SAMM50 binds to ATG8 proteins via an LIR motif in the NTS. (A and B) GST pulldowns with GST-hATG8 proteins and in vitro translated WT or LIR mutant Myc-SAMM50. (C) Affinities (K_d values) of SAMM50 LIR peptides to hATG8 proteins determined by BLI. Color code indicates fold changes relative to WT GABARAP. (D and E) Structure of the SAMM50 LIR bound to GABARAP and GABARAPL1. (D) Close-up of chimera structure of SAMM50 LIR bound to GABARAP. SAMM50 LIR (aa 24–38) is in green ribbon with interacting residues as sticks and GABARAP in white cartoon and transparent surface with HP1 and HP2 colored in pink and blue surfaces, respectively. (E) Close-up of SAMM50 LIR bound to GABARAPL1. The LIR (aa 24–38) is in pink ribbon with interacting residues as sticks. GABARAPL1 is in white cartoon, and transparent surface with HP1 and HP2 colored in pink and blue, respectively. (F) Superposition of chimera structure of SAMM50 LIR chimera (green) bound to GABARAP and SAMM50 LIR peptide (pink) bound to GABARAPL1. Both LIRs are in cartoon with interacting residues as sticks. GABARAP and GABARAPL1 are in white cartoon, and transparent surface with HP1 and HP2 colored in pink and blue, respectively. (G and H) GST pulldowns of in vitro translated Myc-MTX1 and Myc-MTX2 with recombinant GST-hATG8s (G) or with GST-GABARAP WT and the Y49A mutant (H). (I) GST pulldowns of in vitro translated Myc-SAMM50 and Myc-MTX1 with recombinant GST-GABARAP and indicated mutants. (J) Immunoblots of lysates of WT, SAMM50 KD, and SAMM50 KD HeLa cells reconstituted with Myc-SAMM50 WT and Myc-SAMM50 Δ24–35 mutant untreated or treated with BafA1 for 24 h. (K) Relative protein levels in J from three independent experiments. Values are mean ± SD. **, P < 0.005; †, NS; one-way ANOVA. UDS, ubiquitin-interacting motif docking site.