Figure 2Domain 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 the new ATG2 LIR motif that directs GABARAP interaction (orange) and the previously identified WIPI4 interaction motif (YFS; purple box). Approximately 30 amino acids separate these two motifs in human ATG2A and ATG2B. Multiple sequence alignment of multiple species using Jalview highlights the conservation of these regions. Abbreviations for species: Hs - Homo sapiens; Mm - Mus musculus; Rn - Rattus norvegicus; Pt - Pan troglodytes; Xt - Xenopus tropicalis Dr - Danio rerio; Dm - Drosophila melanogaster; Ce - Caenorhabditis elegans; Sp - Schizosaccharomyces pombe; Sc - Saccharomyces cerevisiae. Myc-tagged ATG2A-wild type (WT) and ATG2A-mLIR (orange) were co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP in HEK293T cells, lysed and anti-GFP nanobodies coupled to agarose were used to immunoprecipitate GFP-tagged proteins. Samples were subjected to western blotting and probed for the presence of Myc-ATG2A in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   As in (B) but using Myc-tagged ATG2B-WT or ATG2B-mLIR (orange) co-expressed with GFP-alone, GFP-LC3B or GFP-GABARAP. Samples were subjected to western blotting and probed for the presence of Myc-ATG2B in immunoprecipitated samples. Anti-p62/SQSTM1 was used as an internal control for the immunoprecipitated samples.   HA-tagged ATG2A-WT, -mLIR (orange) and -mYFS (YFS/AAA; purple) were stably expressed in U2OS ATG2A/B double knock-out cells using retrovirus transduction. Cells were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and anti-HA beads were used to immunoprecipitate HA-tagged ATG2A and processed for western blot. Blots were then probed with antibodies against HA-tag (ATG2A), anti-WIPI4, and anti-GFP for the presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of WIPI4 and GFP-GABARAP-ii co-precipitation with HA-ATG2A from (D). bands were normalized against HA-ATG2A immunoprecipitate and expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) and error bars mean ± SD.   ATG2A/B do8uble knock-out cells (DKO) stably expressing HA-tagged ATG2A-WT, -mLIR (orange) or -mYFS (YFS/AAA; purple) were transfected with GFP-GABARAP and 24 h later grown in complete medium (CM) or starved for 2 h (EBSS). Cells were lysed and GFP-GABARAP was immunoprecipitated using anti-GFP trap beads. Samples were processed for western blot and probed with anti-HA-tag (ATG2A), anti-WIPI4, and anti-GFP for their presence/absence in immunoprecipitated samples. All blots are representative of at least n=3 independent experiments.   Quantification of HA-ATG2A and WIPI4 co-precipitation with GFP-GABARAP from (F). Results were expressed as a fold change compared to HA-ATG2A-WT (complete media; CM). Each symbol represents an independent experiment (n=3) with error bars mean ± SD.   GFP alone, GFP-GABARAP or GFP-GABARAP with increasing concentrations of mCherry-WIPI4 were expressed in HEK293T cells for 24 h, lysed and GFP-trap beads used to immunoprecipitate GFP alone or GFP-GABARAP. Samples were then probed with antibodies to detect endogenous ATG2A or ATG2B in immunoprecipitated samples. Blots are representative of n=3 independent experiments.   Quantification of ATG2A (green line, round symbols) and ATG2B (grey line and squares) co-precipitation with GFP-GABARAP in the presence of increasing concentrations of mCherry-WIPI4 from (H). Co-precipitation was normalised to GFP-GABARAP alone. Line and error bars are mean ±SD of n=3 independent experiments.