Fig. 2: IpaH1.4 can mediate the ubiquitination and proteasomal degradation of RNF213.

a In vitro ubiquitination assays of IpaH1.4 with the RNF213(371-5207) C4516A mutant using a mixture of the un-labeled ubiquitin (Ub) and the fluorescent Cy5-labeled ubiquitin (Cy5-Ub) in the presence E1 (UBA1), E2 (UBE2D1), E3 (IpaH1.4), and ATP or in the absence of E1 (UBA1), E2 (UBE2D1), E3 (IpaH1.4), or ATP. The below panels showing the red fluorescent Cy5-labeled ubiquitin signals near the gel loading wells, which are corresponding to the ubiquitinated RNF213 proteins. b In vitro ubiquitination assays of IpaH1.4 with the RNF213(371-5207) C4516A mutant using un-labeled ubiquitin (Ub). Notably, the ubiquitinated RNF213 band used for MS-based analysis to identify ubiquitination sites is indicated with a red dashed box. c The summary of the IpaH1.4-mediated ubiquitination sites of RNF213 identified by mass spectrometry-based analysis. d The distribution of the identified ubiquitination sites in c on the domain schemes of human RNF213. The domain boundaries and the potential ubiquitination sites are labeled with black and blue, respectively. e Immunoblot analyses of lysates from HeLa cells expressing AcGFP-tagged RNF213 without or with the mCherry-tagged wild type IpaH1.4, IpaH1.4 C368A mutant, or IpaH1.4 NEL domain in the absence or in the presence of proteasomal inhibitor MG132 or autophagy inhibitor bafilomycin A1 (BafA1). For MG132 or BafA1 treated cells, a final concentration of 20 μM for MG132 or 100 nM for BafA1 were added into the culture medium 12 h before cell harvest. f Immunoblot analyses of lysates from HeLa cells over-expressing the mCherry-tagged wild type IpaH1.4 or IpaH1.4 C368A mutant. The endogenous RNF213 protein levels were monitored by a specific RNF213 antibody. g Immunoblot analyses of the cell lysates from HeLa cells infected for 24 h with the wild type (WT) S. flexneri strain, the ipaH1.4/ipaH2.5 double knock-out (ΔipaH1.4/ΔipaH2.5) S. flexneri strain, or the ΔipaH1.4/ΔipaH2.5 S. flexneri strain rescued with ipaH1.4, respectively. The endogenous RNF213 protein levels were monitored by a specific RNF213 antibody. Representative confocal micrographs of RNF213-knockout HeLa cells stably expressing AcGFP-tagged wild type RNF213 at 4 h after infection with the wild type S. flexneri M90T strain (h), the ipaH1.4 knockout (ΔipaH1.4) S. flexneri (i), the ipaH2.5 knockout (ΔipaH2.5) S. flexneri (j) or the ipaH1.4/ipaH2.5 double-knockout (ΔipaH1.4/ΔipaH2.5) S. flexneri (k). HeLa cells were stained with a specific antibody to ubiquitin (red), and DAPI (blue) to show the invaded bacteria and the nuclei of cells. WT wild type; Scale bars, 10 µm. Statistical result related to the percentage of cells containing cytosolic S. flexneri with RNF213-coat (l), or ubiquitin-coat (m) decorated on the surface at 4 h after infection. The data represent the mean ± SEM of 47, 48, 40, and 37 cells for each group, presented in the same order as in (l) or in (m). An unpaired ordinary one-way ANOVA analysis followed by Sidak multiple comparisons test was used to define a statistically significant difference.