Fig. 2.

Ats-1 nucleates autophagosomes involving ER autophagy proteins. (A and B) Co-IP assay for protein interaction between Ats-1–GFP and HA-Atg14L (A) or Myc-UVRAG (B). Cotransfected HEK293 cells were immunoprecipitated (IP) with preimmune IgG or anti (α)–Ats-1. Precipitates were immunoblotted (IB) with anti–Ats-1 and anti-HA or anti-Myc. (C) The diffuse pattern of Ats-1(∆N17)-GFP in transfected RF/6A cells. (D and E) Co-IP assay for protein interaction between HA-Atg14L, and Ats-1(∆N17)-GFP (D) or Cox8A-Ats-1(∆N17)-GFP (E). Cotransfected HEK293 cells were IP with preimmune IgG or α–Ats-1. Precipitates were IB with anti–Ats-1 and anti-HA. (F) Localization of Cox8A-Ats-1(∆N17)-GFP (green) with mitochondria labeled with anti–Mn-Sod (red). The boxed area is magnified on the right. (G–I) Localization of HA-Atg14L (G), HA-DFCP1 (H), and HA-LC3 (I) to Ats-1–GFP vesicles. Boxed areas are magnified on the right. (Scale bars: C, E, and G–I, 10 μm.) (J) Correlative light-electron microscopy for RF/6A cells expressing Ats-1–GFP or GFP alone. (i and iv) Fluorescence micrographs of cells expressing Ats-1–GFP and GFP, respectively. (Scale bars: 10 μm.) (ii and v) TEM of the fluorescent cells in i and iv. (Scale bars: 10 μm.) (iii and vi) Magnified boxed area in ii and v. Yellow pseudocolored areas denote autophagosomes. (Scale bars: 1.0 μm.) (K) Phosphorylated mTOR level in Ats-1–GFP-expressing cells. RF/6A cells transfected with plasmid encoding GFP or Ats-1–GFP were subjected to Western blot analysis using antibodies against actin, mTOR, and phosphorylated mTOR (p-mTOR). Rapamycin-treated RF/6A cells were used as positive control. The values under the bands show the relative ratios of band intensities of p-mTOR to actin, with the ratio in the GFP lane set as one.