Crude Mitochondrial Isolation from Yeast

Crude mitochondrial isolation was performed as described previously (Van Vranken et al., 2018 (link)). Cell pellets were resuspended in TD buffer (100 mM Tris–SO₄, pH 9.4 and 100 mM DTT) and incubated for 15 min at 30°C. Cells were then washed once in SP buffer (1.2 M sorbitol and 20 mM potassium phosphate, pH 7.4) and incubated in SP buffer with 0.3 mg/ml lyticase (Sigma, L4025) for 1 hr at 30°C to digest the cell wall. Spheroplasts were washed once and homogenized in ice-cold SEH buffer (0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.4, 1 mM PMSF, yeast protease inhibitor cocktail [Sigma, P8215]) by applying 20 strokes in a dounce homogenizer. Crude mitochondria were isolated by differential centrifugation at 3000 × g first to remove larger debris and 10,000 × g to pellet down mitochondria. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific, 23225).