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.