Fig 2: ATG2A and ATG2B contain a highly conserved LC3 interaction region (LIR). Related to Fig 2 ADomain structure of ATG2A (green) and ATG2B (grey) proteins. Both ATG2s contain an N-terminal VPS13/chorein domain, ATG2 C-terminal autophagy domain (CAD motif) and ATG2 C-terminal domain. Position and sequence of putative ATG2 LC3 interaction regions (LIRs) as identified by iLIR and manual annotation. ATG2A has 5 potential LIRs, and ATG2B has 6 potential LIRs. See Table 1 for details.B–EMyc-tagged ATG2A wild type (WT) and putative LIR mutants, where the potential core motif was mutated to alanine, were used in a pull-down assay with GST-tagged mammalian ATG8 proteins. Shown are ATG2-mLIR#1 (B), ATG2-mLIR#2 (C), ATG2-mLIR#3 (D) and ATG2-mLIR#4 (E). ATG2-mLIR#5 is shown on (H). ATG2A-WT or ATG2A-mLIRs were overexpressed in HEK293T cells, and lysates were incubated with purified GST alone or GST-tagged LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 or GABARAP-L2. Samples were spun, washed and blotted for the presence/absence of Myc-tagged ATG2A using anti-Myc antibody. Anti-p62/SQSTM1 was used as an internal control for the GST pull-down samples. GST proteins were visualized by Ponceau S staining of membranes.F, GAs in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR proteins. Shown are ATG2B mLIR#1 (F) and ATG2B mLIR#4 (G). ATG2B-mLIR#2 was present on an alpha helix, whereas ATG2B-mLIR#3 and ATG2B-mLIR#5 were not expressed. Anti-p62/SQSTM1 was used as an internal control for the GST pull-down samples. All blots are representative of at least n = 3 independent experiments.HMyc-tagged ATG2A wild type (WT), ATG2A-mutant LIR #5 (mLIR; FCIL/AAAA) (upper blots) or Myc-tagged ATG2B-WT or ATG2B-mLIR (FCIL/AAAA; lower blots) were overexpressed in HEK293T cells, and lysates were incubated with purified GST alone or GST-tagged LC3A, LC3B, LC3C, GABARAP, GABARAP-L1 or GABARAP-L2. Samples were spun, washed and blotted for the presence/absence of Myc-tagged ATG2s using anti-Myc antibody. GST proteins were visualized by Ponceau S staining of membranes.IU2OS WT and ATG2A/B double-knockout cell total lysates, analysed for the presence/absence of autophagy marker proteins including ATG2A, ATG2B, p62/SQSTM1, LC3A, LC3B, LC3C, GABARAP-L1, GABARAP-L2 and vinculin used as loading control.JGFP alone, GFP-WIPI4 or GFP-WIPI4 with increasing concentrations of mCherry-GABARAP was expressed in HEK293T cells for 24 h, lysed and GFP-TRAP beads used to immunoprecipitate GFP alone or GFP-WIPI4. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B, anti-GFP and anti-GABARAP. Blots are representative of n = 3 independent experiments.KQuantification of ATG2A (blue line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-WIPI4 in the presence of increasing concentrations of mCherry-GABARAP from (J). Co-precipitation was normalized to GFP-WIPI4 alone. Line and error bars are mean ± SD of n = 3 independent experiments.