UPR proteins IRE1 and PERK switch BiP from chaperone to ER stress sensor


BiP is a major endoplasmic reticulum (ER) chaperone and is suggested to act as primary sensor in the activation of the unfolded protein response (UPR). How BiP operates as a molecular chaperone and as an ER stress sensor is unknown. Here, by reconstituting components of human UPR, ER stress and BiP chaperone systems, we discover that the interaction of BiP with the luminal domains of UPR proteins IRE1 and PERK switch BiP from its chaperone cycle into an ER stress sensor cycle by preventing the binding of its co-chaperones, with loss of ATPase stimulation. Furthermore, misfolded protein-dependent dissociation of BiP from IRE1 is primed by ATP but not ADP. Our data elucidate a previously unidentified mechanistic cycle of BiP function that explains its ability to act as an Hsp70 chaperone and ER stress sensor.

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Fig. 1: IRE1 and PERK LDs cause loss of BiP ATPase stimulation by co-chaperone and NEF effectors, but BiP retains its inherent activity.
Fig. 2: Folded and misfolded IRE1 LDs have different effects on BiP interaction and activity.
Fig. 3: UPR proteins and co-chaperones have mutually exclusive binding sites on the BiP NBD that are impacted by the BiPK294F mutant.
Fig. 4: The effect of nucleotides on BiP binding to UPR proteins and co-chaperones.
Fig. 5: ATP—but not ADP—primes and facilitates the release of BiP from IRE1 LD.
Fig. 6: Mechanism of BiP function, encompassing its chaperone and ER stress sensor cycles.

Data availability

Source data for Figs. 15 and Extended Data figures are provided in the online version of the paper.


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We thank P. Nowak for assistance with protein purifications. We thank A. Mouskidis for assistance with FRET ADP measurements. We also thank the Freemont and Zhang laboratories, along with J. Wilson, for use of MST equipment. This work was funded by a Cancer Research UK senior research fellowship awarded to M.M.U.A. (C33269/A20752 and C33269/A23215).

Author information




M.C.K. designed experiments, expressed and purified proteins, conducted experiments including ATPase assays, competitive pulldown assays, MST and FRET assays, analyzed results, presented data and contributed to preparing the figures and manuscript. N.L. expressed and purified proteins (including mutant BiP), conducted MST experiments, analyzed data and contributed to preparing the figures and manuscript. V.D. carried out some initial protein preps and ATPase assay measurements. C.J.A. assisted in the expression of CH1 and MST experiments, and contributed to the manuscript. M.M.U.A. conceptualized and designed experiments, analyzed results, prepared figures and wrote the manuscript, supervised the project and obtained funding.

Corresponding author

Correspondence to Maruf M. U. Ali.

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Peer review information Inês Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Comparison of BiP and GST-BiP stimulation and inhibition.

(a) BiP ATPase activity showing stimulation by co-chaperones and inhibition by IRE1 and PERK LDs. (b) same as (a), but using GST tagged BiP. (c) A comparison of the rate of ATPase activity for BiP and GST tagged BiP. Both tagged and untagged BiP are stimulated by co-chaperones and inhibited by IRE1 and PERK LDs to the same level, indicating that the attachment of GST to BiP had no effect on BiP ATPase stimulation or inhibition. Statistics as in Fig. 1, source data available online. Source data

Extended Data Fig. 2 Mutant BiPK294F has same basal ATPase activity as BiPWT.

(a) BiPK294F ATPase activity on addition of co-chaperones. (b) A comparison of the rate of ATPase activity for BiP and BiPK294F. The K294F mutation based within the BiP NBD, had no effect on inherent BiP ATPase, but prevents ATPase stimulation consistent with-it inhibiting co-chaperone binding. Statistics as in Fig. 1, source data available online. Source data

Extended Data Fig. 3 Assessment of BiPK294F affinity for CH1 in presence of nucleotides.

BiPK294F has same binding affinity for CH1, in different nucleotide bound states, as BiPWT. (a) MST profile measuring the binding affinity between BiP and BiPK294F for CH1. (b) same as (a), but in the presence of ADP. (C) same as (a), but with ATP. The experiments indicate that the mutation had no effect on BiP interaction with misfolded substrate protein or affected BiP nucleotide bound conformations. Statistics as in Fig. 4, source data available online. Source data

Extended Data Fig. 4 Attachment of YFP had no effect on BiP functionality.

(a) ATPase activity of BiP and YFP tagged BiP. (b) Comparison of the rate of ATPase activity for BiP and YFP- tagged BiP on addition of co-chaperones and CFP-IRE1 LD. The attachment of YFP had no effect on BiP basal activity, or stimulation by co-chaperones, or inhibition by CFP tagged IRE1 LD. Statistics as in Fig. 1, source data available online. Source data

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Kopp, M.C., Larburu, N., Durairaj, V. et al. UPR proteins IRE1 and PERK switch BiP from chaperone to ER stress sensor. Nat Struct Mol Biol 26, 1053–1062 (2019). https://doi.org/10.1038/s41594-019-0324-9

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