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. 2017 May 15:6:e27187.
doi: 10.7554/eLife.27187.

The Sec61 translocon limits IRE1α signaling during the unfolded protein response

Arunkumar Sundaram  1   2 , Rachel Plumb  1 , Suhila Appathurai  1 , Malaiyalam Mariappan  1
Affiliations

The Sec61 translocon limits IRE1α signaling during the unfolded protein response

Arunkumar Sundaram et al. Elife. .

Abstract

IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.

Keywords: ER stress; cell biology; endoplasmic reticulum; none; unfolded protein response.

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Conflict of interest statement

The authors declare that no competing interests exist.

Figures

Figure 1.
Figure 1.. IRE1α complexes are regulated by an interaction with the Sec61 translocon.
(A) IRE1α -/- HEK293 cells complemented with wild-type IRE1α-HA, wIRE1α-HA (Δ434–443), or sIRE1α-HA (S439A/T446A/S450A/T451A) were treated with 2.5 μg/ml thapsigargin (Tg) for the indicated hours (hr), lysed with digitonin, and analyzed by BN-PAGE immunoblotting (top) as well as phos-tag based immunoblotting to probe phosphorylated IRE1α (bottom). A denotes a ~500 kDa complex of IRE1α in BN-PAGE immunoblotting. B denotes a ~720 kDa complex of IRE1α. (B) The cells expressing IRE1α-HA or wIRE1α-HA were treated with 2.5 ug/ml Tg for the indicated hours and analyzed by both BN-PAGE immunoblotting and standard immunoblotting with a PERK antibody. (C) IRE1α-HA or wIRE1α-HA expressing cells were treated with either control siRNA or Sec61α siRNA followed by treatment with 2.5 μg/ml Tg for the indicated times. The samples were analyzed as in panel A. (D,E) The samples from the panel C were analyzed by BN-PAGE immunoblotting with either PERK or Sec61α antibodies. DOI: http://dx.doi.org/10.7554/eLife.27187.002
Figure 1—figure supplement 1.
Figure 1—figure supplement 1.. IRE1α mutants that either disrupt the interaction or improve the interaction with Sec61 translocon.
(A) Comparison of the IRE1α sequences from amino acid 434 to 452 in vertebrates. Mutations in yellow indicated the region of IRE1α that disrupts the interaction with the Sec61 translocon. Mutations in the blue region of IRE1α improve the interaction with the Sec61 translocon. (B) The cell lysates from transiently transfected HA-tagged Ire1a variants were immunoprecipitated with anti-HA antibodies, eluted with sample buffer and analyzed by immunoblotting. (C) An immunoblot comparing the endogenous IRE1α in HEK293 cells (Control) with wild-type IRE1α-HA, wIRE1α-HA (Δ434–443), or sIRE1α-HA (S439A/T446A/S450A/T451A) complemented into IRE1α -/- HEK293 cells. While wIRE1α refers to an IRE1α mutant that interacts weakly with the Sec61 translocon, sIRE1α refers to an IRE1α mutant that interacts strongly with the Sec61 translocon. DOI: http://dx.doi.org/10.7554/eLife.27187.003
Figure 1—figure supplement 2.
Figure 1—figure supplement 2.. Endogenous IRE1α exists as preformed complexes in HEK293 and INS-1 cells.
(A) The digitonin lysate of HEK293 cells treated with 2.5 μg/ml Tg or INS-1 cells treated with 0.5 μg/ml Tg were analyzed by BN-PAGE immunoblotting with IRE1α antibodies. (B) Samples from the panel A were analyzed by a BN-PAGE immunoblotting with PERK antibodies. DOI: http://dx.doi.org/10.7554/eLife.27187.004
Figure 1—figure supplement 3.
Figure 1—figure supplement 3.. BN-PAGE analysis of the Sec61 translocon.
IRE1α -/- HEK293 cells complemented with wild-type IRE1α-HA, wIRE1α-HA, or sIRE1α-HA were treated with 2.5 μg/ml thapsigargin (Tg) for the indicated hours (hr), lysed with digitonin, and analyzed by BN-PAGE immunoblotting with Sec61β antibodies. DOI: http://dx.doi.org/10.7554/eLife.27187.005
Figure 2.
Figure 2.. IRE1α forms a hetero-oligomeric complex with the Sec61 translocon.
(A) Coomassie blue stained gels showing IRE1α variants that were purified from HEK293 cells stably expressing 2X strep-tagged IRE1α. (B) The indicated concentration of purified IRE1α proteins was analyzed by BN-PAGE based immunoblotting with IRE1α antibodies. (C) The purified IRE1α proteins were analyzed as in panel B using Sec61α antibodies. (D) The purified IRE1α proteins were analyzed by standard immunoblotting with BiP and Sec61α antibodies. DOI: http://dx.doi.org/10.7554/eLife.27187.006
Figure 2—figure supplement 1.
Figure 2—figure supplement 1.. XBP1u mRNA cleavage by purified IRE1α variants.
