Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor, IRE1β

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1 alpha and IRE1 beta, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1 beta senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1 beta clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1 beta complex is robustly increased upon ER stress. Moreover, unlike IRE1 alpha, the luminal domain of IRE1 beta showed antiaggregation activity in vitro, and IRE1 beta was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1 beta, while IRE1 alpha was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1 alpha, the luminal event mediated by IRE1 beta involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.