TY - JOUR TI - Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers AU - Belyy, Vladislav AU - Zuazo-Gaztelu, Iratxe AU - Alamban, Andrew AU - Ashkenazi, Avi AU - Walter, Peter A2 - Brodsky, Jeffrey L A2 - Malhotra, Vivek VL - 11 PY - 2022 DA - 2022/06/22 SP - e74342 C1 - eLife 2022;11:e74342 DO - 10.7554/eLife.74342 UR - https://doi.org/10.7554/eLife.74342 AB - 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. KW - UPR KW - IRE1 KW - endoplasmic reticulum KW - single-molecule KW - stress signaling JF - eLife SN - 2050-084X PB - eLife Sciences Publications, Ltd ER -