(A) The purified IRE1α proteins were independently incubated with 32P-labeled unspliced XBP1u mRNA prepared from in vitro transcription reactions. The cleaved fragments were separated by 6 M urea PAGE and exposed for autoradiography. (B) Immunoblots of purified Ire1a proteins. Note that both wild type IRE1α and strong Ire1a (sIRE1α) contain the Sec61 translocon, whereas weak Ire1a (wIRE1α) lacks the Sec61 translocon. DOI: http://dx.doi.org/10.7554/eLife.27187.007
Figure 3.
Figure 3.. The Sec61 translocon inhibits IRE1α higher-order oligomer or cluster formation in cells.
(A) IRE1α -/- HEK293 cells complemented with IRE1α-HA, wIRE1α-HA or sIRE1α-HA were treated with 5 μg/ml Tunicamycin (TM) for 4 hr. Scale bars are 10 μm. Subsequently, cells were processed using an immunostaining procedure to label IRE1α (green) with rabbit anti-HA antibodies as well as a Hoechst stain to label nuclei (blue) and imaged using a confocal microscope. (B) IRE1α-HA or wIRE1α-HA expressing cells were induced with various amounts of doxycycline, treated with TM and analyzed as in panel A. (C) Quantification of the number of cells with IRE1α clusters from the panel C. Error bar represents standard deviation. (D) Immunoblots show the expression of IRE1α in response to varying concentrations of doxycycline. DOI: http://dx.doi.org/10.7554/eLife.27187.008
Figure 3—figure supplement 1.
Figure 3—figure supplement 1.. IRE1α and wIRE1α are localized to the ER in HEK293 cells.
IRE1α-/- HEK293 cells stably expressing IRE1α -HA or wIRE1α -HA were induced with 2 ng/ml doxycycline for at least 12 hr before the treatment with 5 μg/ml TM for 4 hr. Subsequently, the cells were processed for an indirect immunofluorescence by laser scanning confocal microscopy. The localization of the endogenous Sec61β, which marks the ER, was probed with rabbit anti-Sec61β antibodies followed by goat anti-rabbit secondary antibodies conjugated to Cy3. IRE1α was probed with mouse anti-HA antibody followed by goat anti-mouse secondary antibodies conjugated to Cy2. Nuclei were stained with Hoechst. Scale bars are 10 μm. DOI: http://dx.doi.org/10.7554/eLife.27187.010
Figure 3—figure supplement 2.
Figure 3—figure supplement 2.. The Sec61 translocon interaction defective IRE1α mutant form clusters in both MEF and HEK293 cells.
(A) IRE1α-/- MEF cells stably complemented with either wild type IRE1α-HA or w IRE1α-HA were treated or untreated with 5 μg/ml of tunicamycin (TM) for 4 hr. Subsequently, cells were processed for an indirect immunofluorescence. While the localization of the endogenous Sec61β was probed with rabbit anti-Sec61β antibodies and Cy3, IRE1α was probed with mouse anti-HA antibodies and Cy2. (B) IRE1α-/- HEK293 cells were complemented with IRE1α (V437A/D443A) mutant, which does not interact with the Sec61translocon (Figure 1—figure supplement 1), and were treated with 5 μg/ml TM for 4 hr. Subsequently, cells were processed using an immunostaining procedure to label IRE1α (green) with rabbit anti-HA antibodies as well as a Hoechst stain to label nuclei (blue) and imaged using a confocal microscope. DOI: http://dx.doi.org/10.7554/eLife.27187.011
Figure 4.
Figure 4.. The Sec61 translocon regulates the activation of IRE1α during ER stress.
(A) IRE1α -/- HEK293 cells complemented with either wild type IRE1α -HA, wIRE1α-HA, or sIRE1α-HA were induced with the indicated amounts of doxycycline, treated with 2.5 μg/ml Tg for 2 hr where indicated and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (B) Quantification of IRE1α, wIRE1α, and sIRE1α phosphorylation from panel A. (C) IRE1α-HA, wIRE1α-HA, or sIRE1α-HA expressing cells were treated with 1 μg/ml of Tg for the indicated time points and analyzed as in panel A. (D) Quantification of IRE1α, wIRE1α, and sIRE1α phosphorylation from panel C. DOI: http://dx.doi.org/10.7554/eLife.27187.012
Figure 5.
Figure 5.. The Sec61 translocon regulates the attenuation of IRE1α activity during ER stress.
(A) IRE1α -/- HEK293 cells complemented with either wild type IRE1α-HA or wIRE1α-HA were treated with 2.5 μg/ml of Tg for the indicated time points and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (B) Quantification of IRE1α and wIRE1α phosphorylation from panel A. (C) IRE1α-HA or wIRE1α-HA cells were treated with 10 μg/ml of TM for the indicated time points and analyzed as in panel A. (D) Quantification of IRE1α and wIre1 phosphorylation from panel C. (E) IRE1α-HA or wIRE1α-HA cells were transfected with XBP1s plasmid and treated with 1 μg/ml of Tg for the indicated time points and analyzed as in panel A. (F) Quantification of IRE1α and wIRE1α phosphorylation from panel E. (G) IRE1α-HA or sIRE1α-HA cells were treated with 2.5 μg/ml of Tg for the indicated time points and analyzed as in panel A. (H) Quantification of IRE1α and sIRE1α phosphorylation from panel G. DOI: http://dx.doi.org/10.7554/eLife.27187.015
