Fig. 1: VDAC2 is a phospholipid scramblase.

a Schematic showing phospholipid transport from the ER to all bilayer leaflets of the mitochondrial double membrane. Non-vesicular mechanisms deliver ER-synthesized phospholipids to the cytoplasmic face of the outer mitochondrial membrane (OMM). The lipids are scrambled across the OMM by a lipid transporter (scramblase) before moving through the intermembrane space (IMS) and across the inner mitochondrial membrane (IMM). b Scramblase assay using [³H]phosphatidylinositol ([³H]PI). Protein-free-liposomes (L) or VDAC2-proteoliposomes (P) are reconstituted with [³H]PI and probed with PI-specific phospholipase C (PI-PLC), which hydrolyzes [³H]PI to diacylglycerol (DAG) and [³H]inositol cyclic phosphate ([³H]Inos-P). Only [³H]PI molecules in the outer leaflet are hydrolyzed in protein-free-liposomes, whereas all [³H]PI will be hydrolyzed in scramblase-containing vesicles where [³H]PI from the inner leaflet is scrambled to the outer leaflet. c Time-course of PI-PLC-mediated hydrolysis of [³H]PI in protein-free-vesicles (liposome) versus vesicles reconstituted with VDAC2 at a theoretical protein/vesicle copy number of 30. Data points are shown as mean and 95% CI, n = 4, and fitted to a single exponential function. d Channel assay using NBD-PC. Vesicles are reconstituted as in panel b, but with fluorescent NBD-PC instead of [³H]PI. The membrane-impermeant reductant dithionite bleaches approximately 50% of the NBD-PC fluorescence in protein-free liposomes (L), corresponding to those molecules located in the outer leaflet. For vesicles containing a VDAC2 channel, all NBD-PC molecules are expected to be bleached as dithionite can enter the vesicles. e Time-course of NBD-PC fluorescence (normalized to the starting value) in liposomes and VDAC2-vesicles on adding dithionite. f Scramblase assay using NBD-PC. Vesicles are reconstituted as in panel d and treated with fatty acid-free BSA which extracts NBD-PC from the outer leaflet. In complex with BSA, NBD-PC fluorescence is partly quenched, ~60% lower than when it is in the membrane. For protein-free liposomes, fluorescence is expected to drop by ~30%, whereas for scramblase-containing vesicles fluorescence is expected to drop by ~60%. g Time-course of NBD-PC fluorescence (normalized to the starting value) in liposomes and VDAC2-vesicles on adding BSA. Assays similar to those shown in panels e and g were performed 12 times with similar results.