Docking, Not Fusion, as the Rate-Limiting Step in a SNARE-Driven Vesicle Fusion Assay
Docking, Not Fusion, as the Rate-Limiting Step in a SNARE-Driven Vesicle Fusion Assay
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DOI：
10.1016/j.bpj.2011.03.015
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发表时间：
2011-05-04
影响因子：
3.4
通讯作者：
Weisshaar, James C.
中科院分区：
文献类型：
--
作者：
Smith, Elizabeth A.;Weisshaar, James C.
体外囊泡融合实验显示，在几十分钟到几小时的非生理性时间尺度上，监测t-SNARE和V-SNARE囊泡在原液中的脂质混合的融合非常缓慢。在这里，基于单囊泡、荧光共振能量转移的分析清楚地将包含全长陷阱的单个囊泡对的对接和融合步骤分开。对接效率极低，而且是速率限制步骤。重要的是，在两种检测中，对接和融合动力学是相似的(一种是将v-圈套囊泡拴在表面，另一种是v-圈套小泡在溶液中游离)。V(C)肽Synaptobrevin-2(Syb(57-92))的加入使对接效率提高了约30倍，但对接仍然是限速的。在V(C)肽存在下，融合步骤发生的时间尺度类似于10 S。在以往涉及批量融合分析的实验中，加入突触素/Ca(2+)、MUNC-18或复合蛋白加速了观察到的脂混合速度，这种增强可能来自对接步骤，而不是融合步骤。
In vitro vesicle fusion assays that monitor lipid mixing between t-SNARE and v-SNARE vesicles in bulk solution exhibit remarkably slow fusion on the nonphysiological timescale of tens of minutes to several hours. Here, single-vesicle, fluorescence resonance energy transfer-based assays cleanly separate docking and fusion steps for individual vesicle pairs containing full-length SNAREs. Docking is extremely inefficient and is the rate-limiting step. Of importance, the docking and fusion kinetics are comparable in the two assays (one with v-SNARE vesicles tethered to a surface and the other with v-SNARE vesicles free in solution). Addition of the V(C) peptide synaptobrevin-2 (syb(57-92)) increases the docking efficiency by a factor of similar to 30, but docking remains rate-limiting. In the presence of V(C) peptide, the fusion step occurs on a timescale of similar to 10 s. In previous experiments involving bulk fusion assays in which the addition of synaptotagmin/Ca(2+), Munc-18, or complexin accelerated the observed lipid-mixing rate, the enhancement may have arisen from the docking step rather than the fusion step.