Both Atg8 homologue–binding activity and GAP activity of OATL1 were required for the delayed autophagosomal maturation. (A) Schematic representation of the OATL1 mutants used in this study. (B) MEF cells stably expressing T7-OATL1, T7–OATL1-WA, T7–OATL1-ED/AA, or none of these (control) were cultured under nutrient-rich (N), starved (S), or replenished (R) conditions. The cells cultured under each condition were fixed and stained with anti-LC3 antibody. The mean numbers of LC3-positive dots per cell are shown. Error bars represent the means ± SEM of representative data (n ≥ 100) from two independent experiments. ***, P < 0.001; one-way analysis of variance and Tukey posthoc test (compared with the control under the same conditions). (C) The same as in B, except that T7–OATL1-RK was used instead of T7–OATL1-WA or T7–OATL1-ED/AA. (D) Cell lysates from control, T7-OATL1–, T7–OATL1-WA–, and T7–OATL1-ED/AA–expressing cells cultured under the same conditions as in B were analyzed by immunoblotting with anti-T7 tag antibody (top), antiactin antibody (middle), and anti-LC3 antibody (bottom). (E) The same as in D, except that T7–OATL1-RK was used instead of T7–OATL1-WA or T7–OATL1-ED/AA.