Fig. 7
Rendering GABARAP more LC3B-like impairs NDP52 degradation. a Control (wild-type) and hexa ATG8 CRISPR knockout (KO) HeLa cell lines stably expressing indicated MYC-ATG8 constructs or empty vector (EV) in full medium, Earle’s balanced salt solution (EBSS) (SM) or EBSS + bafilomycin A1 (BafA1) (SMB) for 7 h prior to lysis and immunoblot with indicated antibodies. Expression of MYC-ATG8 constructs was induced by 1 µg/ml doxycycline for 6 days. Note, immunoblot of non-induced cells showing equal p62 and NDP52 protein levels in the reconstituted hexa ATG8 CRISPR KO HeLa cell lines is included as Supplementary Fig. 7a. b, c Quantification of p62 (b) and NDP52 (c) protein levels (normalized to actin) shown in a. Statistical analysis of (SM) using one-way analysis of variance (ANOVA) test; mean ±s.d.; data from four (p62) and three (NDP52) independent experiments. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. d Structure of FYCO1 LIR motif bound to LC3B (PDB ID 5WRD). Non-conserved LIR docking site (LDS) residues of LC3B are displayed in white sticks. LC3B is displayed in white cartoon and the FYCO1 LIR in green transparent cartoon. Hydrophobic pocket (HP) 1 and HP2 are indicated in pink and blue, respectively. e Structure of PCM1 LIR motif bound to GABARAP (P212121). Non-conserved LDS residues of GABARAP are displayed in white sticks. GABARAP is displayed in white cartoon and the PCM1 LIR motif is shown in orange transparent cartoon. f Surface electrostatic potential of FYCO1:LC3B structure in the same orientation as shown in d. g Surface electrostatic potential of PCM1:GABARAP (P212121) structure with ULK1 LIR superimposed (pink cartoon). f, g Projections shown are −5 (red) and +5 (blue) kT/e using pymol apbs plugin with red and blue representing negative and positive potential, respectively. Red dashed lines encircle basic patch in LC3B (d, f) and extension of HP2 in GABARAP (e, f)
