Congress of multiple dimers is needed for cross-phosphorylation of IRE1α and its RNase activity

The unfolded protein response can switch from a pro-survival to a maladaptive, pro-apoptotic mode. During ER stress, IRE1 alpha sensors dimerize, become phosphorylated, and activate XBP1 splicing, increasing folding capacity in the ER protein factory. The steps that turn on the IRE1 alpha endonuclease activity against endogenous mRNAs during maladaptive ER stress are still unknown. Here, we show that although necessary, IRE1 alpha dimerization is not sufficient to trigger phosphorylation. Random and/or guided collisions among IRE1 alpha dimers are needed to elicit cross-phosphorylation and endonuclease activities. Thus, reaching a critical concentration of IRE1 alpha dimers in the ER membrane is a key event. Formation of stable IRE1 alpha clusters is not necessary for RNase activity. However, clustering could modulate the potency of the response, promoting interactions between dimers and decreasing the accessibility of phosphorylated IRE1 alpha to phosphatases. The stepwise activation of IRE1 alpha molecules and their low concentration at the steady state prevent excessive responses, unleashing full-blown IRE1 activity only upon intense stress conditions.