Compiled data for all Figures_PNAS_2025_Teo_et al_TMEM16F_headgroup_selectivity.xlsx

In this study, we report a cell-based microplate phospholipid (PL) scrambling assay by monitoring the fluorescence polarization values of nitrobenzoxadiazole-labeled PLs (NBD-PLs) in the presence of albumin. The interaction between albumin and the outer leaflet-residing NBD-PLs on the plasma membrane results in a higher FP value than inner leaflet-residing NBD-PLs that are not accessible to albumin binding, hence enabling the distinction of NBD-PLs transbilayer distribution. When comparing the albumin-associated FP value with and without scrambling stimulation, it is possible to infer the net movement of PLs on the plasma membrane. We employed this method to investigate the headgroup preference of TMEM16F, a calcium-activated PL scramblase, using cells with or without endogenous TMEM16F expression, and revealed TMEM16F preferentially scrambles phospholipids with PC and PS headgroups over PE., we deciphered its headgroup preference on the plasma membrane. Coarse-grained molecular dynamics (CGMD) simulations support these findings.