Figure 5—figure supplement 1.
Figure 5—figure supplement 1.. Attenuation of the endogenous IRE1α activity during ER stress.
(A) HEK293 cells were treated with 5 ug/ml tunicamycin (TM) for the indicated times and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (B) Quantification of endogenous IRE1α phosphorylation from panel A. DOI: http://dx.doi.org/10.7554/eLife.27187.020
Figure 5—figure supplement 2.
Figure 5—figure supplement 2.. Accumulation of misfolded proteins is required for the activation of IRE1α.
IRE1α -/- HEK293 cells stably expressing IRE1α or wIRE1α were pretreated with 5 μg/ml TM for 3 hr, washed, and chased without TM but including 100 μg/ml cycloheximide (CHX) for the indicated time points and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. DOI: http://dx.doi.org/10.7554/eLife.27187.021
Figure 6.
Figure 6.. Severe ER stress causes higher-order oligomer formation and extended activation of wild type IRE1α.
(A) IRE1α-HA, sIRE1α-HA, and wIRE1α-HA complemented IRE1α -/- HEK293 cells were treated with 2.5 μg/ml Tg for 4 hr or 10 μg/ml Tg for 2 hr. Subsequently, cells were processed using an immunostaining procedure to label IRE1α (green) with rabbit anti-HA as well as a Hoechst stain to label nuclei (blue). Scale bars are 10 μm. (B) Images from A were analyzed to determine the number of cells containing IRE1α, wIRE1α clusters, or sIRE1α clusters. Error bar represents S.E.M. (C) IRE1α-HA or wIRE1α-HA expressing cells were treated with 10 μg/ml Tg for the indicated time points and analyzed by phos-tag immunoblotting for IRE1α and standard immunoblotting for the indicated antigens. (D) Quantification of IRE1α and wIRE1α phosphorylation from panel C. (E) IRE1α or wIRE1α expressing cells were treated with either 2.5 μg/ml Tg or 10 μg/ml Tg for 18 hr and analyzed by immunoblots as well as the XBP1 mRNA splicing assay. XBP1u - Unspliced XBP1 mRNA, XBP1s - spliced XBP1 mRNA. DOI: http://dx.doi.org/10.7554/eLife.27187.022
Figure 6—figure supplement 1.
Figure 6—figure supplement 1.. The IRE1α interaction with the Sec61 translocon is stable during severe ER stress conditions.
HEK293 cells were either treated with 2.5 μg/ml Thapsigargin (Tg) or 10 μg /ml Tg for the indicated times and harvested for immunoprecipitation with the indicated antibodies. GFP antibodies were used as a control. The immunoprecipitates were analyzed by immunoblotting with antibodies against IRE1α or Sec61β. The star symbol indicates the Sec61β bands that were distorted by the high concentration of digitonin in the input samples. DOI: http://dx.doi.org/10.7554/eLife.27187.025
Figure 6—figure supplement 2.
Figure 6—figure supplement 2.. Co-localization of IRE1α and Sec61 during severe ER stress.
IRE1α-/- HEK293 cells stably expressing IRE1α-HA, wIRE1α-HA, or sIRE1α-HA were induced with 5 ng/ml doxycycline for 18 hr before treatment with 10 μg/ml Tg for 30 min. Subsequently, the cells were processed for an indirect immunofluorescence by laser scanning confocal microscopy. The localization of the endogenous Sec61β, which marks the ER, was probed with rabbit anti-Sec61β antibodies followed by goat anti-rabbit secondary antibodies conjugated to Cy3. IRE1α was probed with mouse anti-HA antibody followed by goat anti-mouse secondary antibodies conjugated to Cy2. Nuclei were stained with Hoechst. Scale bars are 10 μm. DOI: http://dx.doi.org/10.7554/eLife.27187.026
Author response image 1.
Author response image 1.. BN-PAGE immunoblotting analysis of the Sec61 translocon complex.
IRE1α -/- HEK293 cells complemented with wild-type IRE1α-HA or wIREα-HA were treated with 2.5 μg/ml Tg for the indicated hours (hr), lysed with digitonin, and analyzed by BN-PAGE immunoblotting as well as a protein marker lane from the same BN-PAGE gel was excised. DOI: http://dx.doi.org/10.7554/eLife.27187.027
Author response image 2.
Author response image 2.. Domain mapping of the IRE1α interaction region in Sec61α.
The lysates prepared from HEK293 cells transiently expressing the indicated 3x-HA tagged Sec61a variants were immunoprecipitated with anti-HA antibodies and analyzed by immunoblotting. Δ denotes deletion of indicated amino acid residues from canine Sec61a. DOI: http://dx.doi.org/10.7554/eLife.27187.028
See this image and copyright information in PMC

