Measuring Phospholipid Flippase Activity by NBD-Lipid Uptake in Living Yeast Cells

Phospholipid flippases in the P4-ATPase family are essential for establishing membrane asymmetry. These ATP-powered pumps translocate specific lipids from the exofacial leaflet to the cytosolic leaflet of the plasma membrane, thereby concentrating substrate lipids, such as phosphatidylserine, in the cytosolic leaflet while non-substrate lipids populate the exofacial leaflet. Here, we describe a method for measuring P4-ATPase transport activity in the yeast plasma membrane by using flow cytometry to quantify the uptake of lipids derivatized with a fluorescent [7-nitro-2-1,3-benzoxadiazol-4-yl)amino] (NBD) group on a short (C6) fatty acyl chain. The NBD-lipid uptake assay quantitatively measures P4-ATPase transport activity and substrate selectivity in the native membrane environment.