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J. Biol. Chem., Vol. 279, Issue 36, 37951-37955, September 3, 2004

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SNAREs Prefer Liquid-disordered over "Raft" (Liquid-ordered) Domains When Reconstituted into Giant Unilamellar Vesicles^*

Kirsten Bacia, Christina G. Schuette, Nicoletta Kahya, Reinhard Jahn, and Petra Schwille¶

From the Dresden University of Technology, Institute of Biophysics, Tatzberg 47-51, 01307 Dresden, Germany and Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany

Membrane domains ("rafts") have received great attention as potential platforms for proteins in signaling and trafficking. Because rafts are believed to form by cooperative lipid interactions but are not directly accessible in vivo, artificial phase-separating lipid bilayers are useful model systems. Giant unilamellar vesicles (GUVs) offer large free-standing bilayers, but suitable methods for incorporating proteins are still scarce. Here we report the reconstitution of two water-insoluble SNARE proteins into GUVs without fusogenic additives. Following reconstitution, protein functionality was assayed by confocal imaging and fluorescence auto- and cross-correlation spectroscopy. Incorporation into GUVs containing phase-separating lipids revealed that, in the absence of other cellular factors, both proteins exhibit an intrinsic preference for the liquid-disordered phase. Although the picture from detergent resistance assays on whole cells is ambiguous, reconstitutions of components of the exocytic machinery into GUVs by this new approach should yield insight into the dynamics of protein complex associations with hypothesized liquid-ordered phase microdomains, the correspondence between detergent-resistant membranes and liquid-ordered phase, and the mechanism of SNARE-mediated membrane fusion.

Received for publication, June 23, 2004

^* 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 Figs. S1-S4.

^¶ To whom correspondence should be addressed. Tel.: 49-351-463-40-328; Fax: 49-351-463-40-342; E-mail: pschwil{at}gwdg.de.

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