The intracellular trafficking pathway, macroautophagy, is a recycling and disposal service that can be upregulated during periods of stress to maintain cellular homeostasis. An essential phase is the elongation and closure of the phagophore to seal and isolate unwanted cargo prior to lysosomal degradation. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore elongation and closure, but little mechanistic detail is known about their function. We have identified a highly conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2‐WIPI4 interaction site. We show that the ATG2A‐GABARAP interaction mutants are unable to form and close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double‐knockout cells. In contrast, the ATG2A‐WIPI4 interaction mutant fully restored phagophore formation and autophagy flux, similar to wild‐type ATG2A. Taken together, we provide new mechanistic insights into the requirements for ATG2 function at the phagophore and suggest that an ATG2‐GABARAP/GABARAP‐L1 interaction is essential for phagophore formation, whereas ATG2‐WIPI4 interaction is dispensable.

The intracellular trafficking pathway, macroautophagy, acts as a recycling and disposal service that can be upregulated during periods of stress, to maintain cellular homeostasis.An essential transition point in the pathway is the sealing of the immature phagophore to form an autophagosome, isolating unwanted cargo prior to lysosomal degradation. However, little mechanistic detail is known about phagophore closure. Human ATG2A and ATG2B proteins, through their interaction with WIPI proteins, are thought to be key players during phagophore closure. We have identified a highly-conserved motif driving the interaction between human ATG2 and GABARAP proteins that is in close proximity to the ATG2-WIPI4 interaction site. We show that the ATG2-GABARAP interaction mutants are unable to close phagophores resulting in blocked autophagy, similar to ATG2A/ATG2B double knock-out cells. In contrast, the ATG2-WIPI4 interaction mutant fully restored phagophore closure and autophagy flux, similar to wild type ATG2. Taken together, we provide new mechanistic insights to the requirements for ATG2 function at the phagophore and suggest that an ATG2-GABARAP interaction is essential for phagophore closure, whereas ATG2-WIPI4 interaction is dispensable. Abbreviations: ATG2 -Autophagy-related protein 2; ESCRT -endosomal sorting complex required for transport; GABARAP -Gamma-aminobutyric acid receptor-associated protein: GIM -GABARAP Interaction Motif; GST -Glutathione S-transferase; LIR -LC3 interaction region; MAP1LC3 (LC3) -Microtubule-associated proteins 1A/1B light chain; PtIns3Pphosphatidylinositol-3-phosphate; PI3K -PtsIns3P kinase; SQSTM1 -Sequestosome-1; VPS34 -Vacuolar Protein sorting 34; WIPI -WD repeat domain phosphoinositide-interacting protein;