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

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 f...

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Bibliographic Details
Published ineLife Vol. 7
Main Authors Kopp, Megan C, Nowak, Piotr R, Larburu, Natacha, Adams, Christopher J, Ali, Maruf Mu
Format Journal Article
LanguageEnglish
Published England eLife Sciences Publications Ltd 05.01.2018
eLife Sciences Publications, Ltd
Subjects
Allosteric properties
Allosteric Regulation
Bandwidths
Biochemistry and Chemical Biology
Deoxyribonucleic acid
DNA
Endoplasmic reticulum
Endoplasmic Reticulum Chaperone BiP
Endoplasmic Reticulum Stress
Endoribonucleases - metabolism
ER stress
Fluorescence Resonance Energy Transfer
FRET
Heat-Shock Proteins - metabolism
Homeostasis
Humans
in vitro protein analysis
IRE1
Protein Binding
Protein folding
Protein Serine-Threonine Kinases - metabolism
Proteins
Research Advance
Structural Biology and Molecular Biophysics
unfolded protein response
unfolded protein response
in vitro protein analysis
IRE1
biochemistry
ER stress
FRET
structural biology
biophysics
human
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Summary: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.
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ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.30257