A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.
A Chemical Controller of SNARE-Driven Membrane Fusion That Primes Vesicles for Ca(2+)-Triggered Millisecond Exocytosis.
复制标题
DOI：
10.1021/jacs.5b13449
复制
发表时间：
2016-04-06
影响因子：
15
通讯作者：
Kweon DH
中科院分区：
文献类型：
--
作者：
Heo P;Yang Y;Han KY;Kong B;Shin JH;Jung Y;Jeong C;Shin J;Shin YK;Ha T;Kweon DH
膜融合由通过拉链过程形成的SNARE复合物介导。在这里，我们开发了一种化学控制器的膜融合的进展。半融合状态被结合到SNARE复合物的多酚杨梅素所阻止。通过从SNARE复合物中去除杨梅素的漆酶来拯救膜融合的停滞。获救的半融合状态是亚稳态和长寿命的衰减常数为39分钟。这膜融合控制器被施加到描绘如何钙离子刺激融合孔形成在毫秒的时间尺度。我们发现，使用单囊泡融合试验，这种杨梅素引发囊泡与synaptotagmin 1同步响应生理浓度的Ca2+。当向半融合囊泡中加入10 µM Ca2+时，大多数囊泡以16.2 ms的时间常数迅速进入融合孔。因此，结果表明，当建立适当的囊泡引发时，由SNARE和突触结合蛋白1组成的最小胞吐膜融合机制能够在毫秒时间尺度内驱动膜融合。SNARE驱动的膜融合的化学控制器应该作为一个多功能的工具，研究不同的突触蛋白在离散的融合步骤中的不同作用。
Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca2+ stimulates fusion-pore formation in a millisecond timescale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca2+. When 10 µM Ca2+ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.