The unfolded protein response is a key signaling system that regulates protein homeostasis within the ER. The response is induced when there is an accumulation of misfolded protein—due to increase in protein load or aberrant protein folding, which results in activation of a cellular program that aims to restore correctly folded protein levels, in order to ensure a properly functioning ER (Schneider and Bertolotti, 2015; Wang and Kaufman, 2016; Hetz and Papa, 2017).

A critical step in this process is the initial detection of misfolded proteins that leads to UPR induction, the molecular mechanism of which is unclear. We have previously reported an allosteric model for UPR induction (Carrara et al., 2015). Our model was based on the observation that a interaction between the luminal domain (LD) of the key UPR protein, IRE1, and the ATPase domain of BiP, an ER Hsp70 chaperone, dissociates upon the binding of C_H1 misfolded protein to the canonical BiP substrate-binding domain. To observe this important step, we utilized a pull-down assay that qualitatively measured noncanonical dissociation (Carrara et al., 2015). In the present study, we developed a Förster resonance energy transfer (FRET) UPR induction assay that quantifies the association and subsequent dissociation of IRE1 LD with BiP, upon addition of misfolded protein. This new experimental technique reconstitutes in vitro the most crucial mechanistic step in UPR signaling, namely, the detection of misfolded protein and UPR induction. We reassess our previous observations, and extend our measurements to other general ER misfolded proteins and their native folded states. Moreover, we evaluate the important BiP substrate-binding domain mutant BiP^V461F, in the presence of nucleotide and in combination with a mutation that renders BiP ATPase deficient. The new experimental approach significantly enhances the evidence to suggest an allosteric model for UPR induction upon ER stress.

In order to generate a quantitative measure of association and dissociation between IRE1 LD and BiP, we decided to make use of FRET between two fluorophores: cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (Martin et al., 2008; Pollok and Heim, 1999; Felber et al., 2004; Bajar et al., 2016).

As we have previously measured an interaction between the N-terminal ATPase domain of BiP and IRE1 LD (K_d = 1.33 μM), and our pull-down assays utilized an N-terminus-positioned affinity tag that had no impact on interaction (Carrara et al., 2015), we attached fluorescent proteins to the N-terminus of both constructs, with YFP connected via short linker to BiP, upstream of the ATPase domain; and similarly, CFP to IRE1 LD (Figure 1A).

Figure 1

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Figure 1—source data 1

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Measurement of an interaction using this technique would require excitation of CFP at 430 nm wavelength, and observation of emission at the longer wavelength of 480 nm. If YFP is in close proximity, approximately 1–10 nm distance (Bajar et al., 2016), then energy transfer will occur between CFP and YFP, resulting in an emission peak at 530 nm wavelength. This scenario will only occur if there is a direct interaction between the IRE1 LD and the BiP ATPase domain that brings the fluorescent proteins into close proximity (Figure 1B). For our assay, we excited at 430 nm wavelength with bandwidth 10 nm (430-10 nm), and observed the fluorescence emission intensity at 530 nm wavelength with a bandwidth of 10 nm (530-10 nm), which was then divided by the emission intensity at 480 nm also with bandwidth 10 nm (480-10 nm) to give a FRET ratio (530-10 nm/480-10nm), and this was used to measure signal output. This ratio-metric measurement produces less noise then individual fluorescent intensity observations, and is a more reliable FRET signal measure (Bajar et al., 2016; Pollok and Heim, 1999; Martin et al., 2008). We mixed CFP-IRE1 LD and YFP-BiP protein in equimolar ratio, and compared it to non-binding controls. We used two non-binding controls: YFP with CFP-IRE1 LD, and BiP-YFP (with YFP positioned at the C-terminal, beyond the substrate-binding domain) with CFP-IRE1 LD.

Upon excitation, we observed a FRET ratio (530-10 nm/480-10nm) of ~0.62 for CFP-IRE1 LD with YFP-BiP. For our non-binding controls, we measured a FRET ratio of ~0.34 (Figure 1C). The data show an almost doubling of the FRET ratio for CFP-IRE1 LD with YFP-BiP when compared to non-binding controls. This represents a significant FRET signal between the two fluorescent proteins (Pollok and Heim, 1999) that was easily reproducible. Moreover, it indicates a direct interaction between the IRE1 LD and the BiP ATPase domain that we can quantifiably measure.

