Figure 5. CipB family recruits LC3 and... | Rockefeller University Press

Figure 5.

$CipB family recruits LC3 and ubiquitin to mitochondria during C. violaceum infection. (A and B) Confocal microscopy analysis of endogenous ubiquitin colocalization with MitoTracker in HeLa GSDMD−/− cells. Cells infected with C.v. WT (MOI = 10) for 3.5 h were incubated with 100 nM MitoTracker (magenta) for 45 min, then cell were stained with anti-ubiquitin antibody (cyan). Nuclei (gray) were stained with DAPI (A).Scale bar, 10 µm. Quantification of Pearson’s colocalization coefficient between ubiquitin and MitoTracker (B). Data are representative from three different experiments (mean ± SEM, n = 30 cells in each group). Unpaired two-sided Student’s t tests were performed. ****P < 0.0001. (C) Denature-IP analysis of endogenous ubiquitin and Flag-CipB or Flag-SipB in 293T cells. Cell lysates were subjected to denaturing immunoprecipitation (IP), and immunoprecipitates were eluted with SDS loading buffer for immunoblotting. (D) Colocalization of GFP-ubiquitin (cyan) with RFP-CipB truncations (magenta) in HeLa cells. Cells were transfected with RFP, RFP-CipB, RFP-CipB (1-424), or RFP-CipB (425-583) for 14 h. As a positive control, cells transfected with RFP/RFP-Parkin were treated with CCCP (10 µM, 6 h). Cells were analyzed by confocal microscopy. Scale bar, 10 μm. (E) Denaturing-IP analysis of HA-ubiquitin binding to Flag-tagged CipB mutants in 293T cells. Cells were lysed and subjected to denature-IP. The immunoprecipitate was eluted with FLAG peptide for immunoblotting. (F and G) Immunoblot analysis of LC3B-II (F) and Tom20 (G) levels in 293T cells transfected with the indicated CipB mutations. (H) Confocal microscopy analysis demonstrating the colocalization of RFP-CipB (magenta) with GFP-LC3C (cyan), BFP-ubiquitin (yellow), or Tom20 (gray) in HeLa cells. (I) Mitochondrial ubiquitin recruitment requires CipB ubiquitination. 293T cells transfected with RFP, RFP-CipB, or RFP-CipB (K218R) for 21 h were fractionated into cytosolic and mitochondrial component. Ubiquitin levels in whole-cell lysates, cytosolic, and mitochondrial fractions were determined by immunoblotting. (J) Colocalization analysis of CipB family effectors (magenta) with GFP-LC3C (cyan) and BFP-ubiquitin (yellow) in HeLa cells. Scale bar, 10 µm. All data are representative from three different experiments. Source data are available for this figure: SourceData F5.$

CipB family recruits LC3 and ubiquitin to mitochondria during C. violaceum infection. (A and B) Confocal microscopy analysis of endogenous ubiquitin colocalization with MitoTracker in HeLa GSDMD^−/− cells. Cells infected with C.v. WT (MOI = 10) for 3.5 h were incubated with 100 nM MitoTracker (magenta) for 45 min, then cell were stained with anti-ubiquitin antibody (cyan). Nuclei (gray) were stained with DAPI (A).Scale bar, 10 µm. Quantification of Pearson’s colocalization coefficient between ubiquitin and MitoTracker (B). Data are representative from three different experiments (mean ± SEM, n = 30 cells in each group). Unpaired two-sided Student’s t tests were performed. ****P < 0.0001. (C) Denature-IP analysis of endogenous ubiquitin and Flag-CipB or Flag-SipB in 293T cells. Cell lysates were subjected to denaturing immunoprecipitation (IP), and immunoprecipitates were eluted with SDS loading buffer for immunoblotting. (D) Colocalization of GFP-ubiquitin (cyan) with RFP-CipB truncations (magenta) in HeLa cells. Cells were transfected with RFP, RFP-CipB, RFP-CipB (1-424), or RFP-CipB (425-583) for 14 h. As a positive control, cells transfected with RFP/RFP-Parkin were treated with CCCP (10 µM, 6 h). Cells were analyzed by confocal microscopy. Scale bar, 10 μm. (E) Denaturing-IP analysis of HA-ubiquitin binding to Flag-tagged CipB mutants in 293T cells. Cells were lysed and subjected to denature-IP. The immunoprecipitate was eluted with FLAG peptide for immunoblotting. (F and G) Immunoblot analysis of LC3B-II (F) and Tom20 (G) levels in 293T cells transfected with the indicated CipB mutations. (H) Confocal microscopy analysis demonstrating the colocalization of RFP-CipB (magenta) with GFP-LC3C (cyan), BFP-ubiquitin (yellow), or Tom20 (gray) in HeLa cells. (I) Mitochondrial ubiquitin recruitment requires CipB ubiquitination. 293T cells transfected with RFP, RFP-CipB, or RFP-CipB (K218R) for 21 h were fractionated into cytosolic and mitochondrial component. Ubiquitin levels in whole-cell lysates, cytosolic, and mitochondrial fractions were determined by immunoblotting. (J) Colocalization analysis of CipB family effectors (magenta) with GFP-LC3C (cyan) and BFP-ubiquitin (yellow) in HeLa cells. Scale bar, 10 µm. All data are representative from three different experiments. Source data are available for this figure: SourceData F5.