Fig. 5: E3 ligase RNF213 interacts with PorB and catalyzes polyubiquitination.

a MS/MS spectrum of RNF213 peptide identified by mass spectrometry analysis of proteins co-immunoprecipitated with PorB from HeLa cells. b Western blots after immunoprecipitation of PorB from HeLa cells show co-immunoprecipitation of RNF213. c HeLa cells expressing PorB show colocalization between PorB, HSP60 and RNF213. Scale bar, 5 μm. d Live HeLa cell microscopy and 3D image reconstruction shows RNF213 surrounding PorB on MitoBright-labeled mitochondria. Scale bar, 5 μm. The fluorescence colocalization profile of the line is shown. e Western blots showing PorB K171-dependent enhanced co-immunoprecipitation of ubiquitin from HeLa cells transfected with an RNF213 expression vector. f Western blots showing that transfection of HeLa cells with RNF213 siRNA abolishes PorB K171-dependent co-immunoprecipitation of ubiquitin. g Western blots showing PorB K117Q, but not PorB K171Q, enhances PorB-induced degradation of TOM20 and TIM23 in HeLa cells transfected with an RNF213 expression vector. h Western blots showing that transfection of HeLa cells with RNF213 siRNA prevents PorB-induced degradation of TOM20 and TIM23 for PorB K117Q, while degradation remains unaffected for PorB K171Q. Western blots in b, e–h are representative of 3 independent experiments. Images in c and d are representative of 3 independent experiments. Source data are provided as a Source Data file.