1. MITOL prevents ER stress‐induced apoptosis by IRE1α ubiquitylation at ER–mitochondria contact sites
AMITOL added polyubiquitin chains to K481 of IRE1α. Lysates of HEK293 cells transfected with each lysine mutant of IRE1α and indicated vectors were immunoprecipitated with anti‐FLAG antibody, followed by immunoblotting with indicated antibodies. 481: K481R; 545: K545R; 568: K568R. BIRE1α K481R interacted with MITOL. Lysates of HEK293 cells transfected with indicated vectors were immunoprecipitated with anti‐FLAG antibody, followed by immunoblotting with indicated antibodies. C, DOverexpression of IRE1α K481R‐induced apoptosis. MEFs were transfected with indicated vectors. After 24 h, these cells were stained with Annexin V‐FITC (C) or subjected by immunoblotting with indicated antibodies (D). Error bars represent SD (n = 3). *P < 0.05 (Student's t‐test). E, FEnhanced RIDD activity in IRE1α K481R. MEFs were transfected with indicated vectors 24 h prior to analysis (E, F). The RIDD activity was measured by qRT–PCR. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01 (Student's t‐test). GHyper‐oligomerization of IRE1α K481R. MEFs were transfected with IRE1α‐GFP or IRE1α K481R‐GFP. After 24 h, these cells were exposed with Tu for indicated periods and GFP signals were observed. The right panels show 5.5‐fold magnification images of the boxed regions. Percentages of cells with IRE1α foci were calculated from 100 cells by visual inspection in each independent experiment. Scale bar represents 10 μm. Error bars represent SD (n = 3). *P < 0.05, **P < 0.01 (Student's t‐test). HIncreased interaction of IRE1α K481R with BIM. Cells transfected with indicated vectors 24 h prior to immunoprecipitation, followed by immunoblotting with indicated antibodies.
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EMBO J, Volume: 38, Issue: 15, First published: 13 June 2019, DOI: ( /embj )