We have previously demonstrated that misfolded protein, C_H1, binds to the canonical SBP of BiP, and effects dissociation of BiP from IRE1 LD. We reasoned that the addition of C_H1 to our assay should cause significant reduction in the FRET signal. To this end, we added 10-fold molar excess of C_H1 to CFP-IRE1 LD and YFP-BiP, and measured the FRET signal ratio. To make interpretation easier, we took the CFP-IRE1 LD with YFP-BiP sample to represent 100% FRET signal, and the non-binding controls to represent 0% FRET signal. Upon addition of C_H1, the FRET signal was reduced to ~4% (Figure 1D), clearly indicating that the binding of C_H1 misfolded protein to the canonical BiP SBD caused the dissociation of the BiP ATPase domain from the IRE1 LD.

In order to give us a better understanding of C_H1’s ability to inhibit the FRET signal, we added varying concentrations of C_H1 to CFP-IRE1 LD and YFP-BiP, and measured the FRET signal. We found that the addition of C_H1 reduced the FRET signal in a dose-dependent fashion (inhibition constant K_i = 0.51 ± 0.01 μM) (Figure 2), with 10-fold molar excess reducing the FRET signal to almost non-binding control levels. This suggests that a 10-fold molar excess of C_H1 was sufficient for almost complete inhibition of FRET signal.

Figure 2

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Figure 2—source data 1

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To confirm that the FRET signal was a consequence of IRE1 binding to the BiP ATPase domain, we generated YFP-BiP ATPase domain and YFP-BiP SBD proteins (Figure 3A), and measured the FRET signal upon their interaction with CFP-IRE1 LD. We observed a 100% FRET signal equivalent to that resulting from full-length YFP-BiP interaction with CFP-IRE1 LD, whereas we measured no FRET signal when using the SBD (Figure 3B–C). This reaffirms the notion that the ATPase domain of BiP is necessary for the interaction with IRE1 LD, consistent with microscale thermophoresis (MST) and pull-down assay measurements (Carrara et al., 2015); furthermore, this interaction has also been suggested by another study (Todd-Corlett et al., 2007).

Figure 3

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In addition, we previously demonstrated that non-canonical IRE1 LD and BiP interaction was independent of nucleotides, and hence BiP chaperoning activity. We tested this observation again using our FRET UPR induction assay. We added 5 mM ATP, ADP and AMP-PNP to CFP-IRE1 LD and YFP-BiP, and compared these to samples examined in the absence of nucleotide. We observed no significant difference upon the addition of various nucleotides, or in the absence of nucleotide, with all samples measuring almost 100% FRET signal (Figure 3D). This result again suggests that IRE1 LD–BiP association is independent of nucleotide binding.

To gain further mechanistic insights, we wanted to address whether other ER misfolded proteins could cause dissociation of BiP from IRE1; thereby, applying our model to other general ER misfolded proteins. A literature survey identified a number of ER misfolded protein substrates that had been used previously, including α1-antitrypsin (Tsutsui and Wintrode, 2007), RNase A (Petrova et al., 2008), apolipoprotein (Morrow et al., 1999), and transthyretin mutant D18G (TTR^D18G) (Sato et al., 2007). Interestingly, some of these misfolded protein substrates were generated from their correctly folded state in vitro. This allowed us to evaluate not only the misfolded protein, but also the same proteins in their folded native state, thus providing an important control that we were previously unable to assess using the inherently misfolded protein C_H1. We added both folded and misfolded proteins to our assay, and compared output to 100% FRET signal sample and 0% non-binding control. The addition of folded antitrypsin, RNase A, and apolipoprotein had no affect on the FRET signal, with all three measurements close to or equal to 100% (Figure 4A–C). The addition of misfolded versions of the same ER proteins: antitrypsin, RNase A, and apolipoprotein, reduced the signal to ~6% (Figure 4A–C). For TTR^D18G, which lacked a folded control, the FRET signal was reduced to ~2% (Figure 4—figure supplement 1). First, the data clearly indicate that only the misfolded state of the ER proteins induce an effect, by binding to canonical BiP SBD, resulting in IRE1 and BiP dissociation via the ATPase domain, with resultant loss in FRET signal. Second, dissociation of IRE1 and BiP upon binding of misfolded protein is not specific to C_H1 substrate, rather other ER misfolded proteins can induce dissociation by binding to BiP SBD, consistent with BiP being a HSP70-type chaperone.

