In vitro FRET analysis of IRE1 and BiP association and dissociation upon endoplasmic reticulum stress.

Megan C Kopp,Piotr R Nowak,Christopher J Adams,Natacha Larburu,Maruf Mu Ali

doi:10.7554/elife.30257

Abstract

The unfolded protein response (UPR) is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER). The primary step in UPR activation is the detection of misfolded proteins, the mechanism of which is unclear. We have previously suggested an allosteric mechanism for UPR induction (Carrara et al., 2015) based on qualitative pull-down assays. Here, we develop an in vitro Förster resonance energy transfer (FRET) UPR induction assay that quantifies IRE1 luminal domain and BiP association and dissociation upon addition of misfolded proteins. Using this technique, we reassess our previous observations and extend mechanistic insight to cover other general ER misfolded protein substrates and their folded native state. Moreover, we evaluate the key BiP substrate-binding domain mutant V461F. The new experimental approach significantly enhances the evidence suggesting an allosteric model for UPR induction upon ER stress.

Highlights

The unfolded protein response is a key signaling system that regulates protein homeostasis within the endoplasmic reticulum (ER)
In order to generate a quantitative measure of association and dissociation between IRE1 luminal domain (LD) and BiP, we decided to make use of Forster resonance energy transfer (FRET) between two fluorophores: cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) (Martin et al, 2008; Pollok and Heim, 1999; Felber et al, 2004; Bajar et al, 2016)
As we have previously measured an interaction between the N-terminal ATPase domain of BiP and IRE1 LD (Kd = 1.33 mM), and our pull-down assays utilized an N-terminus-positioned affinity tag that had no impact on interaction (Carrara et al, 2015), we attached fluorescent proteins to the N-terminus of both constructs, with YFP connected via short linker to BiP, upstream of the ATPase domain; and CFP to IRE1 LD (Figure 1A)

Summary

Introduction

The unfolded protein response is a key signaling system that regulates protein homeostasis within the ER. Our model was based on the observation that a interaction between the luminal domain (LD) of the key UPR protein, IRE1, and the ATPase domain of BiP, an ER Hsp chaperone, dissociates upon the binding of CH1 misfolded protein to the canonical BiP substrate-binding domain. To observe this important step, we utilized a pull-down assay that qualitatively measured noncanonical dissociation (Carrara et al, 2015). The new experimental approach significantly enhances the evidence to suggest an allosteric model for UPR induction upon ER stress

Methods

Results

Conclusion