Clustering of IRE1α in the mammalian ER membrane is independent of its enzymatic activities
Authors: Ricci Daniela , Marroco Ilaria , Vargas Jade , Eletto Daniela , Dibos Miriam , Boyle Sarah , Iwamoto Yuichiro , Argon Yair 
Published: November 2023

Under mild endoplasmic reticulum (ER) stress, the unfolded protein response (UPR) machinery restores homeostasis by increasing the ER protein‐folding capacity. Under such conditions, the transmembrane kinase/endoribonuclease sensor of UPR, IRE1α, is activated to perform unique splicing of the pre‐transcription factor XBP1 as well as to degrade selective mRNAs and microRNAs. Remarkably, this activation is transient and returns to the basal level even in the continuous presence of the stress condition (1). ER stress also induces clustering of IRE1α in the plane of the ER membrane and this behavior is also transient, tracking closely the enzymatic activity of IRE1α with time (1,2). It is still not clear how the activity and clustering of IRE1 are related and whether they lead to coping with stress or to cell death.To answer these questions we monitored the changes in IRE1α distribution and activity using doxycyclin‐induced expression of GFP‐tagged wild type IRE1α or mutants thereof, in an IRE1α‐deficient HAP‐1 cell line, with and without various small molecule ER stress inducers and IRE1α ligands.The transient nature of the enzymatic activity of IRE1α (either XBP1 splicing or RIDD activity) was observed in low doses of each of the stressors thapsigargin, tunicamycin and DTT, but IRE1α activities did not attenuate under higher doses of each stressor. Clustering of IRE1αGFP, on the other hand, was transient even at high doses and did not require an active RNase domain: wild type IRE1αGFP still clustered in the presence of the RNase inhibitor 4m8c, albeit into bigger structures, and the catalytically inactive mutant K907A also displayed clustering. Clustering of RNase‐dead IRE1α did not attenuate over time. Furthermore, activating the RNase activity in an ER‐stress independent manner with the flavonoid luteolin was not sufficient to induce the clustering behavior and in fact inhibited the clustering induced by tunicamycin.Clustering appeared to depend on the ability of the luminal domains of IRE1α to interact, because the oligomerization‐deficient mutant D123P was incapable of clustering and because co‐expression of the N‐terminal luminal domain of IRE1α as a soluble ER protein inhibited the clustering of full‐length IRE1αGFP.Initiation of both clustering and XBP1 splicing depended on the kinase activity of IRE1α, but the conformation changes in the cytosolic domain that are needed for XBP1 splicing were not necessary for clustering. Dispersal of clusters was independent of phosphorylation, as shown by treatment with the PP1 inhibitor, okadaic acid.IRE1α clustering per se was insufficient to specify death as the fate of the cell, since there was no difference after 24hr in the percentage of live/dead cells between cells that could not cluster and cells that formed large clusters.In conclusion, our data suggest that IRE1α clustering is not needed for RNase activity, and that even persistent splicing and hyper‐clustering do not directly cause apoptosis and are only a readout of cell fate.Support or Funding InformationNIH AG18001