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 remain 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.

Stress-induced clustering of the UPR sensor IRE1α is driven by disordered regions within its ER lumenal domain

“…We anticipate that these long-lived interactions are crucial for providing sufficient time for transmitting the information through the membrane bilayer to initiate the autophosphorylation of the kinase domains leading to activation of its RNase domain. This model is in line with the recent data, which showed a lag between IRE1α oligomerization and its trans-autophosphorylation activity in cells 31 . In our experiments, we used a simple membrane composition, and thus future studies are necessary to assess how changes in the membrane composition during ER stress might regulate IRE1α clustering 25,26,43 .…”

“…Semi-quantitative polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR) analyses revealed that IRE1α LNYL-mNG mutant exhibited impaired XBP1 mRNA splicing activity compared to wild type IRE1α-mNG, indicating that the interfaces formed by the LNYL region in IRE1α LD play an important role in forming active IRE1α assemblies in cells (Fig 5B,C). Instead, IRE1α TLPL-mNG splicing activity was only slightly diminished 31,42 (Fig 5B,C). These data suggested that the mutation in the interface formed by TLPL segment can be compensated by formation of other protein interfaces in cells.…”

“…In the absence of doxycycline, cells expressed low levels of IRE1α due to the inherent leakiness of the doxycycline-inducible system (Fig. The size of IRE1α clusters in cell depends on the protein concentration 28,31,42 . While clusters formed at endogenous levels of IRE1α in most tissues are generally too small to overcome the diffraction limit of light, IRE1α forms microscopically visible clusters when it is ectopically expressed to levels 2-20 times over endogenous protein levels Next, we investigated whether the mutants would impact IRE1α activity monitored by its ability to splice XBP1 mRNA.…”

“…IRE1 activation highly correlates with its assembly into microscopically visible clusters in cells 8,22,[28][29][30] . Clustering of IRE1 is initiated by its ER-lumenal sensor domain 7,9,22,29,31 . Importantly, mutations introduced to the oligomerization interface in IRE1's LD impair the formation of high-order oligomers and abolish IRE1 signaling in cells 7,22,29,31 .…”

Tube geometry controls protein cluster conformation and stability on the endoplasmic reticulum surface

“…Many of the experiments that observe IRE1 clustering overexpressed IRE1. Despite work indicating that IRE1 clusters are important for UPR signaling, 13,14,20,38 recent work 50,51 indicates that endogenous IRE1 levels in certain mammalian cells (approximately 1 mm À2 ) are significantly lower than when IRE1 is overexpressed, and that large IRE1 clusters may not be required for UPR signaling. However, it is noted that IRE1 concentration can vary between cells and for cells in pathological states.…”