Dynamic changes in complexes of IRE1α, PERK, and ATF6α during endoplasmic reticulum stress

The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1 alpha, PERK, and ATF6 alpha, are activated by the accumulation of misfolded proteins in the ER. It is unclear how the endogenous UPR sensors are regulated by both ER stress and the ER luminal chaperone BiP, which is a negative regulator of UPR sensors. Here we simultaneously examined the changes in the endogenous complexes of UPR sensors by blue native PAGE immunoblotting in unstressed and stressed cells. We found that all three UPR sensors exist as preformed complexes even in unstressed cells. While PERK complexes shift to large complexes, ATF6 alpha complexes are reduced to smaller complexes on ER stress. In contrast, IRE1 alpha complexes were not significantly increased in size on ER stress, unless IRE1 alpha is overexpressed. Surprisingly, depletion of BiP had little impact on the endogenous complexes of UPR sensors. In addition, overexpression of BiP did not significantly affect UPR complexes, but suppressed ER stress mediated activation of IRE1 alpha, ATF6 alpha and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1 alpha and misfolded secretory proteins in cells, which suggests that the binding of unfolded proteins to preformed complexes of UPR sensors may be crucial for activation.