title: Cell-Based Lipid Flippase Assay Employing Fluorescent Lipid Derivatives.
abstract: P-type ATPases in the P4 subfamily (P4-ATPases) are transmembrane proteins unique for eukaryotes that act as lipid flippases, i.e., to translocate phospholipids from the exofacial to the cytofacial monolayer of cellular membranes. While initially characterized as aminophospholipid translocases, studies of individual P4-ATPase family members from fungi, plants, and animals show that P4-ATPases differ in their substrate specificities and mediate transport of a broader range of lipid substrates. Here, we describe an assay based on fluorescent lipid derivatives to monitor and characterize lipid flippase activities in the plasma membrane of cells, using yeast as an example.
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Maria S
authors.initials: MS
authors.lastname: Jensen
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Sara
authors.initials: S
authors.lastname: Costa
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark.
authors.forename: Thomas
authors.initials: T
authors.lastname: Günther-Pomorski
authors.affiliation: Department of Plant and Environmental Sciences, Centre for Membrane Pumps in Cells and Disease (PUMPKIN), University of Copenhagen, Thorvaldsensvej 40, 1871, Frederiksberg C, Denmark. rlo@plen.ku.dk.
authors.forename: Rosa L
authors.initials: RL
authors.lastname: López-Marqués
mesh_terms: D001393:Azoles / Q000737:chemistry*; D057075:Enzyme Assays / Q000379:methods*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D009578:Nitrobenzenes / Q000737:chemistry*; D049294:Phospholipid Transfer Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology
chemical_list: C510797:7-nitrobenz-2-oxa-1,3-diazol-4-yl; D001393:Azoles; D005456:Fluorescent Dyes; D008055:Lipids; D009578:Nitrobenzenes; D049294:Phospholipid Transfer Proteins
keywords: Back-exchange; Flow cytometry; Fluorescence; Heterologous expression; Phospholipid probes; Saccharomyces cerevisiae