title: Flippase activity detected with unlabeled lipids by shape changes of giant unilamellar vesicles.
abstract: Transbilayer movement of phospholipids in biological membranes is mediated by energy-dependent and energy-independent flippases. Available methods for detection of flippase mediated transversal flip-flop are essentially based on spin-labeled or fluorescent lipid analogues. Here we demonstrate that shape change of giant unilamellar vesicles (GUVs) can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Insertion of lipids into the external leaflet created an area difference between the two leaflets that caused the formation of a bud-like structure. Under conditions of negligible flip-flop, the bud was stable. Upon reconstitution of the energy-independent flippase activity of the yeast endoplasmic reticulum into GUVs, the initial bud formation was reversible, and the shapes were recovered. This can be ascribed to a rapid flip-flop leading to relaxation of the monolayer area difference. Theoretical analysis of kinetics of shape changes provides self-consistent determination of the flip-flop rate and further kinetic parameters. Based on that analysis, the half-time of phospholipid flip-flop in the presence of endoplasmic reticulum proteins was found to be on the order of few minutes. In contrast, GUVs reconstituted with influenza virus protein formed stable buds. The results argue for the presence of specific membrane proteins mediating rapid flip-flop.
authors: Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstrasse 42, D-10115 Berlin, Germany.
authors: Andreas
authors: A
authors: Papadopulos
authors: Stefanie
authors: S
authors: Vehring
authors: Iván
authors: I
authors: López-Montero
authors: Lara
authors: L
authors: Kutschenko
authors: Martin
authors: M
authors: Stöckl
authors: Philippe F
authors: PF
authors: Devaux
authors: Michael
authors: M
authors: Kozlov
authors: Thomas
authors: T
authors: Pomorski
authors: Andreas
authors: A
authors: Herrmann
mesh_terms: D001693:Biological Transport, Active; D002474:Cell-Free System; D004721:Endoplasmic Reticulum / Q000201:enzymology; D007700:Kinetics; D008567:Membranes, Artificial; D008954:Models, Biological; D009975:Orthomyxoviridae / Q000737:chemistry / Q000378:metabolism; D049294:Phospholipid Transfer Proteins / Q000737:chemistry* / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D008567:Membranes, Artificial; D049294:Phospholipid Transfer Proteins; D010743:Phospholipids; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins