Atg9 localizes around the autophagosome in ypt7Δ cells. (A) ATG9-2×GFP atg1^D211A cells (top), ATG9-2×GFP atg1^D211A atg11Δ VPH1-TagRFPT cells (middle), and ATG9-2×GFP atg1^D211A atg11Δ atg17Δ cells (bottom) were starved for 1 h (Stv., 1 h) in the presence of 1 mg/ml cycloheximide (Chx.). After starvation, nutrient-rich medium was added to the culture and incubated for 15 min (re-add, 15 min). Nut., nutrient. (B) ATG9-2×GFP atg1^D211A atg11Δ cells were treated with rapamycin for 1 h. Atg1^D211A-mCherry (PAS), Vph1-TagRFPT (vacuole), Anp1-TagRFPT (Golgi), and Idh1-TagRFPT (mitochondria) were used as organelle markers. The Atg9-2×GFP clusters observed in these cells represent the PAS (labeled with Atg1^D211A-mCherry) but not clusters accumulated at the Golgi apparatus (labeled with Anp1-TagRFPT). (C) Cytoplasmic Atg9 vesicles individually assemble to the PAS. ATG9-2×GFP atg1^D211A-mCherry atg11Δ cells were treated with rapamycin for 1 h and observed at 32 ms/frame (see also Video 8). Arrows and arrowheads indicate cytoplasmic mobile Atg9 vesicles and Atg9 clusters assembled at the PAS, respectively. Outlines indicate the edges of cells. (D) Intracellular behavior of Atg9-2×GFP is altered in a manner dependent on autophagosome formation. ATG9-2×GFP ypt7Δ cells and ATG9-2×GFP ypt7Δ atg11Δ atg17Δ cells were starved for 4 h and observed at 32 ms/frame (see also Video 9). Arrowheads indicate immobile Atg9-positive structures. (E) Atg9 localizes around the autophagosome. ATG9-2×GFP mCherry-Atg8 ypt7Δ atg11Δ cells were starved for 4 h and observed at 3,000 ms/frame. The rectangle indicates the autophagosome analyzed in F. (F) The autophagosome highlighted in E was analyzed using the line scan analysis function of MetaMorph software. (G) The cells used in E were starved for 4 h and subjected to EM. AP, autophagosomes.