Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity
Differential Interaction of Tomosyn with Syntaxin and SNAP25 Depends on Domains in the WD40 β-Propeller Core and Determines Its Inhibitory Activity
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DOI：
10.1074/jbc.m113.515296
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发表时间：
2014-06-13
影响因子：
4.8
通讯作者：
Ashery, Uri
中科院分区：
文献类型：
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作者：
Bielopolski, Noa;Lam, Alice D.;Ashery, Uri
神经元胞外分泌依赖于可溶性n -乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物的有效形成，并受SNARE结合蛋白tomosyn的调节。为了获得关于tomosyn活性的新信息，我们研究了它在质膜（PM）上的迁移和组织与其他SNARE蛋白和胞吐抑制的关系。通过直接随机光学重建显微镜（dSTORM），我们发现tomosyn被组织成与syntaxin簇相邻的小簇。此外，我们发现tomosyn存在于syntaxin-tomosyn复合物和syntaxin-SNAP25-tomosyn复合物中。缺少β -螺旋桨核心537-578或897-917残基的Tomosyn突变体在PM上扩散更快，并且与SNAP25的结合减少，这表明这些突变体将PM上的Tomosyn -syntaxin-SNAP25复合物之间的平衡转移到Tomosyn -syntaxin复合物上。由于这些缺失突变体对胞吐的抑制作用较小，我们认为tomosyn的抑制作用是通过tomosyn-syntaxin- snap25复合物介导的，而不是通过tomosyn-syntaxin复合物介导的。这些发现首次描述了tomosyn在PM的动力学及其与胞吐抑制的关系。
Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537-578 or 897-917 from its beta-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.