plasmid was around 65% (Figure 7F). These findings thus confirmed that BiP contributes to reducing Ca2+ leakage from the ER via the Sec61 complex.

Figure 7
Effect of ER stress inducers and SEC61A1Y344H expression on Ca2+ leakage from the ER. HeLa cells were treated with the indicated siRNAs and plasmids for 48 or 96 h and loaded with the calcium indicator Fura-2 AM as described in Materials and methods. ...

BiP limits ER calcium leakage via a conserved tyrosine residue of the Sec61α subunit

To elucidate the possible mechanism of BiP action, we investigated how a SEC61A1 cDNA with a specific point mutation behaves in the complementation experiment. Recent work on a mouse model for diabetes led to the conclusion that mutation of the conserved tyrosine residue in position 344 of murine Sec61α to histidine leads to a deficient Sec61 complex that can support protein transport into the ER and normal insulin secretion but still leads to β-cell death and diabetes (Lloyd et al, 2010). The mutated Sec61α was introduced into HeLa cells via the SEC61A1 expression plasmid as described above. According to western blot analysis, the complementation efficiency of the mutated SEC61A1 expression plasmid was around 60%, that is, comparable to complementation with the wild-type protein (Figure 7F). In contrast to wild-type Sec61α, however, mutant Sec61αY344H failed to restore the effects of DTT or tunicamycin in the presence of the SEC61A1-UTR siRNA (Figure 7D and E). Furthermore, mutant Sec61αY344H led to an increased Ca2+ leakage from the ER in the presence of thapsigargin per se, that is, even in the absence of any folding antagonists (buffer controls showed 25% increase, Figure 7E). We suggest that this outcome results from the presence of Sec61 channels that fail to be gated by BiP because of a mutated ER lumenal loop 7 of Sec61α.

BiP interaction with loop 7 of Sec61α also modulates Sec61 channel opening in protein translocation

Besides its role in Sec61 channel closure, mammalian BiP has two functions in protein translocation into the ER. It is involved in the insertion of precursor polypeptides into the Sec61 complex, that is, opening of the Sec61 channel (Klappa et al, 1991; Dierks et al, 1996; Alder et al, 2005), and it binds to the precursor polypeptide in transit and acts as a molecular ratchet, thus mediating completion of translocation (Nicchitta and Blobel, 1993; Tyedmers et al, 2003; Shaffer et al, 2005). Based on the above-described experiments, we reasoned that BiP and loop 7 of Sec61α may also be involved in the initial phase of protein translocation in a precursor-specific manner. This question was addressed using the established protein transport assay (Wilson et al, 1995). In addition to the expected negative-control protein preprolactin (ppl; co-translationally transported presecretory protein) (Görlich and Rapoport, 1993), several precursors were employed in the transport assay that were expected to show BiP dependence (Lang et al, 2012): pERj3 (ER lumenal protein), preprocecropin A (ppcecA; post-translationally transported presecretory protein), and two variants of prion protein (pPrP) that lack the central hydrophobic domain (amino-acid residues 113–133; ΔHD) and the GPI acceptor region (residues 231–254, ΔGPI), respectively (Winklhofer et al, 2003). Initial insertion of precursors into the Sec61 complex was assayed as modification by signal peptidase and/or oligosaccharyl transferase, completion of translocation as protease protection in the absence of detergent. In addition, membrane insertion of the model tail anchored (TA) membrane protein Sec61β-ops13 that does not involve the Sec61 complex for its integration into the ER membrane (Lang et al, 2012) was analysed as N-glycosylation and served as a control for the integrity of the membrane.

BiP-depleted cells were obtained by treating HeLa cells with the subtilase cytotoxin SubAB, which specifically inactivates BiP (Paton et al, 2006) (average reduction 98±0%). Respective control cells were treated with SubAA272B, an inactive mutant form of SubAB. After 2 h, the two different cell types were converted to semi-permeabilized cells and assayed for transport of various precursor polypeptides (Figure 8A). As expected, we observed precursor-specific involvement of BiP: the precursor of pl was not affected by the absence of BiP (97±5% processing as compared with the SubAA272B treated cells; n=5), while the transport of pERj3, ppcecA, and the two prion precursors was reduced (to between 2±1 and 55±10%, n=6, 4, and 2). Thus, the lack of BiP led to a substrate-specific defect in transport of proteins into the ER that correlated with the defect that was observed after SEC63 gene silencing (Lang et al, 2012). The TA protein Sec61β-ops13 was not affected by BiP depletion (105±8%, n=5). Hence, BiP depletion had no overall effects on the integrity of the ER membrane, as can be concluded from the activity of oligosaccharyl transferase that depends on a lipid-linked oligosaccharide.

