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

Abstract

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

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.

References

  1. 1.

    Hetz, C. & Papa, F. R. The unfolded protein response and cell fate control. Mol. Cell 69, 169–181 (2017).

    Article  Google Scholar 

  2. 2.

    Wang, M. & Kaufman, R. J. Protein misfolding in the endoplasmic reticulum as a conduit to human disease. Nature 529, 326–335 (2016).

    CAS  Article  Google Scholar 

  3. 3.

    Walter, P. & Ron, D. The unfolded protein response: from stress pathway to homeostatic regulation. Science 334, 1081–1086 (2011).

    CAS  Article  Google Scholar 

  4. 4.

    Adams, C. J., Kopp, M. C., Larburu, N., Nowak, P. R. & Ali, M. M. U. Structure and molecular mechanism of ER stress signaling by the unfolded protein response signal activator IRE1. Front. Mol. Biosci. 6, 11 (2019).

    CAS  Article  Google Scholar 

  5. 5.

    Zhou, J. et al. The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response. Proc. Natl Acad. Sci. USA 103, 14343–14348 (2006).

    CAS  Article  Google Scholar 

  6. 6.

    Credle, J. J., Finer-Moore, J. S., Papa, F. R., Stroud, R. M. & Walter, P. On the mechanism of sensing unfolded protein in the endoplasmic reticulum. Proc. Natl Acad. Sci. USA 102, 18773–18784 (2005).

    CAS  Article  Google Scholar 

  7. 7.

    Carrara, M., Prischi, F., Nowak, P. R. & Ali, M. M. U. Crystal structures reveal transient PERK luminal domain tetramerization in endoplasmic reticulum stress signaling. EMBO J. 34, 1589–1600 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Shamu, C. E. & Walter, P. Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J. 15, 3028–3039 (1996).

    CAS  Article  Google Scholar 

  9. 9.

    Tirasophon, W., Welihinda, A. A. & Kaufman, R. J. A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells. Genes Dev. 12, 1812–1824 (1998).

    CAS  Article  Google Scholar 

  10. 10.

    Ali, M. M. U. et al. Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response. EMBO J. 30, 894–905 (2011).

    CAS  Article  Google Scholar 

  11. 11.

    Harding, H. P., Zhang, Y. & Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271–274 (1999).

    CAS  Article  Google Scholar 

  12. 12.

    Cox, J. S. & Walter, P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404 (1996).

    CAS  Article  Google Scholar 

  13. 13.

    Calfon, M. et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature 415, 92–96 (2002).

    CAS  Article  Google Scholar 

  14. 14.

    Prischi, F., Nowak, P. R., Carrara, M. & Ali, M. M. U. Phosphoregulation of Ire1 RNase splicing activity. Nat. Commun. 5, 3554 (2014).

    Article  Google Scholar 

  15. 15.

    Bakunts, A. et al. Ratiometric sensing of BiP-client versus BiP levels by the unfolded protein response determines its signaling amplitude. eLife 6, e27518 (2017).

    Article  Google Scholar 

  16. 16.

    Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Kampinga, H. H. & Craig, E. A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat. Rev. Mol. Cell Biol. 11, 579–592 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    Behnke, J., Feige, M. J. & Hendershot, L. M. BiP and its nucleotide exchange factors Grp170 and Sil1: mechanisms of action and biological functions. J. Mol. Biol. 427, 1589–1608 (2015).

    CAS  Article  Google Scholar 

  19. 19.

    Carrara, M., Prischi, F., Nowak, P. R., Kopp, M. C. & Ali, M. M. U. Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling. eLife 4, e03522 (2015).

    Article  Google Scholar 

  20. 20.

    Kopp, M. C., Nowak, P. R., Larburu, N., Adams, C. J. & Ali, M. M. U. In vitro FRET analysis of IRE1 and BiP association and dissociation upon endoplasmic reticulum stress. eLife 7, e30257 (2018).

    Article  Google Scholar 

  21. 21.

