Disordered regions in the IRE1α ER lumenal domain mediate its stress-induced clustering

Conserved signaling cascades monitor protein-folding homeostasis to ensure proper cellular function. One of the evolutionary conserved key players is IRE1, which maintains endoplasmic reticulum (ER) homeostasis through the unfolded protein response (UPR). Upon accumulation of misfolded proteins in the ER, IRE1 forms clusters on the ER membrane to initiate UPR signaling. What regulates IRE1 cluster formation is not fully understood. Here, we show that the ER lumenal domain (LD) of human IRE1 alpha forms biomolecular condensates in vitro. IRE1 alpha LD condensates were stabilized both by binding to unfolded polypeptides as well as by tethering to model membranes, suggesting their role in assembling IRE1 alpha into signaling-competent stable clusters. Molecular dynamics simulations indicated that weak multivalent interactions drive IRE1 alpha LD clustering. Mutagenesis experiments identified disordered regions in IRE1 alpha LD to control its clustering in vitro and in cells. Importantly, dysregulated clustering of IRE1 alpha mutants led to defects in IRE1 alpha signaling. Our results revealed that disordered regions in IRE1 alpha LD control its clustering and suggest their role as a common strategy in regulating protein assembly on membranes. Proteotoxic stress causes the endoplasmic reticulum (ER) stress sensor IRE1 alpha to cluster, but the mechanistic basis of its cluster formation remains poorly understood. This study reveals that disordered regions within the protein's ER lumenal domain (LD) mediate its assembly into signaling clusters.IRE1 alpha LD forms biomolecular condensates in solution.Disordered regions within IRE1 alpha LD drive the formation of condensates through weak multivalent interactions.Binding of unfolded polypeptide ligands and membrane-tethering leads to the formation of stable IRE1 alpha LD clusters.The integrity of the disordered regions in its lumenal domain is critical for IRE1 alpha clustering and unfolded protein response (UPR) signaling in cells. The disordered regions of the ER luminal domain of IRE1 alpha form condensates in vitro and control IRE1 alpha clustering and stress signalling in cells.