SAMM50 interacts with hATG8 proteins to regulate basal mitophagy. (A) Immunoblots of extracts of WT and HeLa cells with KO of all six hATG8 proteins untreated or treated with BafA1 for 24 h. (B) Relative protein levels for A from three independent experiments. Values are mean ± SD. **, P < 0.005; *, P < 0.01; †,NS; one-way ANOVA. (C) GST-pulldown assays with in vitro translated Myc-SAMM50 and recombinant GST, GST-LC3A, and GST-GABARAP. Values are mean ± SD from three independent experiments. **, P < 0.005; *, P < 0.01; one-way ANOVA. (D) Coimmunoprecipitation of endogenous SAMM50 from HeLa cells transiently transfected with 3x-Flag-LC3A or 3x-Flag-GABARAP. (E) Live-cell images of HeLa cells stably expressing EGFP-GABARAP and MIC19-mCherry. Arrows indicate colocalization between mitochondrial fragments and GABARAP. Scale bars, 10 µm. See Video 3. (F) Live-cell images of HeLa cells stably expressing EGFP-LC3A and MIC19-mCherry. Arrows indicate colocalization between mitochondrial fragments and LC3A. Scale bars, 10 µm. See Video 4. (G) GST-pulldown assay with in vitro translated Myc-SAMM50 and recombinant GST, GST-GABARAP, and GST-GABARAP F49A LDS mutant. Values are mean ± SD from three independent experiments. **, P < 0.005; one-way ANOVA. (H) Domain architecture of SAMM50 with positions of LIR, POTRA, and β-barrel domains. Peptide array of 20-mer peptides spanning full-length SAMM50 probed with GST-GABARAP and developed with GST antibody. Each peptide was moved three amino acids relative to the previous one. (I) Sequence alignment of canonical LIR motifs in human and mouse SAMM50 and other mitophagy receptors FKBP8, BCL2-L-13, FUNDC1, and autophagy receptors p62 and NBR1. (J and K) GST pulldowns with GST-tagged hATG8s and in vitro translated Myc-SAMM50 WT and the F28A/V31A mutant (J) quantified in K from three independent experiments. Values are mean ± SD. **, P < 0.005; *, P < 0.01; one-way ANOVA.
Figure 6.

SAMM50 interacts with hATG8 proteins to regulate basal mitophagy. (A) Immunoblots of extracts of WT and HeLa cells with KO of all six hATG8 proteins untreated or treated with BafA1 for 24 h. (B) Relative protein levels for A from three independent experiments. Values are mean ± SD. **, P < 0.005; *, P < 0.01; †,NS; one-way ANOVA. (C) GST-pulldown assays with in vitro translated Myc-SAMM50 and recombinant GST, GST-LC3A, and GST-GABARAP. Values are mean ± SD from three independent experiments. **, P < 0.005; *, P < 0.01; one-way ANOVA. (D) Coimmunoprecipitation of endogenous SAMM50 from HeLa cells transiently transfected with 3x-Flag-LC3A or 3x-Flag-GABARAP. (E) Live-cell images of HeLa cells stably expressing EGFP-GABARAP and MIC19-mCherry. Arrows indicate colocalization between mitochondrial fragments and GABARAP. Scale bars, 10 µm. See Video 3. (F) Live-cell images of HeLa cells stably expressing EGFP-LC3A and MIC19-mCherry. Arrows indicate colocalization between mitochondrial fragments and LC3A. Scale bars, 10 µm. See Video 4. (G) GST-pulldown assay with in vitro translated Myc-SAMM50 and recombinant GST, GST-GABARAP, and GST-GABARAP F49A LDS mutant. Values are mean ± SD from three independent experiments. **, P < 0.005; one-way ANOVA. (H) Domain architecture of SAMM50 with positions of LIR, POTRA, and β-barrel domains. Peptide array of 20-mer peptides spanning full-length SAMM50 probed with GST-GABARAP and developed with GST antibody. Each peptide was moved three amino acids relative to the previous one. (I) Sequence alignment of canonical LIR motifs in human and mouse SAMM50 and other mitophagy receptors FKBP8, BCL2-L-13, FUNDC1, and autophagy receptors p62 and NBR1. (J and K) GST pulldowns with GST-tagged hATG8s and in vitro translated Myc-SAMM50 WT and the F28A/V31A mutant (J) quantified in K from three independent experiments. Values are mean ± SD. **, P < 0.005; *, P < 0.01; one-way ANOVA.

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