Figure 4.
CipB binds to LC3 via LIR motifs to promote mitophagy. (A) Domain architecture of CipB showing 4 LIR motifs (red) and the predicted transmembrane domain (TMD, black). LIR sequences: LIR1 (FESV), LIR2 (FVEL), LIR3 (FQAL), and LIR4 (FQRL). (B) Co-IP analysis of Flag-CipB with RFP-tagged human ATG8 proteins (hATG8s) in 293T cells. Cells were co-transfected with Flag-CipB and RFP-hATG8s for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (C) Colocalization of RFP-CipB with GFP-hATG8s in HeLa cells co-transfected with RFP-CipB (magenta) and GFP-hATG8s (cyan). Scale bar, 10 µm. (D) Quantification of Pearson’s correlation coefficients for RFP-CipB and GFP-hATG8s colocalization shown in (C). Mean ± SEM, n = 25 cells in each group. (E) Quantification of GFP-hATG8s puncta from (C). Mean ± SEM, n = 25 cells in each group. (F) Co-IP analysis of RFP-LC3C and Flag-tagged CipB family proteins in 293T cells treated as in B. (G) aa sequence of the LIR motif in CipB or CipB mFXXL(V). CipB mFXXL(V): CipB indicated site mutated to alanine. The LIR motif in CipB is highlighted in magenta font, while the LIR motif in CipB mFXXL(V) is highlighted in blue font. These abbreviations and symbols maintain consistent meaning throughout the article. (H) Co-IP of Flag-CipB or Flag-CipB mFXXL(V) with RFP-LC3C in 293T cells treated as in B. (I)In vitro pull-down assay of His-LC3C with GST-CipB or GST-CipB mFXXL(V). (J and K) Immunofluorescence of GFP-LC3B puncta. 293T cells stably expressing GFP-LC3B (cyan) were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V) (magenta) for 14 h and then analyzed by confocal microscope (J). Scale bar, 10 μm. (K) Quantification of GFP-LC3B puncta (n = 30 cells/group; ****P < 0.0001). (L) Immunoblot analysis of Tom20 levels in 293T cells transfected with indicated vectors for 21 h. (M) Co-IP of RFP-LC3C with Flag-CCT2, Flag-BFP, Flag-CipB, and Flag-CipB LIR mutants in 293T cells. Immunoprecipitation were performed with anti-FLAG M2 affinity gel, and precipitated proteins were detected with antibodies to Flag and RFP. mLIR1 (mutant of LIR1): F23A&V26A; mLIR2 (mutant of LIR2): F260A&L263A; mLIR3 (mutant of LIR3); F279A&L282A; mLIR4 (mutant of LIR4): F469A&L472A. (N–P) Western blot images showing levels of LC3B-II (N), Tom20 (O), and Tim23 (O) in 293T cells. Cells were transfected with indicated plasmid or treated with 10 µM CCCP plus 0.5 µM Baf A1 for 21 h. Cells were lysed and immunoblotted with indicated antibodies. Quantification of normalized Tom20 and Tim23 (P). (Q and R) Immunofluorescence of GFP-LC3B in 293T cells expressing GFP-LC3B (cyan). Cells were transfected with the RFP-tagged (magenta) plasmid and then analyzed by confocal microscope (Q). Scale bar, 10 µm. Quantification of the number of GFP-LC3B puncta per cell (R). Data are shown as mean ± SEM (n = 25 cells in each group). The data are representative of three independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (D, E, K, P, and R). ns, not significant; *P <0.05; **P <0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F4.

CipB binds to LC3 via LIR motifs to promote mitophagy. (A) Domain architecture of CipB showing 4 LIR motifs (red) and the predicted transmembrane domain (TMD, black). LIR sequences: LIR1 (FESV), LIR2 (FVEL), LIR3 (FQAL), and LIR4 (FQRL). (B) Co-IP analysis of Flag-CipB with RFP-tagged human ATG8 proteins (hATG8s) in 293T cells. Cells were co-transfected with Flag-CipB and RFP-hATG8s for 14 h. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity gel. The immunoprecipitate was eluted with SDS loading buffer and analyzed by immunoblotting. (C) Colocalization of RFP-CipB with GFP-hATG8s in HeLa cells co-transfected with RFP-CipB (magenta) and GFP-hATG8s (cyan). Scale bar, 10 µm. (D) Quantification of Pearson’s correlation coefficients for RFP-CipB and GFP-hATG8s colocalization shown in (C). Mean ± SEM, n = 25 cells in each group. (E) Quantification of GFP-hATG8s puncta from (C). Mean ± SEM, n = 25 cells in each group. (F) Co-IP analysis of RFP-LC3C and Flag-tagged CipB family proteins in 293T cells treated as in B. (G) aa sequence of the LIR motif in CipB or CipB mFXXL(V). CipB mFXXL(V): CipB indicated site mutated to alanine. The LIR motif in CipB is highlighted in magenta font, while the LIR motif in CipB mFXXL(V) is highlighted in blue font. These abbreviations and symbols maintain consistent meaning throughout the article. (H) Co-IP of Flag-CipB or Flag-CipB mFXXL(V) with RFP-LC3C in 293T cells treated as in B. (I)In vitro pull-down assay of His-LC3C with GST-CipB or GST-CipB mFXXL(V). (J and K) Immunofluorescence of GFP-LC3B puncta. 293T cells stably expressing GFP-LC3B (cyan) were transfected with RFP, RFP-CipB, or RFP-CipB mFXXL(V) (magenta) for 14 h and then analyzed by confocal microscope (J). Scale bar, 10 μm. (K) Quantification of GFP-LC3B puncta (n = 30 cells/group; ****P < 0.0001). (L) Immunoblot analysis of Tom20 levels in 293T cells transfected with indicated vectors for 21 h. (M) Co-IP of RFP-LC3C with Flag-CCT2, Flag-BFP, Flag-CipB, and Flag-CipB LIR mutants in 293T cells. Immunoprecipitation were performed with anti-FLAG M2 affinity gel, and precipitated proteins were detected with antibodies to Flag and RFP. mLIR1 (mutant of LIR1): F23A&V26A; mLIR2 (mutant of LIR2): F260A&L263A; mLIR3 (mutant of LIR3); F279A&L282A; mLIR4 (mutant of LIR4): F469A&L472A. (N–P) Western blot images showing levels of LC3B-II (N), Tom20 (O), and Tim23 (O) in 293T cells. Cells were transfected with indicated plasmid or treated with 10 µM CCCP plus 0.5 µM Baf A1 for 21 h. Cells were lysed and immunoblotted with indicated antibodies. Quantification of normalized Tom20 and Tim23 (P). (Q and R) Immunofluorescence of GFP-LC3B in 293T cells expressing GFP-LC3B (cyan). Cells were transfected with the RFP-tagged (magenta) plasmid and then analyzed by confocal microscope (Q). Scale bar, 10 µm. Quantification of the number of GFP-LC3B puncta per cell (R). Data are shown as mean ± SEM (n = 25 cells in each group). The data are representative of three independent experiments. P values were determined by one-way ANOVA with Tukey’s test. (D, E, K, P, and R). ns, not significant; *P <0.05; **P <0.01; ***P < 0.001; ****P < 0.0001. Source data are available for this figure: SourceData F4.

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