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The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 Measurement of SUPER template membrane release SUPER template membrane shedding experiments were performed according to the protocol of Neumann et al . 10 µL of SUPER templates were gently pipetted into the top of a 90 µL solution of protein at specified concentrations in 20 mM MOPS pH 7.35, 150 mM NaCl, 0.5 mM EGTA and EDTA, 5 mM TCEP buffer. SUPER templates were allowed to slowly settle for 30 min at room temperature without disturbing. SUPER templates containing unreleased membrane were then sedimented by gentle centrifugation at 300 g for 2 min in a swinging bucket rotor. 75 µL of supernatant containing released membrane was collected and mixed in a 96-well plate with Triton X-100 at a final concentration of 0.1% and volume of 100 µL. In order to measure the total fluorescence of SUPER template membrane, a detergent control consisting of SUPER templates added directly to 0.1% Triton X-100, which solubilized all SUPER template membrane, was run. The fluorescence intensity of released membrane was measured in a plate reader using 590 nm excitation light and an emission filter centered at 620 nm. After subtracting the fluorescence of 0.1% Triton X-100 in buffer alone from all measurements, membrane release was calculated by dividing the fluorescence intensity after protein exposure by the fluorescence intensity of the detergent control. The background level of membrane release in the absence of protein was also measured by incubating SUPER templates in buffer alone. This buffer control was subtracted from all measurements as background.