References

    1. Acosta-Alvear D, Zhou Y, Blais A, Tsikitis M, Lents NH, Arias C, Lennon CJ, Kluger Y, Dynlacht BD. XBP1 controls diverse cell type- and condition-specific transcriptional regulatory networks. Molecular Cell. 2007;27:53–66. doi: 10.1016/j.molcel.2007.06.011. - DOI - PubMed
    1. Adamson B, Norman TM, Jost M, Cho MY, Nuñez JK, Chen Y, Villalta JE, Gilbert LA, Horlbeck MA, Hein MY, Pak RA, Gray AN, Gross CA, Dixit A, Parnas O, Regev A, Weissman JS. A multiplexed single-cell CRISPR screening platform enables systematic dissection of the unfolded protein response. Cell. 2016;167:1867–1882. doi: 10.1016/j.cell.2016.11.048. - DOI - PMC - PubMed
    1. Back SH, Kaufman RJ. Endoplasmic reticulum stress and type 2 diabetes. Annual Review of Biochemistry. 2012;81:767–793. doi: 10.1146/annurev-biochem-072909-095555. - DOI - PMC - PubMed
    1. Benhamron S, Hadar R, Iwawaky T, So JS, Lee AH, Tirosh B. Regulated IRE1-dependent decay participates in curtailing immunoglobulin secretion from plasma cells. European Journal of Immunology. 2014;44:867–876. doi: 10.1002/eji.201343953. - DOI - PubMed
    1. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biology. 2000;2:326–332. doi: 10.1038/35014014. - DOI - PubMed