    Karagoz, G. E., Acosta-Alvear, D. & Walter, P. The unfolded protein response: detecting and responding to fluctuations in the protein-folding capacity of the endoplasmic reticulum. Cold Spring Harb. Perspect. Biol. 11, a033886 (2019).

    Article  Google Scholar 

  22. 22.

    Acosta-Alvear, D. et al. The unfolded protein response and endoplasmic reticulum protein targeting machineries converge on the stress sensor IRE1. eLlife 7, e43036 (2018).

    Article  Google Scholar 

  23. 23.

    Amin-Wetzel, N. et al. A J-protein co-chaperone recruits BiP to monomerize IRE1 and repress the unfolded protein response. Cell 171, 1625–1637.e13 (2017).

    CAS  Article  Google Scholar 

  24. 24.

    Feige, M. J. et al. An unfolded CH1 domain controls the assembly and secretion of IgG antibodies. Mol. Cell 34, 569–579 (2009).

    CAS  Article  Google Scholar 

  25. 25.

    Todd-Corlett, A., Jones, E., Seghers, C. & Gething, M.-J. Lobe IB of the ATPase domain of Kar2p/BiP interacts with Ire1p to negatively regulate the unfolded protein response in Saccharomyces cerevisiae. J. Mol. Biol. 367, 770–787 (2007).

    CAS  Article  Google Scholar 

  26. 26.

    Petrova, K., Oyadomari, S., Hendershot, L. M. & Ron, D. Regulated association of misfolded endoplasmic reticulum lumenal proteins with P58/DNAJc3. EMBO J. 27, 2862–2872 (2008).

    CAS  Article  Google Scholar 

  27. 27.

    Laufen, T. et al. Mechanism of regulation of hsp70 chaperones by DnaJ cochaperones. Proc. Natl Acad. Sci. USA 96, 5452–5457 (1999).

    CAS  Article  Google Scholar 

  28. 28.

    Yan, M., Li, J. & Sha, B. Structural analysis of the Sil1–Bip complex reveals the mechanism for Sil1 to function as a nucleotide-exchange factor. Biochem. J. 438, 447–455 (2011).

    CAS  Article  Google Scholar 

  29. 29.

    Kityk, R., Kopp, J. & Mayer, M. P. Molecular mechanism of J-domain-triggered ATP hydrolysis by Hsp70 chaperones. Mol. Cell 69, 227–237.e4 (2018).

    CAS  Article  Google Scholar 

  30. 30.

    Martin, S. F., Tatham, M. H., Hay, R. T. & Samuel, I. D. W. Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway. Protein Sci. 17, 777–784 (2008).

    CAS  Article  Google Scholar 

  31. 31.

    Craig, E. A. Hsp70 at the membrane: driving protein translocation. BMC Biol. 16, 11 (2018).

    Article  Google Scholar 

  32. 32.

    Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P. & Ron, D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat. Cell Biol. 2, 326–332 (2000).

    CAS  Article  Google Scholar 

  33. 33.

    Boisvert, F. M. et al. A quantitative spatial proteomics analysis of proteome turnover in human cells. Mol. Cell Proteomics 11, M111.011429 (2012).

    Article  Google Scholar 

  34. 34.

    Preissler, S. & Ron, D. Early events in the endoplasmic reticulum unfolded protein response. Cold Spring Harb. Perspect. Biol 11, a033894 (2019).

    Article  Google Scholar 

Download references

Acknowledgements

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

Affiliations

Authors

Contributions

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.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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

Supplementary information

Source data

Source Data Fig. 1

Statistical Source data

Source Data Fig. 2

Statistical Source data

Source Data Fig. 3

Statistical Source data

Source Data Fig. 3

Unprocessed gels

Source Data Fig. 4

Statistical Source data

Source Data Fig. 5

Statistical Source data

Source Data Extended Data Fig. 1

Statistical Source data

Source Data Extended Data Fig. 2

Statistical Source data

Source Data Extended Data Fig. 3

Statistical Source data

Source Data Extended Data Fig. 4

Statistical Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

Further reading