Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers

Abstract

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remained unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α

“…In order to investigate if IRE1α clusters were the sites of XBP1 mRNA splicing on the ER, we imaged IRE1α-GFP in the HeLa cell lines stably expressing XBP1 reporter transcripts along with NLS-stdMCP-stdHalo and Sec61b-SNAP introduced above ( Figure 4E ). In agreement with earlier reports ( Li et al, 2010 ) as well as a parallel study ( Belyy et al, 2021 ), we detected IRE1α-GFP foci (defined as a ≥fivefold enrichment of GFP signal over background) in 21.89 ± 7.31% of cells expressing high levels of the fusion protein already after relatively short induction of ER stress with TM (5 µg/ml) for 2 hours ( Figure 4F ). Surprisingly, this was not the case for cells expressing Emi1-IRE1α-GFP at low levels, where we were unable to detect IRE1α-GFP clusters even after prolonged exposure to TM (5 µg/ml) for up to 7 hours.…”

“…These observations are in good agreement with an earlier study, in which the pharmacological activation of IRE1α with the flavonol luteolin promoted strong splicing of XBP1 in the absence of IRE1α clustering ( Ricci et al, 2019 ). In addition, and directly related to our work, a parallel study shows that endogenously tagged IRE1α also fails to assemble into large clusters upon induction of ER stress ( Belyy et al, 2021 ). In this work, the authors characterize IRE1α oligomerization during ER stress and find that the resting pool of IRE1α in the ER membrane is dimeric, while in response to stress transient IRE1α tetramers are assembled as the functional subunits that are required for trans-autophosphorylation and XBP1 splicing.…”

“…Instead, when expressed at endogenous levels, IRE1α-GFP simply outlines the ER and cleaves XBP1 mRNA in the absence of cluster formation during ER stress. This finding is further confirmed by a complimentary study that images single IRE1α molecules to characterize their oligomerization dynamics in response to ER stress and also demonstrates that large IRE1α clusters are not required for splicing activity ( Belyy et al, 2021 ).…”

“…Based on this hypothesis, we sought to further investigate and visualize the sites of XBP1 processing on the ER membrane. To this aim, we developed an approach that allowed us to detect IRE1α in the reporter transcript-expressing HeLa cell lines introduced above and that was complementary to the single-molecule imaging approach, which detects endogenously tagged IRE1α protein molecules and is published in parallel to this study ( Belyy et al, 2021 ). We knocked out the endogenous IRE1α using CRISPR/Cas9 and reconstituted its expression with a GFP-tagged IRE1α protein ( Figure 4A ).…”

Signaling by the integrated stress response kinase PKR is fine-tuned by dynamic clustering

“…However, recent crystallographic evidence indicates that PKR could form high-order associations through front-to-front interfaces in PKR’s kinase domain ( Mayo et al, 2019 ). This observation suggests that PKR forms high-order associations in living cells, similar to the ER stress sensors PERK and IRE1 ( Carrara et al, 2015 ; Korennykh et al, 2009 ; Li et al, 2010 ; Bertolotti et al, 2000 ; Cui et al, 2011 ; Belyy et al, 2021 ) and the innate immunity effector RNase L ( Han et al, 2012 ). These independent lines of evidence hint at a conserved mechanistic principle of dynamic clustering of stress sensors upon activation.…”