Figure 1

ATTO488-PE is an effective reporter of phospholipid scrambling in large unilamellar vesicles reconstituted with ER membrane proteins. (a) Schematic illustration of the scramblase activity assay. The assay makes use of large unilamellar vesicles that contain a trace amount of ATTO488-PE distributed equally between both leaflets and exploits the ability of dithionite to chemically reduce the ATTO488 fluorophore, thereby irreversibly eliminating its fluorescence. In protein-free liposomes (left), ATTO488-PE in the outer leaflet is bleached upon addition of membrane-impermeant dithionite, whereas the inner leaflet pool is protected, resulting in 50% loss of fluorescence. For proteoliposomes with a functional scramblase (right), ATTO488-PE exchanges rapidly between the leaflets, resulting in 100% loss of fluorescence upon dithionite addition. (b) Photostability of ATTO488-PE in giant unilamellar vesicles (GUVs). Fluorescence intensity of individual ATTO488-PE labelled GUVs (color-matched between the image cycles, n = 60) before and after five confocal imaging scans. The mean ± s.d. is indicated. (c) Representative traces showing fluorescence loss on adding dithionite to protein-free liposomes (green) and proteoliposomes (purple) reconstituted with ER membrane proteins. The arrowhead indicates dithionite addition. The horizontal dashed line corresponds to 50% loss of fluorescence. (d) Half-time of dithionite-mediated fluorescence reduction of ATTO488-PE and C6-NBD-PC reconstituted into liposomes. Results are presented as mean ± s.d. of at least three independent reconstitutions (unpaired t test with Welch’s correction, *P < 0.0005).