Lai Ying, Lou Xiaochu, Wang Chuqi, Xia Tian, Tong Jiansong
1] Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, United States [2].
Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University, Ames, Iowa 50011, United States.
Sci Rep. 2014 Apr 3;4:4575. doi: 10.1038/srep04575.
Synaptotagmin 1 (Syt1) is a major Ca(2+)-sensor that evokes neurotransmitter release. Here we used site-specific fluorescence resonance energy transfer (FRET) assay to investigate the effects of Syt1 on SNAREpin assembly. C2AB, a soluble version of Syt1, had virtually no stimulatory effect on the rate of the FRET at N-terminus of SNARE complex both with and without Ca(2+), indicating C2AB does not interfere with the initial nucleation of SNARE assembly. However, C2AB-Ca(2+) accelerated the FRET rate significantly at membrane proximal region, indicating C2AB-Ca(2+) promotes the transition from a partially assembled SNARE complex to the fusion-competent SNAREpin. Similar enhancement was also observed at the end of the transmembrane domain of SNARE proteins. The stimulatory effect disappeared if there was no membrane or only neutral membrane present.
突触结合蛋白1（Syt1）是一种引发神经递质释放的主要钙离子传感器。在此，我们使用位点特异性荧光共振能量转移（FRET）测定法来研究Syt1对SNAREpin组装的影响。C2AB是Syt1的一种可溶性形式，无论有无钙离子，它对SNARE复合物N端的FRET速率几乎没有刺激作用，这表明C2AB不会干扰SNARE组装的初始成核过程。然而，C2AB - Ca²⁺在膜近端区域显著加速了FRET速率，表明C2AB - Ca²⁺促进了从部分组装的SNARE复合物向具有融合能力的SNAREpin的转变。在SNARE蛋白跨膜结构域末端也观察到了类似的增强作用。如果没有膜或仅存在中性膜，则刺激作用消失。
