SGD Paper 
Oikawa D and Kimata Y (2011) Experimental Approaches for Elucidation of Stress-Sensing Mechanisms of the IRE1 Family Proteins. Methods Enzymol 490():195-216
Abstract: Endoplasmic reticulum (ER) stress, which is often regarded as the accumulation of unfolded proteins in the ER, triggers cellular protective events including the unfolded protein response (UPR). In the yeast S. cerevisiae, the UPR signaling pathway starts from the ER-located transmembrane protein Ire1, the activation of which eventually leads to transcriptional induction of various genes including those encoding ER-located molecular chaperones. Mammals have two Ire1 paralogues, of which IRE1alpha exhibits ubiquitous tissue expression. Here, we show how we have approached study of the molecular mechanisms by which ER stress activates the Ire1 family proteins. Immunoprecipitation analyses indicated that the ER-located chaperone BiP associates with IRE1alpha and yeast Ire1, while ER stress dissociates these complexes. We also devised experimental systems for exogenous expression of wild-type or mutant versions of IRE1alpha and yeast Ire1 at appropriate levels, in order to monitor correctly their activity in evoking downstream events. An IRE1alpha partial deletion mutant with which BiP poorly associates showed considerable activity even under nonstress conditions, whereas a BiP-nonbinding mutant of yeast Ire1 was almost normally regulated in an ER-stress dependent manner. This finding suggests that the dissociation of BiP is the principal determinant of IRE1alpha's activation upon ER stress, while yeast Ire1 is largely controlled by another factor(s). Based on in vitro ability to inhibit aggregation of denatured proteins, we deduce that the luminal domain of yeast Ire1, but not that of IRE1alpha, is capable of direct interaction with unfolded proteins. Since this ability of yeast Ire1 was abolished by a mutation impairing its cellular activity, we propose that yeast Ire1 is fully activated by its direct interaction with unfolded proteins.CI - Copyright A(c) 2011 Elsevier Inc. All rights reserved.
| Status: Published | Type: Journal Article | PubMed ID: 21266252 |
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