The Sec61 translocon limits IRE1α signaling during the unfolded protein response
- Nanobiology Institute, Yale School of Medicine, United States
- School of Health Sciences, University of Science Malaysia, Malaysia
Figures
Figure 1 with 3 supplements
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.
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.
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.
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.
Figure 2 with 1 supplement
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.
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.
Figure 3 with 2 supplements
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.
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Figure 3—source data 1
Doxycycline titration and quantification of IRE1α clusters as described Figure 3C.
- https://doi.org/10.7554/eLife.27187.009
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.
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.
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.
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Figure 4—source data 1
Doxycycline titration and activation of IRE1α, wIRE1α or sIRE1α as described Figure 4B.
- https://doi.org/10.7554/eLife.27187.013
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Figure 4—source data 2
Activation of IRE1α, wIRE1α or sIRE1α in Tg-treated cells as described Figure 4D.
- https://doi.org/10.7554/eLife.27187.014
Figure 5 with 2 supplements
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.
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Figure 5—source data 1
Attenuation of IRE1α and wIRE1α in Tg-treated cells as described in Figure 5B.
- https://doi.org/10.7554/eLife.27187.016
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Figure 5—source data 2
Attenuation of IRE1α and wIRE1α in TM-treated cells as described Figure 5D.
- https://doi.org/10.7554/eLife.27187.017
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Figure 5—source data 3
Attenuation of IRE1α and wIRE1α in XBP1s expressing cells as described Figure 5F.
- https://doi.org/10.7554/eLife.27187.018
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Figure 5—source data 4
Attenuation of IRE1α and sIRE1α in Tg-treated cells as described in Figure 5H.
- https://doi.org/10.7554/eLife.27187.019
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.
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.
Figure 6 with 2 supplements
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.
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Figure 6—source data 1
Quantification of IRE1α clusters under sever stress as described Figure 6B.
- https://doi.org/10.7554/eLife.27187.023
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Figure 6—source data 2
Attenuation of IRE1α or wIRE1α under severe stress as described Figure 6D.
- https://doi.org/10.7554/eLife.27187.024
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.
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.
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.
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.
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The Sec61 translocon limits IRE1α signaling during the unfolded protein response
eLife 6:e27187.
https://doi.org/10.7554/eLife.27187
