Nanodrop 1000 instrument

Yeast transformants were inoculated in 4 ml of SD medium and grown at 28°C with shaking at 140 rev./min. After 24 h, cells were inoculated into 200 ml of SD medium and grown for 24 h under the same conditions. Cells were harvested and induced for protein expression in 1 litre of YPG medium at 24°C with shaking at 140 rev./min. After 18–19 h of induction, cells were collected by centrifugation at 3000 g for 5 min at 4°C and washed in 25 ml of ice-cold water. After centrifugation, cells were resuspended in 10 ml of lysis buffer [40 mM Hepes/NaOH, pH 7.5, 150 mM NaCl, 26% (v/v) glycerol, 0.1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 5 μg/ml antipain and 157 μg/ml benzamidine HCl (Sigma–Aldrich)] and vortex-mixed with glass beads (200 μm; Sigma–Aldrich). The cell lysate was clarified by two consecutive centrifugation steps at 1000 g for 10 min at 4°C and 10000 g for 15 min at 4°C. Membranes were subsequently collected by centrifugation at 125000 g for 1 h at 4°C. Pellets were homogenized in 1.5 ml of S-buffer (same as lysis buffer but containing 20% (v/v) glycerol) and the protein concentration was measured spectrophotometrically using a Nanodrop™ 1000 instrument (Thermo Fisher Scientific). Total yeast membranes were snap-frozen in liquid nitrogen and stored at −80°C.