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To reinforce these observations, we repeated the experiment utilizing our previously reported qualitative pull-down assay, in which GST- or His₆-tagged BiP forms a complex with IRE1 LD that was visualized on aSDS-PAGE gel. In the presence of native folded ER proteins—antitrypsin, RNase A, or apolipoprotein—the interaction between IRE1 LD and BiP remained intact. Upon addition of misfolded versions of the same ER proteins, the interaction between the IRE1 LD and the ATPase domain of BiP dissociated, leaving BiP bound to the misfolded protein via its SBD (Figure 4D–F). Therefore, the qualitative pull-down assay reproduced the observations made with the FRET UPR induction assay.

V461F is a key mutation of BiP that prevents misfolded substrate engagement (Laufen et al., 1999; Petrova et al., 2008); it does this by inserting a bulky side chain that sterically hinders access to the active site of the SBD. Our allosteric model predicts that dissociation of the IRE1 LD and the ATPase of this mutated BiP will not occur upon addition of misfolded protein, with no subsequent loss in the FRET signal. We generated YFP-BiP^V461F protein, and compared it to YFP-BiP^WT by observing the FRET signal in the absence and presence of misfolded protein. For YFP-BiP^WT, we observed a 95% reduction in the FRET signal upon addition of misfolded protein. By contrast, the addition of misfolded protein to the YFP-BiP^V461F sample showed no significant loss in FRET signal (Figure 5A–C). Furthermore, to emphasize the point that the interaction between the IRE1 LD and BiP was not regulated by ATP, but that the primary determinant for dissociation was misfolded proteins, we observed the FRET signal upon the addition of ATP and using BiP^V461F in combination with a mutation that renders BiP deficient in its ATPase activity, T229A (Gaut and Hendershot, 1993; Petrova et al., 2008). In the presence of 5 mM ATP, both BiP^WT and BiP^T229A interacted with CFP-IRE1 LD; moreover, dissociation of complex was caused by addition of misfolded protein in a manner that was independent of ATP (Figure 5—figure supplement 1). However, the double mutant, BiP^{V461F, T229A}, associated with CFP-IRE1 LD in the presence of ATP, but failed to dissociate upon the addition of misfolded protein (Figure 5—figure supplement 1). Thus this mutant behaved in exactly the same way as the SBD single mutant, BiP^V461F (Figure 5A–C), indicating again that the BiP–IRE1 association and dissociation were independent of nucleotides..

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Next, to support our in vitro data, we attempted to measure an interaction in cells using co-immunoprecipitation. We co-transfected HEK293 cells with IRE1 and either BiP wildtype or BiP V461F in the absence and presence of ER stress. In the absence of ER stress, we observed an interaction both between IRE1 and BiP wildtype and between IRE1 and BiP V461F. Upon addition of ER stress, however, the interaction between IRE1 and BiP wildtype was significantly reduced, whereas no loss in interaction was observed with BiP V461F, thus supporting the in vitro FRET data. Taken together, both the in vitro and cellular data indicate that when BiP forms an interaction with the IRE1 LD, via its ATPase domain, misfolded proteins can bind to the canonical active site of the BiP SBD to cause dissociation of BiP, resulting in loss of FRET signal. The BiP^V461F mutant, in which access to the active site is blocked, was unable to interact with misfolded protein and hence could not effect the dissociation of BiP from IRE1, resulting in no reduction of FRET signal. Moreover, as misfolded protein binding to the BiP substrate-binding site caused BiP ATPase domain to dissociate from IRE1, an allosteric mechanism is clearly suggested, supporting our model.

