Fig. 1.

The identification of the BATS domain required for autophagosome targeting and autophagy activation. (A). Schematic representation of the deletion mutants of Barkor. All mutants are tagged with hrGFP and FLAG. Autophagosome localization is defined as cytosolic puncta overlapping with LC3 upon CQ treatment. (B). Cells stably expressing Myc-LC3 were transfected with various Barkor/Atg14(L) fragments tagged with hrGFP. Cells were treated with 200 μM CQ for 2 h and stained with anti–c-Myc antibody for LC3 and green fluorescence for Barkor/Atg14(L) mutants. (C). U₂OS cells that overexpress full-length Barkor, BarkorΔ10aa and U₂OS parental cells were visualized using a transmission electron microscope. AVs are marked by black arrows. Scale bar, 2 μm. (D). AVs per cross-section were counted (control, 3.45 ± 0.35; Barkor/Atg14(L) Δ10aa, 3.5 ± 0.32; Barkor/Atg14(L) WT: 12.6 ± 0.9). (E). LC3 and tubulin were detected in cells described in C; 2-h treatment with 50 nM Bafilomycin was used to block lysosomal degradation.