Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

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.

Automated summary

A new fluorescence microscopy-based assay was developed to study phospholipid scramblase activity of membrane proteins. This assay involves bleaching the fluorescence of ATTO-dye labeled phospholipids with the membrane-impermeant reductant sodium dithionite. The methodology proved suitable for studying scramblase activity of yeast endoplasmic reticulum at the single vesicle level.