*2.3. Phosphatase Assay*

Pho8Δ60-expressing strains (WT, *mmi1*Δ and *atg8*Δ) were grown in YPD medium to early log phase (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. The cells were collected at indicated time points, and the alkaline phosphatase activity of Pho8Δ60 was carried out as described previously [41,43,44]. In total, five OD600 equivalents of yeas<sup>t</sup> cells were harvested, washed once with ice-cold water, and once with wash buffer (0.85% *w*/*v* NaCl and 1mM PMSF) and resuspended in 500 μL lysis buffer (20 mM Pipes, pH 7.0, 0.5% *v*/*v* Triton X-100, 50 mM KCl, 100 mM potassium acetate, 10 mM MgSO4, 10 μM ZnSO4, and 1 mM PMSF). The cells were lysed with 250 μL equivalents of glass beads using a Fast-prep desintegrator 5 times for 20 s at 4 ◦C and incubated for 2 min on ice in-between. The lysates were centrifuged at 14,000× *g* for 5 min at 4 ◦C. The supernatant was collected and 100 μL of the supernatant was added to 400 μL reaction buffer (250 mM Tris-HCl, pH 8.5, 0.4% *v*/*v* Triton X-100, 10 mM MgSO4, and 1.25 mM p-nitrophenyl phosphate). Samples were incubated for 10 min at 30 ◦C before terminating the reaction by adding 500 μL of stop buffer (2 M glycine, pH 11). Production of p–nitrophenol was monitored by measuring the absorbance at 400 nm (A400) using a spectrophotometer (Helios Gamma Spectrophotometer, Unicam), and the concentration in nmol

of p–nitrophenol in the samples was calculated from a standard curve of commercial p–nitrophenol (Sigma) (0 to 100 nmol). Protein concentration in the extracts was measured with the PierceTM BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA), and the specific activity was calculated as nmol p–nitrophenol/min/mg protein. The statistical evaluation of phosphatase activity was performed by two-way analysis of variance (ANOVA) using R software.
