Figure EV5. Phospholipid balance promotes succession of autophagosome completion.

• A, B
  Δypt7 and Δopi3Δypt7 mutant cells expressing GFP‐Atg8 were grown to log phase in SD‐URA medium and shifted to SD‐N for 3 h, cell lysates were subjected to protease protection assay combined with immunoblot analysis (left panel). GFP‐Atg8 processing (A) or Ape1 Processing (B) were determined with or without addition of proteinase K (Prot. K), in the presence or absence of detergent (Triton). Protection of GFP‐Atg8/ ape1 may be assessed when proteinase K is added without detergent. Right panel (A)‐ protected GFP‐the ratio of GFP‐Atg8 to total GFP (GFP‐Atg8 + free GFP). Statistical analysis was done by Student's t‐test (paired, two tailed; **P ≤ 0.005), error bars represent SEM of at least 3 independent experiments. Right panel (B)‐ protected Ape1‐the ratio of prApe1 to total Ape1 (PrApe1 + mApe1). Statistical analysis was done by ANOVA multiple comparison Tukey's test (****P ≤ 0.0001, ***P ≤ 0.001).
• C
  Representative images of mNG‐tagged Atg8 colocalized with mScarletI‐Ape1 expressed under the CUP1 promoter on the background of Δymr1. WT, Δopi3, Δatg1 and Δopi3Δatg1 cells were grown to log phase in SD medium in the presence of 10 μM copper sulfate, and shifted to SD‐N in the presence of 10 μM copper sulfate. Images were taken during SD‐N (1–3 h) by widefield microscopy. Scale bar 1 μm.

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