Immunoprecipitation of the COPI coat complex

For the TAP-tag immunoprecipitation experiments described in Figure 4, lysates were prepared from a total of 3000 OD₆₀₀ units of cells, pelleted, resuspended in 100 ml of spheroplast buffer (1.4 M sorbitol, 100 mM sodium phosphate, pH 7.5, 0.35% β-mercaptoethanol, and 0.5 mg/ml Zymolyase), and incubated for 30 min at 37°C. The spheroplasts were pelleted and washed twice with 150 ml of 1.7 M sorbitol and 20 mM HEPES (pH 7.4), and the spheroplast pellet was resuspended in 8 ml of lysis buffer (20 mM HEPES, pH 7.4, 4 mM EDTA, 1 mM dithiothreitol [DTT]) and Dounce homogenized. The resulting lysate was brought to 0.1 M NaCl, 1% NP-40, and incubated on ice for 20 min and then centrifuged for 30 min at 60,000 × g. The supernatants (4–12 mg protein) were then incubated with 30 μl of IgG–Sepharose beads (GE Healthcare) for 2 h at 4°C. The beads were washed three times with wash buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM DTT, 2.5 mM MgCl₂, 1% Triton X-100, and a protease inhibitor cocktail that included 1 μg/ml of aprotinin, chymostatin, antipain, pepstatin, and leupeptin) before they were eluted in 20 μl of 3X SDS–PAGE sample buffer and analyzed by Western blotting.