Figure 8
The absence of BiP and the presence of Sec61αY344H affect protein transport into the ER in a precursor-specific manner. (A) Two hours before preparation of semi-permeabilized cells, HeLa cells were treated with SubAB or SubAA272B at final concentrations ...

Next, the strategy was to replace wild-type Sec61α with Sec61αY344H in HeLa cells and to analyse protein transport. HeLa cells were treated with siRNA directed against SEC61A1-UTR or a negative-control siRNA for 48 h. Then, the cells were transfected with wild-type SEC61A1- or mutant SEC61A1Y344H-rescue plasmid or a vector control. According to western blot analysis, the complementation efficiency of both SEC61A1 expression plasmids was between 60 and 65%. After further 48 h, the various cell populations were converted to semi-permeabilized cells and assayed for transport of the precursor with the most pronounced BiP dependence, pERj3. Preprolactin and Sec61β-ops13 served as negative controls. As expected, in the presence of ER from control cells, pERj3 was processed by signal peptidase and oligosaccharyl transferase (Figure 8B). Furthermore, in the presence of SEC61A1-UTR siRNA-treated cells, processing was inhibited (4±1% as compared with the control siRNA-treated cells, n=4) and the inhibition was partially relieved when SEC61A1-rescue plasmid was co-transfected (59±15%, n=5). However, pERj3 transport was not restored to a comparable extent when SEC61A1Y344H-rescue plasmid was co-transfected instead of SEC61A1-rescue plasmid (28±13%, n=5). In contrast, ppl and Sec61β-ops13 were not affected by Sec61αY344H (Figure 8B). Thus, Sec61αY344H was not able to support translocation of pERj3 as efficiently as the wild-type protein. Because of the overlapping effects of BiP depletion and SEC61A1Y344H expression on protein transport, BiP may indeed interact with loop 7 of Sec61α for gating of the Sec61 complex from the closed to the open state.

BiP interacts with loop 7 of Sec61α in a specific manner

To directly demonstrate the putative interaction of BiP with loop 7 of Sec61α and to identify the BiP-binding site within this ER lumenal loop, peptide-binding experiments were carried out. Peptide spots that correspond to loop 7 of the human Sec61α1 were synthesized on cellulose membranes (amino-acid residues 311 through 355; Supplementary Figure S5). The peptides consisted of 15 amino-acid residues with an overlap of 13 residues with adjacent peptides and were incubated with 14C-labelled protein: In addition to BiP, the nucleotide-binding domain (NBD) of BiP and BiP that was saturated with an established substrate peptide were employed and served as negative controls (Awad et al, 2008). The bound proteins were visualized by phosphorimaging. Out of the 16 peptides that covered the entire loop 7, peptides 10 and 15 were preferentially bound by BiP and, therefore, suggested specific binding (Figure 9A). Peptide 15 (corresponding to amino-acid residues 339–353) and the three adjacent spots included the tyrosine residue 344 in central position. In addition, BiP and the two negative-control proteins bound to peptide 6 that has to be dismissed as unspecific binding (corresponding to amino-acid residues 321–335). In surface plasmon resonance spectroscopic (SPR) analysis (Figure 9B), the affinity (determined as Kd) of BiP for peptide 15 was found to be about 500 μM, thus falling into the established affinity range of BiP for its substrates (Fourie et al, 1994). Next, we analysed the nucleotide requirements for the observed interaction of BiP with peptide 15 (Figure 9C). BiP:ADP (right panel) showed more pronounced binding than BiP:ATP (middle panel), as had been observed by Alder et al (2005), supporting the notion of a substrate interaction of BiP. At last, we asked if the effects of the Y344H mutation in loop 7 (Figures 7D and and8B)8B) and the differential effect of BiP and Kar2p (Figure 3B) can be observed at the level of the peptide-binding experiments. Thus, we analysed binding of BiP to the peptide 15 that included the Y344H mutation in comparison to the corresponding wild-type peptide and Kar2p binding to the wild-type peptide. Replacement of tyrosine 344 by histidine led to significantly decreased BiP binding (Figure 9B and C). Furthermore, Kar2p bound less efficiently to peptide 15 as compared with BiP (Figure 9B). Thus, loop 7 indeed contains a BiP interaction site near the tyrosine residue 344. However, future experiments will have to confirm this interaction at the protein level.

