Fig. 2: Native VDAC1 scrambles phospholipids poorly.
From: Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase
Representative traces corresponding to channel assays performed on vesicles reconstituted with different amounts of VDAC2 (a) or VDAC1 (b) (Shown are protein concentrations corresponding to theoretical protein/vesicle copy number of 30, 10 and 2). c Protein-dependence plot showing functionalization of vesicles with channel activity, i.e., fraction of large unilamellar vesicles (LUVs) with at least one channel. PPR*, PPR (protein/phospholipid ratio (mg/mmol)), corrected to eliminate the contribution of empty vesicles41. The data were analyzed according to a Poisson statistics model for reconstitution of proteins into individual vesicles. A similar mono-exponential fit constant was obtained for both proteins (~2 mg/mmol). Representative traces corresponding to scramblase assays performed on vesicles reconstituted with different amounts of VDAC2 (d) or VDAC1 (e) as in (a and b). f Correlation between the fraction of LUVs showing channel activity (dithionite assay) and vesicles with scramblase activity (BSA-back extraction assay). Data points correspond to LUVs reconstituted with different amounts of protein (n = 4, including two independent reconstitutions). g, h Crosslinking of VDAC proteins after reconstitution into vesicles. Reconstituted LUVs were treated with EGS to crosslink proteins in proximity. The samples were analyzed by SDS-PAGE immunoblotting using antibodies against the N-terminal His tag. Reconstituted VDAC2 shows a significant population of dimers/multimers (g) whereas reconstituted VDAC1 is predominantly monomeric (h, side panel shows a brighter signal to enable visualization of a faint dimer band).
