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J. Biol. Chem., Vol. 282, Issue 21, 15559-15568, May 25, 2007

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Flippase Activity Detected with Unlabeled Lipids by Shape Changes of Giant Unilamellar Vesicles^*

Andreas Papadopulos¹, Stefanie Vehring¹², Iván López-Montero³, Lara Kutschenko, Martin Stöckl, Philippe F. Devaux³, Michael Kozlov^¶, Thomas Pomorski⁴, and Andreas Herrmann⁵

From the Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, Institut für Biologie/Biophysik, Invalidenstrasse 42, D-10115 Berlin, Germany, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France, and ^¶Department of Physiology and Pharmacology, Sackler Faculty of Medicine, Tel Aviv University, 69978 Tel Aviv, Israel

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.

Received for publication, May 17, 2006 , and in revised form, March 16, 2007.

^* This work was supported in part by Deutsche Forschungsgemeinschaft Grant He1928 - 6 (to A. H.) and Po748 - 4 (to T. P.). A grant from the European Community, MRTN-CT-2004-005330 (to A. H., P. F. D., M. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4.

¹ These authors contributed equally.

² Received support from a Short Term Scientific Mission from COST D22 action on lipid protein interactions and from the Berliner Chancengleichheits-Programm N-318/06.

³ Supported by research grants from the CNRS (UMR 7099) and by Université Paris 7-Denis Diderot.

⁴ To whom correspondence may be addressed. Tel.: 49-30-2093-8830; Fax: 49-30-2093-8585; E-mail: thomas.pomorski{at}rz.hu-berlin.de. ⁵ To whom correspondence may be addressed. Tel.: 49-30-2093-8830; Fax: 49-30-2093-8585; E-mail: andreas.herrmann{at}rz.hu-berlin.de.

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