A fast, single-vesicle fusion assay mimics physiological SNARE requirements

被引:113
作者
Karatekin, Erdem [1 ,2 ,3 ]
Di Giovanni, Jerome [4 ,5 ]
Iborra, Cecile [4 ,5 ]
Coleman, Jeff [3 ]
O'Shaughnessy, Ben [6 ]
Seagar, Michael [4 ,5 ]
Rothman, James E. [3 ]
机构
[1] CNRS, Lab Dynam Membranaire & Malad Neurol, F-75005 Paris, France
[2] Univ Paris 05, UMR 8192, Inst Biol Physicochim, F-75005 Paris, France
[3] Yale Univ, Sch Med, Dept Cell Biol, New Haven, CT 06520 USA
[4] INSERM, UMR 641, F-13344 Marseille 15, France
[5] Univ Aix Marseille 2, Fac Med Secteur Nord, UMR 641, F-13344 Marseille 15, France
[6] Columbia Univ, Dept Chem Engn, New York, NY 10027 USA
关键词
lipid bilayer; membrane fusion; SNARE mechanisms; supported bilayer; MEMBRANE-FUSION; NEUROTRANSMITTER RELEASE; LIPID-BILAYERS; EXOCYTOSIS; PROTEINS; LIPOSOME; COMPLEX; EVENTS; CELLS; SPECIFICITY;
D O I
10.1073/pnas.0914723107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
Almost all known intracellular fusion reactions are driven by formation of trans-SNARE complexes through pairing of vesicle-associated v-SNAREs with complementary t-SNAREs on target membranes. However, the number of SNARE complexes required for fusion is unknown, and there is controversy about whether additional proteins are required to explain the fast fusion which can occur in cells. Here we show that single vesicles containing the synaptic/exocytic v-SNAREs VAMP/synaptobrevin fuse rapidly with planar, supported bilayers containing the synaptic/exocytic t-SNAREs syntaxin-SNAP25. Fusion rates decreased dramatically when the number of externally oriented v-SNAREs per vesicle was reduced below 5-10, directly establishing this as the minimum number required for rapid fusion. Docking-to-fusion delay time distributions were consistent with a requirement that 5-11 t-SNAREs be recruited to achieve fusion, closely matching the v-SNARE requirement.
引用
收藏
页码:3517 / 3521
页数:5
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