Figure 9
BiP binds at or near the minihelix in loop 7 of Sec61α1. (A) 16 Peptides scanning loop 7 of human Sec61α1 as defined by Van den Berg et al (2004) were synthesized on cellulose membranes via carboxy-terminal attachment as described (Hilpert ...

The putative interaction of BiP with loop 7 was also analysed by molecular modelling. First, a structural model of human BiP was generated by multi-template homology modelling using the software MODELLER (Eswar et al, 2006) based on the structural templates 1YUW, 2KHO and 2V7Y. Next, 41 heptapeptides covering the entire loop 7 were docked into the peptide-binding sites of 14 X-ray structures of Hsp70 proteins plus the homology model of BiP using the Autodock4 programme (Morris et al, 2009). For each peptide, 10 different runs were performed. Docking scores below −8 kcal/mol were observed for the two heptapeptides including residues 339–345 and 347–353, respectively (Figure 9D). For comparison, redocking of 11 heptapeptides was performed that had been observed in the respective X-ray structures. This analysis resulted in scores between −7.3 and −10.8 kcal/mol. Thus, two heptapeptides taken from loop 7 bound to Hsp70 proteins with similar affinities as compared with native Hsp70 substrate heptapeptides, nicely correlating with the peptide spot data (Figure 9A).

Discussion

BiP is involved in gating of the Sec61 channel from the open to the closed state

In the resting mammalian cell, the cytosolic-free Ca2+ concentration is 50–100 nM due to Ca2+ clearance by plasma membrane pumps and exchangers (Yu and Hinkle 2000). At the same time, the free Ca2+ concentration in the ER lumen is 100–800 μM due to the action of SERCA. This Ca2+ distribution is typically compromised by passive Ca2+ efflux from the ER. Indirect evidence has suggested that the Sec61 complex, which mediates signal peptide-dependent protein transport into the ER, contributes to the leak after termination of protein translocation (Lomax et al, 2002; Van Coppenolle et al, 2004; Flourakis et al, 2006; Giunti et al, 2007; Ong et al, 2007). Recently, this concept was directly confirmed by the observations that the Sec61 complex is Ca2+ permeable and that silencing the SEC61A1 gene in HeLa cells prevents the Ca2+ leakage linked to termination of protein synthesis (Lang et al, 2011a). As noted, previous work also identified CaM as closing the Sec61 channel in vitro and limiting Ca2+ leakage in a Ca2+-dependent manner in cells by binding to an IQ motif in the cytosolic aminoterminus of Sec61α (Erdmann et al, 2011). Furthermore, in-vitro experiments have demonstrated that ER lumenal BiP closes the Sec61 channel, suggesting a role for BiP in limiting Ca2+ leakage via the Sec61 complex (Hamman et al, 1998; Wirth et al, 2003).

Here, we asked if the role of BiP in channel closure can be confirmed at the cellular level and if the mechanism can be further elucidated. The concentration of active BiP in cells was manipulated by different means, and the effects of reduced levels of BiP on ER Ca2+ leakage were monitored by live cell Ca2+ imaging. Regardless of the method used to lower the BiP concentration, the absence of functional BiP led to increased Ca2+ leakage from the ER at the level of Sec61 complexes. Recent work on a mouse model led to the implication that a point mutation in a minihelix within the ER lumenal loop 7 of murine Sec61α, formed by amino-acid residues 341 through 344, results in a partially deficient Sec61 complex and β-cell death and diabetes (Lloyd et al, 2010) (Figure 10A). When we replaced wild-type Sec61α with mutant Sec61αY344H in human cells, basal Ca2+ leakage from the ER increased and was unaffected by manipulation of the BiP concentration. Therefore, we conclude that BiP limits ER Ca2+ leakage by contributing to Sec61 complex gating from the open to the closed state via binding to or near the minihelix within loop 7 of Sec61α (Figure 10A).

