BAR scaffolds drive membrane fission by crowding disordered domains

Wilton T. Snead; Wade F. Zeno; Grace Kago; Ryan W. Perkins; J Blair Richter; Chi Zhao; Eileen M. Lafer; Jeanne C. Stachowiak

All proteins were expressed as N-terminal glutathione-S-transferase (GST) fusion constructs in BL21 E. coli cells following induction with 1 mM IPTG. Full-length amphiphysin, N-BAR, Amph CTD ∆SH3, I-BAR, I-BAR-AP180 CTD and F-BAR were induced at 30 °C for 6-8 h. N-BAR-epsin CTD was induced at 16 °C for 20 h. Full-length FCHo1 was induced at 12 °C for 24 h. Cells were harvested and bacteria were lysed using lysis buffer and probe sonication. For full-length FCHo1, lysis buffer was: 100 mM sodium phosphate pH 8.0, 5 mM EDTA, 5 mM DTT, 10% glycerol, 1 mM PMSF, 1% Triton X-100, 1x Roche protease inhibitor cocktail. For all other proteins, lysis buffer was: 500 mM Tris-HCl pH 8.0, 5 mM EDTA, 10 mM β-mercaptoethanol or 5 mM TCEP, 5% glycerol, 1 mM PMSF, 1% Triton X-100, 1x Roche or Pierce protease inhibitor cocktail. Proteins were purified from bacterial extracts by incubating with glutathione resin, followed by extensive washing (at least 10x column volumes). Full-length amphiphysin, N-BAR, Amph CTD ∆SH3, and F-BAR were cleaved directly from the resin using soluble HRV-3C (Thermo Fisher) or thrombin (GE Healthcare Life Sciences) proteases overnight at 4 °C with rocking. HRV-3C, which contained a GST tag, was removed by passage through a glutathione agarose column. Thrombin was removed with p-aminobenzamidine-agarose resin (Sigma-Aldrich). N-BAR-epsin CTD, I-BAR, and I-BAR-AP180 CTD were eluted with 15 mM reduced glutathione in 500 mM Tris-HCl pH 8.0, 5 mM EDTA, 10 mM βmercaptoethanol or 5 mM TCEP, 5% glycerol, 1 mM PMSF buffer. Full-length FCHo1 was eluted with 15 mM reduced glutathione in 100 mM sodium phosphate pH 8.0, 5 mM EDTA, 5 mM DTT, 10% glycerol, 1 mM PMSF buffer. The proteins were concentrated with EMD Millipore Amicon centrifugal filter units, desalted with Zeba Spin Desalting Columns (Fisher), and then incubated with either Thrombin CleanCleave Kit (Sigma-Aldrich), soluble HRV-3C, or soluble thrombin overnight at 4 °C with rocking. Cleaved GST was removed by passage through a glutathione agarose column. I-BAR-AP180 CTD and N-BAR-epsin CTD were further purified by gel filtration chromatography using a Superose 6 column equilibrated with 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 5 mM EGTA, 1 mM PMSF, 5 mM DTT. All proteins were stored as small aliquots or liquid nitrogen pellets at -80 °C.