SNARE-driven, 25-millisecond vesicle fusion in vitro.
Docking and fusion of single proteoliposomes reconstituted with full-length v-SNAREs (synaptobrevin) into planar lipid bilayers containing binary t-SNAREs (anchored syntaxin associated with SNAP25) was observed in real time by wide-field fluorescence microscopy. This enabled separate measurement of the docking rate k(dock) and the unimolecular fusion rate k(fus). On low t-SNARE-density bilayers at 37 degrees C, docking is efficient: k(dock) = 2.2 x 10(7) M(-1) s(-1), approximately 40% of the estimated diffusion limited rate. Full vesicle fusion is observed as a prompt increase in fluorescence intensity from labeled lipids, immediately followed by outward radial diffusion (D(lipid) = 0.6 microm2 s(-1)); approximately 80% of the docked vesicles fuse promptly as a homogeneous subpopulation with k(fus) = 40 +/- 15 s(-1) (tau(fus) = 25 ms). This is 10(3)-10(4) times faster than previous in vitro fusion assays. Complete lipid mixing occurs in <15 ms. Both the v-SNARE and the t-SNARE are necessary for efficient docking and fast fusion, but Ca2+ is not. Docking and fusion were quantitatively similar on syntaxin-only bilayers lacking SNAP25. At present, in vitro fusion driven by SNARE complexes alone remains approximately 40 times slower than the fastest, submillisecond presynaptic vesicle population response.
Liu
Tingting
T
Departments of Chemistry and Physiology, University of Wisconsin-Madison, Madison, WI 53706, USA.
Tucker
Ward C
WC
Bhalla
Akhil
A
Chapman
Edwin R
ER
Weisshaar
James C
JC
D007089:Image Enhancement / Q000379:methods; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry*; D008560:Membrane Fluidity; D008561:Membrane Fusion; D008856:Microscopy, Fluorescence / Q000379:methods*; D011510:Proteolipids / Q000737:chemistry; D013997:Time Factors
D008051:Lipid Bilayers; D008081:Liposomes; D011510:Proteolipids; C020485:proteoliposomes