OATL1 was localized at LC3-positive isolation membranes and autophagosomes. (A and B) GFP-OATL1 was colocalized with autophagic marker proteins. NIH3T3 cells transiently expressing GFP-OATL1 under starved conditions were fixed and stained with anti-LC3 antibody (A) or anti-Atg12 antibody (B). Merged images are shown at the right. (C) GFP-OATL1 was localized at ring-shaped organelles only under starved conditions. MEF cells stably expressing GFP-OATL1 were cultured under nutrient-rich, starved, and replenished conditions. Images of living cells are shown. (D) GFP-OATL1 was clearly colocalized with mStr-LC3 in living cells. MEF cells stably expressing GFP-OATL1 and mStr-LC3 cultured under starved conditions were observed. A merged image is shown at the right. (E) Immuno-EM revealed colloidal gold particles, indicating the presence of GFP-OATL1, on the isolation membranes of autophagosome-like structures (arrows) under starved conditions. M, mitochondria. (F–H) GFP-OATL1 was localized at the Golgi apparatus in autophagy-deficient cells. (F) MEF cells transiently expressing GFP-OATL1 were cultured under starved conditions in the presence (wortmannin) or absence (control) of 100 nM wortmannin, then fixed, and stained with anti-LC3 antibody and anti-GM130 antibody. (G) Wild-type and Atg5-knockout (Atg5-KO) MEF cells transiently expressing GFP-OATL1 were cultured under starved conditions, fixed, and stained with anti-LC3 antibody and anti-GM130 antibody. (H) NIH3T3 cells stably expressing mStr-Atg4B-CA or not expressing mStr-Atg4B-CA (control) were transiently transfected with pEGFP-C1-OATL1 (GFP-OATL1). The cells were cultured under starved conditions, fixed, and stained with anti-Atg16L1 antibody. In F–H, the signal color in the merged images is indicated by the color of the typeface. The insets show magnified views of the boxed areas. Bars: (A–D and F–H) 10 µm; (C, inset) 2 µm; (E) 100 nm.