*2.5. GFP-Atg8 Processing Assay*

BY4741 WT, *mmi1*Δ, and *atg1*Δ strains transformed with plasmid pRS316\_GFPAUT7 [45] carrying GFP-Atg8 were grown in SC medium to OD600 ≈ 0.8 and then shifted to SD-N medium (after double wash with H2O and one wash with SD-N) or rapamycin was added to final concentration 200 nM. In indicated time points, samples were collected and normalized to OD600 ≈ 1 and 100% TCA was added to the final concentration of 12.5%. Samples were frozen at −80 ◦C for at least 1 h, centrifuged for 5 min at 15,000× *g*, and pellets were washed with an ice-cold 80% aceton and dried at room temperature. Dried pellets were resuspended in 50 μL of 0.1N NaOH and 1% *w*/*v* SDS and bath sonicated. Then 50 μL of 2 × SDS loading buffer (100 mM Tris-HCl pH 6.8, 4% *w*/*v* SDS, 20% *v*/*v* glycerol, 0.2% *w*/*v* bromphenol blue) with 0.1M DDT was added and the lysates were resolved by 10% SDS-PAGE and transferred to Protran nitrocellulose membrane (Sigma-Aldrich/Merck, St. Louis, MO, USA). The membrane was blocked with 5% *w*/*v* non-fat dried milk (Regilait, Macon, France) and incubated overnight with a mouse monoclonal anti-GFP antibody conjugated with horseradish peroxidase (sc-9996, Santa Cruz, USA) at 1:1000. As a loading control, detection of Pgk1 with anti-Pgk1 antibody (Abcam, ab113687, 1:10000) and goa<sup>t</sup> anti-mouse IgG conjugated with horseradish peroxidase (Thermo Fisher Scientific, No: 32430, 1:1000) was used. Proteins were detected by SuperSignalTM West Dura Substrate (Thermo Scientific, Rockford, IL, USA). In order to calculate the ratio of free GFP to Pgk1, Western blot signals were detected by G:BOX Chemi-XRQ gel documentary system (Syngene, Cambridge, UK) and quantified by ImageJ program [46].
