Fig. 4: Lysine 63-linked polyubiquitination of PorB induces p62-dependent mitophagy.
From: Gonococcal OMV-delivered PorB induces epithelial cell mitophagy
a Western blots showing that knock-down of p62 in HeLa cells inhibits PorB K117Q-dependent degradation of TOM20 and TIM23. b Reduced p62 and HSP60 colocalization in HeLa cells expressing PorB K5 or Knull compared with PorB WT. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, Kruskal–Wallis with posthoc Dunn test (Empty-WT PorB: P < 10−15; WT PorB-PorB K5: P < 10−15; WT PorB-PorB Knull: P < 10−15). c Western blots of HeLa 4KO cells expressing WT or truncated p62 show that the p62 LIR domain and UBI domain are indispensable for PorB K117Q-induced degradation of TOM20 and TIM23. d Western blots showing co-immunoprecipitation of ubiquitin and p62 after immunoprecipitation of PorB WT and PorB K117Q. e Western blots showing co-immunoprecipitation of both K63-linked and K48-linked polyubiquitin, after immunoprecipitation of PorB from HeLa cells, which is enhanced with inhibitors for autolysosomal degradation (BafA1, 3-MA) for K63-linked ubiquitin or proteasomal degradation (MG132) for K48-linked ubiquitin. f Colocalization between HSP60 and K63-linked polyubiquitin, but not K48-linked polyubiquitin, is induced in HeLa cells expressing PorB. Scale bar, 5 μm. Data are mean ± s.d.; n = 50 cells, two-tailed Mann–Whitney test, K63 only Empty-PorB K117Q: P < 10−15. g Western blots after immunoprecipitation of HA-PorB from HeLa cells show co-immunoprecipitation of K63-linked polyubiquitin is dependent on PorB lysine 171 and co-immunoprecipitation of K48-linked polyubiquitin is dependent on PorB lysine 128. h Western blots showing PorB-induced degradation of TOM20 and TIM23 in HeLa cells is dependent on PorB lysines 117 and 171. i Western blots showing gonococcal OMVs expressing PorB K117Q/K171Q lost the ability to induce degradation of TOM20 and TIM23. Cells in b and f are from 3 independent experiments. Western blots in a, c, d, e, g, h, i are representative of 3 independent experiments. Source data are provided as a Source Data file.
