Upon endoplasmic reticulum (ER) stress, the transmembrane endoribonuclease Ire1α performs mRNA cleavage reactions to increase the ER folding capacity. It is unclear how the low abundant Ire1α efficiently finds and cleaves the majority of mRNAs at the ER membrane. Here, we reveal that Ire1α forms a complex with the Sec61 translocon to cleave its mRNA substrates. We show that Ire1α's key substrate, XBP1u mRNA, is recruited to the Ire1α-Sec61 translocon complex through its nascent chain, which contains a pseudo-transmembrane domain to utilize the signal recognition particle (SRP)-mediated pathway. Depletion of SRP, the SRP receptor or the Sec61 translocon in cells leads to reduced Ire1α-mediated splicing of XBP1u mRNA. Furthermore, mutations in Ire1α that disrupt the Ire1α-Sec61 complex causes reduced Ire1α-mediated cleavage of ER-targeted mRNAs. Thus, our data suggest that the Unfolded Protein Response is coupled with the co-translational protein translocation pathway to maintain protein homeostasis in the ER during stress conditions.DOI: http://dx.doi.org/10.7554/eLife.07426.001

In eukaryotic cells, roughly one-fourth of all mRNAs code for secretory and membrane proteins. This class of proteins must first be segregated to the endoplasmic reticulum, where they are either translocated into the lumen or inserted into the lipid bilayer. The study of these processes has long relied on their successful reconstitution in cell-free systems. The high manipulability of such in vitro systems has allowed the identification of key machinery, elucidation of their functional roles in translocation, and dissection of their mechanisms of action. Here, we provide the basic methodology for (i) setting up robust mammalian-based in vitro translation and translocation systems, (ii) assays for protein translocation, insertion, and topology, and (iii) methods to solubilize, fractionate, and reconstitute ER membranes. Variations of these methods should be applicable not only to forward protein translocation systems but also for dissecting other poorly understood membrane-associated processes such as retrotranslocation.

IRE1α is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1α forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1α complexes in cells with blue native PAGE immunoblotting. We find that IRE1α forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1α oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1α-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1α to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1α complex defines the extent of IRE1α activity and may determine cell fate decisions during ER stress conditions.DOI: http://dx.doi.org/10.7554/eLife.27187.001

The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, PERK, and ATF6α, 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, ATF6α complexes are reduced to smaller complexes on ER stress. In contrast, IRE1α complexes were not significantly increased in size on ER stress, unless IRE1α 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 IRE1α, ATF6α and, to a lesser extent, PERK. Furthermore, we captured the interaction between IRE1α 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.

Highlights d IRE1a forms an ER-stress-independent complex with the Sec61/Sec63 translocon d IRE1a binding to BiP chaperone relies on its association with Sec61/Sec63 d Sec63 attenuates IRE1a signaling by mediating BiP binding to IRE1a during ER stress d IRE1a remains activated in Sec63-deficient cells during prolonged ER stress

20unfolded proteins in the lumen of the endoplasmic reticulum (ER) and propagates the signal to 46 the cytosol. We have previously shown that IRE1α forms a complex with the Sec61 translocon 47 to cleave its substrate mRNAs (Plumb et al., 2015). This complex also regulates IRE1α 48 activation dynamics during ER stress in cells (Sundaram et al., 2017), but the underlying 49 mechanism is unclear. Here, we show that Sec63 is a subunit of the IRE1α/Sec61 translocon 50 complex. Sec63 recruits and activates BiP ATPase through its luminal J-domain to bind onto 51 IRE1α. This Sec63-mediated BiP binding to IRE1α suppresses the formation of higher-order 52 oligomers of IRE1α, leading to proper attenuation of IRE1α RNase activity during persistent ER 53 stress. Thus, our data suggest that the Sec61 translocon bridges IRE1α with Sec63/BiP to 54 regulate the dynamics of IRE1α activity in cells.

The endoplasmic reticulum (ER) localized unfolded protein response (UPR) sensors, IRE1α, 3 PERK, and ATF6α, are activated upon accumulation of misfolded proteins caused by ER 4 stress. It is debated whether these UPR sensors are activated either by the release of their 5 negative regulator BiP chaperone or directly binding to misfolded proteins during ER stress. 6Here we simultaneously examined oligomerization and activation of all three endogenous 7 UPR sensors. We found that UPR sensors existed as preformed oligomers even in unstressed

IRE1a is an endoplasmic reticulum (ER) localized endonuclease activated by misfolded proteins in the ER. Previously, we demonstrated that IRE1a forms a complex with the Sec61 translocon, to which its substrate XBP1u mRNA is recruited for cleavage during ER stress (Plumb et al., 2015). Here, we probe IRE1a complexes in cells with blue native PAGE immunoblotting. We find that IRE1a forms a hetero-oligomeric complex with the Sec61 translocon that is activated upon ER stress with little change in the complex. In addition, IRE1a oligomerization, activation, and inactivation during ER stress are regulated by Sec61. Loss of the IRE1a-Sec61 translocon interaction as well as severe ER stress conditions causes IRE1a to form higher-order oligomers that exhibit continuous activation and extended cleavage of XBP1u mRNA. Thus, we propose that the Sec61-IRE1a complex defines the extent of IRE1a activity and may determine cell fate decisions during ER stress conditions.