In the present study, we have developed an in vitro FRET UPR induction assay that quantifies the association and dissociation of the IRE1 LD from BiP upon ER stress; thereby, describing an important technical tool for dissecting the UPR induction mechanism that measures a three-component interacting system. We extend our observations to cover other general ER misfolded proteins and make comparisons to these proteins in their native folded state. We also evaluate the key BiP SBD mutant BiP^V461F, in the presence of ATP and in combination with an ATPase-deficient BiP mutant. The mutant data, along with the observation that ER misfolded proteins (and not the same proteins in their native folded state) cause dissociation, clearly indicates that misfolded proteins are being recognized or sensed by the BiP SBD when interacting with the IRE1 LD. Interestingly, the present data provide evidence that would discount a BiP competition model (Bertolotti et al., 2000; Kimata et al., 2003; Okamura et al., 2000; Liu et al., 2000). In this model, IRE1 binds to BiP via the SBD, which is the same site that misfolded proteins bind to BiP. Upon ER stress, misfolded protein titre BiP off from IRE1 in a competitive fashion, resulting in dimerization and activation of UPR. This process is ATP dependent (Bertolotti et al., 2000; Kimata et al., 2003), with ATP (and not necessarily misfolded proteins) being sufficient to cause dissociation of IRE1 and BiP. However, as the interaction between IRE1 and BiP is mediated via the SBD, this suggests a chaperone-substrate type interaction (Bertolotti et al., 2000; Kimata et al., 2003); this point is further emphasized by the process being ATP dependent—ATPase activity is an integral part of the Hsp 70 chaperone-substrate mechanism.

Our in vitro data support an allosteric model (Carrara et al., 2015); whereby, the interaction between IRE1 and BiP occurs via the ATPase domain of BiP and is independent of nucleotides; this clearly indicates a UPR signaling productive association and not a chaperone-substrate type interaction. Furthermore, the primary determining factor for dissociation is misfolded protein binding to the SBD of BiP, which engenders an allosteric change that results in the dissociation of the ATPase domain of BiPfrom IRE1 (Carrara et al., 2015). Moreover, as binding between IRE1 and BiP, and the detection of misfolded proteins, involves two different domains of BiP that are coupled by conformational change, there is no requirement for the association with IRE1 and detection of misfolded protein to be a competitive process, thus reconciling the sensitive nature of UPR signaling.

Although our model indicates that the binding and release of BiP from IRE1 is independent of ATPase activity, it could be that nucleotides have influence over other aspects of UPR signaling. It is known that nucleotides greatly impact BiP conformation, with ADP-bound BiP adopting a substantially different conformation to that of ATP-bound BiP. Exactly how this relates to UPR signaling is an area of ongoing research.

The results of the current study are also consistent with our previous data from microscale thermophoresis (MST) and pull-down assays that have shown an interaction between the ATPase domain (and not the SBD) of BiP and IRE1; this observation has been reported before (Todd-Corlett et al., 2007). In summary, we develop an in vitro FRET UPR induction assay that quantifies non-canonical association and dissociation upon ER stress. Furthermore, the data enhance the experimental evidence suggesting an allosteric mechanism for UPR induction.

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Author details

1. Megan C Kopp

   Department of Life Sciences, Centre for Structural Biology, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Visualization

   Competing interests

   No competing interests declared

2. Piotr R Nowak

   Department of Life Sciences, Centre for Structural Biology, Imperial College London, London, United Kingdom

   Contribution

   Methodology, Validation, Formal analysis, Investigation, Data Curation

   Competing interests

   No competing interests declared

3. Natacha Larburu

   Department of Life Sciences, Centre for Structural Biology, Imperial College London, London, United Kingdom

   Contribution

   Validation, Investigation, Resources

   Competing interests

   No competing interests declared

4. Christopher J Adams

   Department of Life Sciences, Centre for Structural Biology, Imperial College London, London, United Kingdom

   Contribution

   Validation, Resources

   Competing interests

   No competing interests declared

5. Maruf MU Ali

   Department of Life Sciences, Centre for Structural Biology, Imperial College London, London, United Kingdom

   Contribution

   Conceptualization, Methodology, Validation, Formal analysis, Writing - original draft, Writing - review and editing, Visualization, Supervision, project administration, Funding aquisition

   For correspondence

   maruf.ali@imperial.ac.uk

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5392-4029

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