Exocyst Complex Purification from Yeast

Exocyst complex purification was performed as described previously (Rossi et al., 2020 (link)). In brief, yeast strains containing a chromosomal copy of C-terminally-tagged Sec8-3xMYC and plasmids expressing wild-type Exo84 or Exo70, dominant mutations of Exo84, or the dominant Exo70-I114F mutant as the sole source of Exo84 or Exo70, respectively, were grown overnight at 30°C in synthetic media to mid-log phase (OD₅₉₉ 1.5). Cells were shifted to YPD (2% glucose) for 2 h to a final OD₅₉₉ of 3.0. To kill the cells, sodium azide and sodium fluoride were added to 20 mM final, then cells were spun at 6,700 × g_max in a JLA10.5 rotor for 6 min and washed with cold 10:20:20 buffer (10 mM Tris, pH 7.5, 20 mM NaN₃, and 20 mM NaF) before freezing on dry ice. Approximately 50 g of cells were lysed in a bead beater in lysis buffer (20 mM Pipes, pH 6.8, 120 mM NaCl, 1 mM EDTA, and 1 mM DTT) with protease inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 2 μg/ml antipain, 14 μg/ml pepstatin A, 2 mM 4-[2-aminoethyl] benzene-sulfonyl fluoride, and HCl). Lysates were cleared by centrifugation at 17,418 × g_max for 12 min at 4°C in a JA25.5 rotor, and the supernatant was removed and spun again at 50,000 × g_max for 30 min in a 41Ti rotor. The final supernatant concentration was adjusted to 30 mg/ml by Bradford assay before preclearing with Sepharose beads for 1 h at 4°C to reduce nonspecific binding. The lysate was spun for 5 min to remove the Sepharose beads and incubated overnight on ice with 9E10 monoclonal anti-myc antibody. Protein A Sepharose beads were added for 2 h at 4°C. The beads were washed three times in lysis buffer and then two times in cleavage buffer (20 mM Tris, pH 7.4, 140 mM NaCl, 0.1 mM EDTA, and 1 mM DTT) before cleaving in 1 ml of cleavage buffer with TEV enzyme for 4 h at 17°C. After removal of the Protein A Sepharose beads, the cleaved exocyst complexes were collected, aliquoted, and frozen at −80°C.