Immunoprecipitation of Mitochondrial Proteins

Mitochondrial isolation and immunoprecipitation were performed as described previously (Van Vranken et al., 2018 (link)). Briefly, cell pellets were washed and lysed as described in the ‘Crude mitochondrial isolation’ section. 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, yPIC) with 10 mM N-ethylmaleimide (NEM) (Sigma, E3876) by applying 20 strokes in a dounce homogenizer. Crude mitochondria were isolated by differential centrifugation. Protein concentrations were determined using a Pierce BCA Protein Assay Kit (Thermo Scientific). 1 mg of crude mitochondria were resuspended in 200 μl of XWA buffer (20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, pH 7.4) with 10 mM NEM and 0.7% digitonin added and incubated on ice for 30 min. After centrifugation at 20,000 × g for 20 min, solubilized mitochondria were incubated with pre-equilibrated anti-HA antibody-conjugated agarose (Sigma, A2095) for 2 hr at 4°C. The agarose was washed three times and eluted by incubating in 2× Laemmli buffer at 65°C for 10 min. Elution was isolated by SDS-PAGE and subjected for immunoblot analysis.