A scissors mechanism for stimulation of SNARE-mediated lipid mixing by cholesterol
- aDepartment of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, IA 50011; and
- bDepartment of Chemistry and Chemical Biology and the Advanced ESR Technology Center, B52 Baker Laboratory, Cornell University, Ithaca, NY 14853; and
- cIntegrative Biology and Biotechnology, Pohang University of Science and Technology, Pohang 790-784, Korea
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Edited by Josep Rizo, University of Texas Southwestern, Dallas, TX, and accepted by the Editorial Board January 26, 2009 (received for review December 23, 2008)
Abstract
Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.
Footnotes
- 1To whom correspondence should be addressed. E-mail: colishin{at}iastate.edu
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Author contributions: Y.-K.S. designed research; J.T. and P.P.B. performed research; J.H.F. and Y.-K.S. contributed new reagents/analytic tools; J.T., P.P.B., J.H.F., and Y.-K.S. analyzed data; and J.T., P.P.B., and Y.-K.S. wrote the paper.
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The authors declare no conflict of interest.
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This article is a PNAS Direct Submission. J.R. is a guest editor invited by the Editorial Board.
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This article contains supporting information online at www.pnas.org/cgi/content/full/0813138106/DCSupplemental.
