*2.6. Fluorescence Microscopy*

Fluorescence microscopy was performed using an Olympus IX-81 inverted microscope equipped with a motorized stage, a 100× PlanApochromat oil-immersion objective (NA = 1.4) and a Hamamatsu Orca-ER-1394 digital camera. Images were processed using Olympus Cell-R™ Xcellence, Adobe CS5 software packages. The same exposure time was used to detect GFP signal in all tested strains. To quantify the cellular distribution of GFP-Atg8 signal, cells were co-stained with a vacuolar marker FM4-64 (1 μg/mL, 1 h, 30 ◦C) and scored into three categories: vacuole & cytosol, cytosol, and vacuole. In the category "vacuole and cytosol" the GFP signal was evenly distributed within the cells and there were no signal gradients between vacuole and cytosol. In the category "cytosol" the higher GFP signal was detected in cytosol compared to vacuole. Finally, in the category "vacuole" the higher GFP signal was detected in vacuole compared to cytosol. The cells with oversaturated signal were excluded from the quantification.

### *2.7. Oxygen Radicals' Measurement by Dihydroethidium Staining*

Dihydroethidium (DHE) staining was performed as described in Neklesa and Davis, 2008 [47]. In total 1 × 10<sup>7</sup> cells/mL were stained with 15 μg/mL of dihydroethidium (DHE) in YPD for 1 h at 30 ◦C under shacking. Cells were washed in PBS, reinoculated in PBS, and in total 20,000 cells were analyzed in a Flow Cytometer BD LSRII (BD Biosciences, USA). To detect specifically the DHE oxidation product hydroxyethidium, an excitation wavelength of 405 nm was used. This wavelength is the closest available wavelength to a distinct hydroxyethidium excitation maximum of 396 nm that is not present for other DHE oxidation products [48]. The signal was collected by using emission filter 576/26, and cell viability was evaluated by Hoechst 33,258 (1 μg/mL). The data were analyzed by FlowJo software and mean fluorescence DHE intensity values were used to compare the total DHE fluorescence of the strains.

### *2.8. Viability Assay for Testing Rapamycin Sensitivity*

To compare the sensitivity of WT and *mmi1*Δ strains to rapamycin, we incubated exponentially growing cells in 24 well plate in YPD with 0, 5, 6, 8, 10, and 15 nM of rapamycin. The cells were seeded to initial OD ≈ 0.1 and incubated overnight at 30 ◦C in EonTM Miroplate Spectrophotometer (BioTek) under agitation. Every 15 min OD600 was determined in each well by Gen5 Microplate Reader and Imager Software. Due to increased concentration of rapamycin were obtained outgrowth curves with a distinct shift in the curves as cells lose viability. For each concentration time shift for OD ≈ 0.5 in the outgrowth curve relative to the curve of untreated cells was determined and viability curves were calculated as described in details in [49].
