TORC2-Ypk1 signaling and mitochondria regulate autophagy flux. (A–D) Cells carrying pRS416 pr^ATG8GFP-ATG8 were grown to log phase in SCD medium (A, C, and D) or synthetic complete galactose medium (B) without uracil and transferred to amino acid starvation medium with 2% glucose (A, C, and D) or galactose (B) as the carbon source. GFP and GFP-Atg8 protein levels were visualized by Western blot analysis as described in Materials and methods. Quantification of autophagy flux at 6 h of starvation is represented as a percentage of the ratio of free GFP to total GFP (GFP + GFP-Atg8) signal. Data are presented as means ± SD of three independent experiments for A, B, and D and four independent experiments for C. (E) WT and ypk1Δ cells were grown and starved as in A, and cells were harvested at the indicated time points. WT cells were also treated with 200 nM rapamycin in SCD medium for 1 h or 25 µg/ml cycloheximide (CHX) for 2 h. Protein extracts were run on 3–8% NuPage Tris-acetate gels, and Western blot analysis was performed using α-Atg13 and α-G6PDH antibodies. Asterisk denotes nonspecific protein band for loading reference.