Figure 10
Model for the mechanisms of BiP- and CaM-mediated gating of the human Sec61 complex. (A, B) Homology model for the human heterotrimeric Sec61 complex. Views from the plane of the membrane (A, lateral gate front) and the cytosol (B) are shown. Transmembrane ...

Hypothesis for BiP-mediated Sec61 channel gating from the open to the closed state

The structure of the Sec61 channel provides a potential mechanism for BiP gating of the Sec61 complex from the open to the closed state (Van den Berg et al, 2004; Zimmermann et al, 2011). The current view for gating of the Sec61 complex in protein translocation is that signal peptides of nascent presecretory polypeptides intercalate between Sec61α transmembrane helices 2 and 7, opening the lateral gate of the Sec61 complex that these two transmembrane helices form (Figure 10B and C) (Gumbart and Schulten, 2007; Hizlan et al, 2012). According to current results, binding of BiP to or near the minihelix in ER lumenal loop 7 of Sec61α facilitates gating from the open to the closed channel. We find this hypothesis attractive because loop 7 connects transmembrane helices 7 and 8 and, is thus close enough to the lateral gate to influence gate movements (Figure 10A and B). Furthermore, this idea is attractive because Sec61 complex gating via loop 7 has also been suggested for gating from the closed to the open state during the early phase of protein translocation for the yeast Sec61 complex (Wilkinson et al, 1997; Trueman et al, 2011). However, this was not linked to BiP action or the diabetes mouse. We note that Y344 is highly conserved in the Sec61α of eukaryotes and the orthologue SecY in Archaea but not in eubacteria (Supplementary Figure S5), which may explain why the mechanism of sealing of the SecYEG complex is different in eubacteria (Park and Rapoport, 2011).

BiP and Ca2+–CaM interplay

The structure of the Sec61 complex may also provide a framework for Ca2+–CaM-mediated gating of the Sec61 complex from the open to the closed state. According to the current view for channel opening, a pore plug that helix 2a forms and that connects transmembrane helices 1 and 2 is displaced concomitant with signal peptide intercalation between transmembrane helices 7 and 8 (Figure 10B and C). Therefore, it is conceivable that binding of Ca2+–CaM to the IQ peptide in the cytosolic domain upstream of transmembrane helix 1 can influence plug movement. We find such a dynamic modulation of the ion permeability of the Sec61 channel by BiP and Ca2+–CaM more plausible than the previous suggestion of a static sealing of the channel by the cytosolic ribosome and the ER lumenal BiP. The latter has also been challenged by the cryo-EM data for ribosome/Sec61 complexes (Menetret et al, 2008; Becker et al, 2009).

Because Ca2+–CaM and BiP can close the Sec61 channel and limit Ca2+ leakage from the ER via the Sec61 complex, the question then becomes one of identifying when these two mechanisms are active. CaM acts only after Ca2+ has started to leak out of the ER and reaches a cytosolic concentration of >10 μM, at least in nanodomains surrounding the Sec61 complexes. Therefore, we assume that under conditions when BiP is available, BiP plays the major role in keeping Ca2+ leakage at bay and that Ca2+–CaM kicks in only when BiP starts to fail. This BiP failure, however, seems to occur rather frequently, because CaM antagonists lead to significantly increased Ca2+ leakage under normal conditions (Erdmann et al, 2011). This proposed order of events will have to be addressed in future experiments.

Hypothesis for BiP-mediated Sec61 channel gating from the closed to the open state

Furthermore, our observations offer a possible explanation for the mechanism of BiP action in the initial phase of protein translocation. The precursor polypeptide that showed the most pronounced BiP dependence also showed sensitivity towards the mutation of Sec61α that relates to the minihelix within the ER lumenal loop 7 (Lloyd et al, 2010). We propose that loop 7 also plays a role in gating of the Sec61 complex from closed to open and that BiP binding to this loop 7 may be required for gating from the closed to the open state in the case of some precursor polypeptides, while others may be able to trigger gating on their own or in collaboration with the ribosome (Figure 10C) (Gumbart et al, 2009). This view is consistent with the suggestion that signal peptides intercalate between transmembrane domains 2 and 7 and that these two transmembrane domains form the so-called lateral gate, possibly releasing signal peptides into the plane of the lipid bilayer in the open state (Van den Berg et al, 2004). Obviously, this hypothesis will have to be thoroughly evaluated in future in-vitro experiments.

