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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 8 4 nm ( Fig. S2G -I, see methods), which corresponds to a radius of gyration of approximately 5 nm (Hofmann et al., 2012; Sherman and Haran, 2006) , comparable to other disordered domains of similar molecular weight Kalthoff et al., 2002) . FCS experiments also showed that the size of Amph CTD ∆SH3 varied with the concentration of monovalent salt in the buffer (Fig. S2J) , consistent with the known sensitivity of highly-charged disordered proteins to changes in ionic strength (Srinivasan et al., 2014) . We next performed tethered vesicle experiments to assess membrane fission by Amph CTD ∆SH3. The protein included an N-terminal hexa-histidine (6his) tag to facilitate binding to membranes by the lipid DOGS-NTA-Ni (Fig. 3A) . Experiments revealed that Amph CTD ∆SH3 indeed drove the formation of highly curved fission products (Fig. 3B,C) . However, 100-fold greater concentration of Amph CTD ∆SH3 in solution (10 µM) was required to generate fission products of similar curvature to those observed with Amph-FL (100 nM) (see Fig. 2D ,F).