Co-immunoprecipitation

Digitonin-extracted mitochondria-enriched fractions of 1 × 10⁸ cells expressing the epitope-tagged protein of interest were solubilized for 15 min on ice in 20 mM Tris-HCl pH7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol containing 1X Protease Inhibitor mix (Roche, EDTA-free) and 1% (w/v) digitonin. After centrifugation (15 min, 20’000 g, 4°C), the lysate (input) was transferred to either 50 μl of HA bead slurry (anti-HA affinity matrix, Roche) or 30 μl c-myc bead slurry (EZview red anti-c-myc affinity gel, Sigma) both of which had been equilibrated in wash buffer (20 mM Tris-HCl pH 7.4, 0.1 mM EDTA, 100 mM NaCl, 10% glycerol, 0.2% (w/v) digitonin). After incubating at 4°C for 1 hr, the supernatant containing the unbound proteins was removed. The bead slurry was washed three times with wash buffer and the bound proteins were eluted by boiling the resin for 5 min in 2% SDS in 60 mM Tris-HCl pH 6.8 (IP). Five percent of both the input and the unbound proteins and 100% of the IP sample were subjected to SDS-PAGE and Western blotting.

For the stalled import intermediate, LDH-DHFR-HA expression was induced by tetracycline and respective cell cultures were supplemented with 1 mM sulfanilamide and 50 μM aminopterine (AMT) 12 hr before the experiment (Harsman et al., 2016). CoIP was performed as described above.