Implications for the link between ER protein misfolding, Ca2+ homeostasis, and apoptosis

As we have described here, reduction of available BiP in cells by either BIP gene silencing or protein misfolding in the ER leads to Ca2+ leakage from the ER. Although it has been known for some time that protein misfolding in the ER initiates the unfolded protein response and, when the latter is insufficient, apoptosis, the underlying mechanisms are not fully understood (Schröder and Kaufman, 2005; Shore et al, 2011). Based on our observations and the fact that the SEC61A1Y344H mutation causes the unfolded protein response and apoptosis in a diabetes mouse model (see below), we propose that the role of BiP in limiting Ca2+ leakage from the ER at the level of the Sec61 complex contributes to the connection between ER protein misfolding and apoptosis: misfolding polypeptides sequester BiP, in the absence of BiP Sec61 complexes become leaky for Ca2+, and elevated Ca2+ efflux including Ca2+ transmission to mitochondria triggers apoptosis.

The SEC61A1Y344H mutation that was found here to lead to increased Ca2+ leakage from the ER was originally described as causing diabetes and hepatosteatosis in mice (Lloyd et al, 2010). Because the authors observed no effects of the mutation on insulin secretion, they concluded that a different mechanism must cause the ER stress, such as ER-associated protein degradation. On the basis of the current results, we suggest altered Ca2+ homeostasis may be an alternative or additional cause for apoptosis in the mutant mice.

Materials and methods

Materials

The quick-change site-directed mutagenesis kit was from Agilent Technologies. Enhanced chemiluminescence (ECL), ECL Plex goat-anti-rabbit IgG-Cy5 and ECL Plex goat-anti-mouse IgG-Cy3 conjugate were purchased from GE Healthcare. Thapsigargin, ionomycin, pluronic F-127, Fluo5N AM and Fura-2 AM were from Invitrogen/Molecular probes. DTT was obtained from Roche, and tunicamycin from Calbiochem. Antibodies against carboxylesterase were from Abcam, against β-actin from Sigma, and against phospho-eIF2α from Cell Signaling. Rabbit antibodies were raised against the COOH-terminal peptides of human Sec61α (14-mer) or Sec62 (11-mer) and the NH2-terminal peptides of human BiP (12-mer) or Grp170 (11-mer) plus an amino- or carboxy-terminal cysteine, and against purified PDI, Grp94, and Calreticulin, respectively.

Cell culture

HeLa cells (ATCC no. CCL-2) and HeLa-CES2 cells (stably transduced with a CES2 lentiviral vector as described by Rehberg et al (2008)) were cultivated at 37°C in Dulbecco's modified Eagle's medium (DMEM) (Gibco) with 10% foetal bovine serum (FBS) (Biochrom) and 1% penicillin/streptomycin (PAA) in a humidified environment with 5% CO2. For live cell calcium imaging, cells were grown on 25-mm cover slips pretreated with poly-L-lysine (1 mg/ml) for 1 h. Cell growth was monitored using the Countess Automated Cell Counter (Invitrogen) and CFSE staining in combination with automated cell sorting in a FACScan cell sorter with FACS Diva software 6.1.3 (Beckton, Dickinson and Company) according to the manufacturer's instructions.

Silencing of gene expression by siRNA

For gene silencing, 5.2 × 105 HeLa cells were seeded per 6 cm culture plate in normal culture. For BIP silencing, the cells were transfected with BIP siRNA (Supplementary Table 1), BIP-UTR siRNA, or control siRNA (AllStars Negative-Control siRNA, Qiagen) using HiPerFect Reagent (Qiagen) according to the manufacturer's instructions (final concentration of siRNAs: 35 nM). After 24 h, the medium was changed and the cells were transfected a second time. SEC61A1 silencing was carried out as described previously (Lang et al, 2011b). Silencing efficiencies were evaluated by western blot analysis using rabbit antibodies directed against BiP and Sec61α and an anti-β-actin-antibody from mouse. The primary antibodies were visualized using ECL Plex goat-anti-rabbit IgG-Cy5- or ECL Plex goat-anti-mouse IgG-Cy3 conjugate and the Typhoon-Trio imaging system in combination with the Image Quant TL software 7.0 (GE Healthcare).

