Project Summary/Abstract: Secretory and membrane proteins, which account for ~30% of all human proteins, are co-translationally translocated across or inserted into the endoplasmic reticulum (ER). These nascent polypeptides are folded into functional proteins with the help of chaperones and folding enzymes in the ER. Defects in protein folding lead to the accumulation of misfolded proteins and the triggering of ER stress, which activates the unfolded protein response (UPR). Of the three major UPR sensors, IRE1α is the most conserved ER-localized transmembrane kinase/RNase that is activated through oligomerization/phosphorylation upon ER stress. Once activated, IRE1α mediates the splicing of XBP1u mRNA to produce an active transcription factor, XBP1s, which drives expression of UPR target genes to mitigate ER stress. Also, IRE1α promiscuously cleaves ER- localized mRNAs through the regulated Ire1-dependent decay (RIDD) pathway to reduce the burden of the incoming protein load. Under chronic ER stress conditions, however, IRE1α switches from the pro-survival mode to pro-apoptotic mode, resulting in cell death, which is associated with human diseases including, type 2 diabetes and cancer. Despite the physiological importance, the factors that control activation and inactivation of IRE1α/XBP1 signaling remain unclear. We have recently discovered that IRE1α forms a complex with the Sec61/Sec63 translocon complex to access its mRNA substrates. In the current funding period, we have shown that the Sec61 translocon bridges IRE1α with the Sec63/BiP complex to turnoff IRE1α signaling during persistent ER stress. Our studies discovered that the Sec63/BiP complex is also responsible for freeing clogged Sec61 translocons as well as promoting protein folding in the ER. These new findings raise the hypothesis that the IRE1α/Sec61/Sec63 complex plays a central role in the activation and inactivation of IRE1α/XBP1 signaling to maintain ER homeostasis in cells. In the next funding period, we will test this hypothesis by (i) determining the role of this complex in making life-or-death decisions during ER stress; (ii) determining the architecture of the IRE1α/Sec61/Sec63/BiP complex; (iii) determining the role of this complex in sensing/responding to protein translocation defects in the ER. In an independent aim, we will establish a novel functional link between a cytosolic quality control and IRE1α/XBP1 signaling. We plan to use a combined approach of CRISPR/Cas9 edited cells, biochemical reconstitution, and structural approaches to address these problems. Overall, we expect these studies will provide a mechanistic insight into how the UPR and protein translocation/quality control pathways work together to maintain ER homeostasis. The knowledge gained from these studies will inform the development of possible treatments for several human diseases including diabetes, cancer, and polycystic liver diseases.