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

The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. . https://doi.org/10.1101/276147 doi: bioRxiv preprint domain of human amphiphysin (residues 2-242) into pGex4T2 using BamHI and EcoRI restriction sites. Epsin CTD was then ligated in frame with N-BAR using SalI and NotI restriction sites. The I-BAR domain of human IRSp53 (residues 1-250) was cloned by using site directed mutagenesis to introduce a stop codon at residue 251 in the pGex6P2 plasmid containing full-length IRSp53. The I-BAR domain of human IRSp53 fused to the C-terminal domain of rat AP180 (I-BAR-AP180 CTD, residues 328-896 of rat AP180) was cloned by first ligating the I-BAR domain of human IRSp53 (residues 1-250) into pGex4T2 using BamHI and EcoRI restriction sites. AP180 CTD was then ligated in frame with I-BAR using SalI and XhoI restriction sites. The pGex6P1 vector containing full-length C. elegans FCHo1 (residues 1-968) was generously provided by the Audhya Lab (University of Wisconsin -Madison) (Wang et al., 2016) . The F-BAR domain of C. elegans FCHo1 (residues 1-276) was cloned into the pGex4T2 vector using BamHI and EcoRI restriction sites.