Whole-cell extracts and mitochondrial isolation

For whole-cell extracts, cells were grown to log phase in YPD or synthetic complete media with Dex or Leu to select for plasmids. Whole-cell lysates of 0.25 OD₆₀₀ cells were obtained by alkaline extraction (0.255 M NaOH and 1% β-mercaptoethanol), precipitated with 9% trichloroacetic acid, washed with acetone, dried, and resuspended in 50 µl MURB protein sample buffer (100 mM MES, pH 7.0, 1% SDS, 3 M urea, and 10% β-mercaptoethanol). 10 µl of sample was used for Western blot analysis.

For isolation of mitochondria, cells were grown to log phase in synthetic complete media with Dex or Leu to select for plasmids. Cells were grown to log phase, and 500 OD₆₀₀ cells were centrifuged at 2,700 rpm. Cell pellets were washed with water, resuspended in NMIB (0.6 M sorbitol, 20 mM Hepes, 100 mM KOAc, 5 mM MgCl₂, 50 mM KCl, and 1 mM PMSF) and 1× Protease Inhibitor Mixture I (Calbiochem), flash-frozen dropwise in liquid N₂, and lysed using a Freezer/Mill (SPEX). Cell lysates were centrifuged at 4,500 rpm for 5 min, and supernatant was centrifuged at 10,500 rpm for 15 min. Pellets were resuspended in 400 µl NMIB with Inhibitor Mixture I. 25 µg protein, determined by Bradford or BCA assay, was added to MURB sample buffer and used for Western blot analysis.