UC San Diego

Ire1 is an ER resident transmembrane protein that functions as a transducer of the unfolded protein response (UPR). Its luminal domain senses unfolded proteins, triggering the activation of the cytosolic domain for the transcriptional up regulation of chaperones and other protein folding enzymes to help restore ER homeostasis. Ire1 is unprecedented in nature with two functional domains, a kinase and an endoribonuclease. Upon activation, Ire1 autophosphorylates, and cleaves an intron from HAC1 mRNA. In the absence of autophosphorylation or lack of nuclease activity, a UPR response cannot be mounted. While the roles of Ire1's kinase and nuclease domains during the activation of UPR are understood to some extent, little is known about the mechanism of attenuation of the response. In this study we used a mutagenesis-based approach, both in vivo and in vitro, to characterize the importance of conserved residues in Ire1 during the attenuation of UPR. We present here that in wild type cells, spliced HAC1 mRNA disappears some time after UPR induction, upon recovery of the ER protein folding capacity. In contrast, cells with a mutation in the conserved DFG kinase motif, Ire1-D828A, show sustained HAC1 splicing, even after recovery of ER protein folding capacity. Mutating the DFG kinase motif inactivated the Ire1 kinase, although ATP binding and RNase activation were normal. Limited proteolysis and further biochemical studies showed that the DFG mutation altered Ire1 conformation such that it was no longer responsive to ATP binding. Taken together, it suggests that Ire1-D828A is rendered conformationally unresponsive to the attenuating signals from the ER luminal domain, causing the sustained HAC1 splicing. In addition, we also observed sustained HAC1 splicing in the Ire1 phosphomimetic activation loop mutant, hinting at the importance of loop dephosphorylation in the attenuation process. Thus, we show that the Ire1 kinase domain in addition to its role in activation of UPR also plays an equally important role in responding to the attenuating signal. In addition, we established that the timely down regulation of UPR is as important an event as activation, because sustained UPR activation was toxic to cellular growth