RT Journal Article
SR Electronic
T1 Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.04.01.437912
DO 10.1101/2021.04.01.437912
A1 Patricia P. M. Mathiassen
A1 Anant K. Menon
A1 Thomas Günther Pomorski
YR 2021
UL http://biorxiv.org/content/early/2021/04/01/2021.04.01.437912.abstract
AB Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.Competing Interest StatementThe authors have declared no competing interest.