Abstract

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.

Highlights

  • The majority of secretory and membrane proteins enter the endoplasmic reticulum (ER) through the Sec61 protein translocation channel (Rapoport, 2007)

  • The concentration of the Sec61 translocon vastly outnumbers the concentration of IRE1a in the ER (Plumb et al, 2015; Kulak et al, 2014), suggesting that it could provide a barrier to IRE1a oligomerization

  • We addressed the question of how IRE1a activity is regulated during ER stress conditions

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Summary

Introduction

The majority of secretory and membrane proteins enter the endoplasmic reticulum (ER) through the Sec protein translocation channel (Rapoport, 2007). During ER stress, signaling pathways, collectively termed the Unfolded Protein Response (UPR), are activated in order to upregulate chaperones and folding enzymes, to reduce the influx of proteins into the ER, and to increase the capacity for ERassociated degradation (Walter and Ron, 2011). In this way, the UPR adapts cells to ER stress conditions and restores ER homeostasis. The UPR can trigger apoptosis during chronic or severe ER stress conditions, suggesting that UPR activity is tightly controlled in order to elicit the appropriate cellular response, whether pro-adaptive or pro-apoptotic (Hetz, 2012). Inappropriate activation of UPR signaling is linked to a number of disease states, including pancreatic beta cell death in diabetes (Back and Kaufman, 2012) and neuronal cell death in certain neurodegenerative diseases (Wang and Kaufman, 2016)

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