Complementation analysis

To rescue the phenotype of BIP or SEC61A1 silencing, the BIP and SEC61A1 cDNAs were inserted into the multi-cloning sites of a pCAGGSM2-IRES–GFP and a pCDNA3-IRES–GFP-vector, respectively. Cells were treated with BIP-UTR siRNA or SEC61A1-UTR siRNA as described above for 48 or 96 h. At 4 h after first transfection, the BIP siRNA-treated cells were transfected with either vector or BIP expression plasmid; 48 h after first transfection, the SEC61A1 siRNA-treated cells were transfected with either vector or SEC61A1 expression plasmid. Plasmid transfections were carried out using Fugene HD (Roche). According to GFP fluorescence, the transformation efficiency was around 80%. The yeast KAR2 gene, BIPR197E cDNA, and SEC61A1Y344H cDNA were inserted into the same vectors. The mutant BIPR197E and SEC61A1 cDNAs had originally been created by quick-change site-directed mutagenesis and confirmed by sequence analysis.

Live cell calcium imaging

Live cell calcium imaging for cytosolic Ca2+ was carried out as previously described (Lang et al, 2011b, 2012) and is described in detail in the Supplementary data. Cytosolic [Ca2+] was estimated from ratio measurements using an established calibration method (Grynkiewicz et al, 1985). Data were analysed using Excel 2007.

ER luminal Ca2+ was determined using the HeLa-CES2 cells as described (Rehberg et al, 2008). These cells were loaded with 4 μM Fluo5N AM (solubilized in pluronic F-127) in HBSS (Gibco) for 15 min at 37°C, washed with HBSS and incubated for further 30 min at 25°C to remove remaining cytosolic dye. During the experiment, cells were treated with thapsigargin (1 μM) and after 9 min with ionomycin (5 μM) to release the total ER Ca2+ of the cells. Where indicated, HeLa-CES2 cells were treated with siRNA directed against BIP or a negative-control siRNA for 48 h prior to calcium imaging. Data were collected on the iMIC microscope by excitation at 490 nm and measurement of the emitted fluorescence at 530 nm. Images containing 10–25 cells/frame were sampled every 6 s.

Protein transport into semi-permeabilized cells

Protein transport into semi-permeabilized cells was carried out as described previously (Lang et al, 2012) and is described in detail in the Supplementary data.

Supplementary Material

Supplementary Data:
Source data for figure 2F:
Source data for figure 5D:
Source data for figure 6D:
Source data for figure 8A:
Source data for figure 8B:
Review Process File:

Acknowledgments

We thank E Krause and J Rettig (Homburg) for supervising the SIM analysis. KAR2 plasmid and antibodies directed against Kar2p were kindly donated by C Stirling (Manchester, UK). We are grateful to T Schenkel (Homburg) for help with the cell sorting and to V Flockerzi (Homburg) for drawing our attention to the diabetes mouse. We acknowledge the expert technical assistance by M Lerner, H Löhr, E Ludes, and M Simon Thomas. This work was funded by the DFG (FOR 967, GRKs 845 and 1326, SFB 894).

Author contributions: NS and SL carried out the Ca2+ imaging experiments. SL performed the protein transport experiments. SC optimized BIP silencing and performed the cell sorting. MJ designed and carried out the peptide spot and SPR experiments. SS carried out the immunofluorescence microscopy, JL the RT–PCR analysis. ÖU and VH performed the bioinformatic analysis. RB produced the HeLa-CES2 cells. AWP and JCP provided the purified SubAB proteins and the expertise how to use them. JD constructed the SEC61A1 expression plasmids. JD, AC and RZ planned and supervised the experiments and wrote the manuscript.

Footnotes

The authors declare that they have no conflict of interest.

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