Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.
Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.
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DOI：
10.1016/s0006-3495(01)75697-4
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发表时间：
2001-07
影响因子：
3.4
通讯作者：
Michael L. Wagner;L. Tamm
中科院分区：
文献类型：
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作者：
Michael L. Wagner;L. Tamm
根据可溶性N-乙基马来酰亚胺敏感因子（NSF）-附着蛋白（SNAP）受体假说（SNARE假说），靶SNARE和囊泡SNARE（t-和v-SNARE）之间的相互作用是细胞内囊泡运输和胞吐作用中膜融合所必需的。 SNARE 在系留、对接和融合中的确切作用仍然存在争议。对平面支撑膜中 SNARE 相互作用的生物物理测量可能会解决有关 SNARE 介导的膜融合机制的一些关键问题。作为实现这一目标的第一步，重组 Syntaxin1A/SNAP25 (t-SNARE) 被重构为聚合物支持的平面脂质双层。支持双层中重建的 t-SNARE 结合可溶性绿色荧光蛋白/囊泡相关膜蛋白 (v-SNARE)，并且 SNARE 复合物可以在 ATP 存在的情况下被 NSF/α-SNAP 特异性解离。因此，SNARE 复合体形成的生理活性在该重建的平面模型膜系统中得到了很好的再现。重建的 t-SNARE 的很大一部分（~75%）在磷脂酰胆碱脂质背景中横向移动，横向扩散系数为 7.5×10−9cm2/s。带负电荷的脂质减少了 t-SNARE 和脂质本身的移动部分。磷脂酰肌醇-4,5-二磷酸在降低复合物的横向移动性方面比磷脂酰丝氨酸更有效。讨论了酸性脂质-SNARE 相互作用如何改变脂质流动性的模型。
According to the solubleN-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/α-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (∼75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5×10−9cm2/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.