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. 2012 Jul 23;198(2):219–233. doi: 10.1083/jcb.201202061

Figure 5.

Figure 5.

Atg9 is embedded in the autophagosomal outer membrane. (A) Atg9 co-migrates with autophagosomes. ypt7Δ cells and ypt7Δ atg11Δ atg17Δ cells expressing GFP-Atg8 were converted to spheroplasts, treated with rapamycin for 5 h, and then ruptured by passage through a membrane filter with 5-µm pores. After removal of cell debris, the total lysates were loaded onto 8–30% (vol/vol) OptiPrep linear gradients and centrifuged at 200,000 g for 1 h. Van1 (Golgi) and Pep12 (endosome) were used as organelle markers. prApe1, a proform of Ape1; Btm, bottom. (B) Atg9 is exposed outside the autophagosomal membrane. After density gradient centrifugation, the autophagosome-enriched fractions (fractions 8–10 in A) were mixed, divided into three aliquots, and then treated with 100 µg/ml proteinase K (PK) for 20 min on ice with or without 0.3% Triton X-100 (TX-100). To exclude free Atg9 vesicles, fractions 8–10 were used as autophagosome-enriched fractions. proc. GFP, the processed form of GFP moiety; deg. prApe1, the degraded form of prApe1. (C) Total lysates were prepared as in A from ypt7Δ cells and ypt7Δ atg11Δ atg17Δ cells treated with rapamycin for 5 h. The total lysates (T) were either treated with 2 M urea or 200 µg/ml proteinase K for 20 min on ice or mock treated (buffer) and then separated into pellet (P17) and supernatant (S17) fractions by centrifugation at 17,400 g for 15 min. (D) ATG9-6×HA ypt7Δ cells were treated with rapamycin for 3 h and then subjected to pre-embedding immuno-EM using anti-HA antibody. Arrowheads indicate gold-enhanced NanoGold particles. (E) Wild-type (WT) cells and atg11Δ atg17Δ cells expressing Atg9-6×FLAG were converted to spheroplasts and treated with rapamycin for 2 h. Total lysates were prepared as in A. Total lysates were centrifuged at 15,000 g for 10 min; supernatant fractions (S15) were subjected to immunoisolation using the anti-FLAG antibody and eluted with the 3×FLAG peptide. The eluted fraction (lane 6) was treated with 1 µM recombinant Atg4 for 10 min at 30°C (lane 8) or mock treated (lane 7). Atg9-6F, Atg9-6×FLAG; prPho8, a proform of Pho8; Un, unbound fractions; E50×, eluted fractions concentrated 50-fold.