Yeast strains were grown in 11 L yeast extract peptone dextrose media (20 g/L peptone, 20 g/L glucose, 10 g/L yeast extract) supplemented with 100 μg/mL ampicillin and 0.02% antifoam in a Microferm fermenter (New Brunswick Scientific) at 30 °C for 1 d (>22 h), with aeration of 34 cubic feet per hour and stirring at 300 rpm. All subsequent steps were performed at 4 °C. Cells were harvested by centrifugation at 4,000 × g for 15 min and resuspended in 1 mL/g lysis buffer (8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, 80 g/L sucrose, 20 g/L sorbitol, 20 g/L glucose, 5 mM 6-aminocaproic acid, 5 mM benzamidine hydrochloride, 5 mM ethylenediaminetetraacetic acid, 10 mg/L phenylmethylsulfonyl fluoride, pH 7.4). Cells were lysed with 0.5 mm glass beads in a bead beater (BioSpec), with six cycles of 1 min of bead beating and 1 min of cooling. Cell debris was removed by centrifugation at 4,000 × g for 15 min. Membranes were then collected by ultracentrifugation (Beckman L-90K, Ti70 rotor) at 145,000 × g for 40 min, resuspended in 0.5 mL/g lysis buffer using a Dounce homogenizer, and stored at −80 °C prior to protein purification.
Frozen membranes were thawed at room temperature, and all the purification steps were performed at 4 °C. Membranes were solubilized with 1% (w/v) n-dodecyl β-D-maltoside (DDM; Anatrace) and mixed for 30 min. Insoluble material was removed by ultracentrifugation at 158,000 × g for 1h (Beckman L-90K, Ti70 rotor), and membranes were filtered with a 0.45 μm syringe filter and applied to a 0.5 mL anti-FLAG M2 affinity gel in a column (Millipore Sigma) pre-equilibrated in DDM Tris-buffered saline (DTBS; 50 mM Tris-HCl, 150 mM NaCl, 0.02% [w/v] DDM, pH 7.4). The column was washed with 10 column volumes of DTBS, and protein was eluted with three column volumes of DTBS containing 150 μg/mL of 3×FLAG peptide and one column volume of DTBS without peptide. Protein was then concentrated to ~200 μL with a 100 kDa molecular weight cutoff (MWCO) Amicon Ultracentrifugal filter (Millipore Sigma) at 1,000 × g, diluted with 4 mL glyco-diosgenin Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, 0.004% [w/v] glyco-diosgenin [Anatrace], pH 7.4) and concentrated to ~200 μL in the same concentrator. For Vma12p-3×FLAG and Vma22p-3×FLAG, samples were further concentrated to ~1 to 2 mg/mL with a 100 kDa MWCO Vivaspin 500 centrifugal concentrator (Sartorius) at 12,000 × g. For Vma21p-3×FLAG, the sample was concentrated at 1,000 × g. Protein concentration was determined by bicinchoninic acid assay (Pierce).
Frozen membranes were thawed at room temperature, and all the purification steps were performed at 4 °C. Membranes were solubilized with 1% (w/v) n-dodecyl β-D-maltoside (DDM; Anatrace) and mixed for 30 min. Insoluble material was removed by ultracentrifugation at 158,000 × g for 1h (Beckman L-90K, Ti70 rotor), and membranes were filtered with a 0.45 μm syringe filter and applied to a 0.5 mL anti-FLAG M2 affinity gel in a column (Millipore Sigma) pre-equilibrated in DDM Tris-buffered saline (DTBS; 50 mM Tris-HCl, 150 mM NaCl, 0.02% [w/v] DDM, pH 7.4). The column was washed with 10 column volumes of DTBS, and protein was eluted with three column volumes of DTBS containing 150 μg/mL of 3×FLAG peptide and one column volume of DTBS without peptide. Protein was then concentrated to ~200 μL with a 100 kDa molecular weight cutoff (MWCO) Amicon Ultracentrifugal filter (Millipore Sigma) at 1,000 × g, diluted with 4 mL glyco-diosgenin Tris-buffered saline (50 mM Tris-HCl, 150 mM NaCl, 0.004% [w/v] glyco-diosgenin [Anatrace], pH 7.4) and concentrated to ~200 μL in the same concentrator. For Vma12p-3×FLAG and Vma22p-3×FLAG, samples were further concentrated to ~1 to 2 mg/mL with a 100 kDa MWCO Vivaspin 500 centrifugal concentrator (Sartorius) at 12,000 × g. For Vma21p-3×FLAG, the sample was concentrated at 1,000 × g. Protein concentration was determined by bicinchoninic acid assay (Pierce).
