Laboratory of Neurobiology, Max-Planck Institute for Multidisciplinary Sciences, Göttingen, Germany.
Department of Chemistry, University of Virginia, Charlottesville, Virginia, USA.
J Biol Chem. 2024 Aug;300(8):107591. doi: 10.1016/j.jbc.2024.107591. Epub 2024 Jul 19.
Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature-dependent with a reduced concentration at 37 °C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25 and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.
神经细胞胞吐需要三个 SNARE 蛋白（突触融合蛋白相关蛋白 25（SNAP25）和突触小泡相关膜蛋白 25（synaptobrevin）位于囊泡膜上，以及 syntaxin 位于质膜上）的组装。然而，该过程的确切步骤以及由调节蛋白控制的组装和融合的关键点尚不清楚。在本工作中，我们使用时间分辨荧光和电子顺磁共振波谱法的组合，在体外研究 SNARE 组装过程中的动力学和中间状态。我们表明，syntaxin 会迅速形成二聚体，然后形成动力学稳定的 2:1 syntaxin:SNAP25 复合物，并且不存在多余的 SNAP25 时不会减少 2:1 复合物。此外，2:1 复合物是温度依赖性的，在 37°C 时浓度降低。SNAP25 的两个片段表现不同。SNAP25 的 N 端 SN1 片段的骨架有序性从 N 端到 C 端显著增加，而 C 端 SNAP25 片段 SN2 则没有。SNAP25 的 SN1 和 SN2 片段都可以与 syntaxin 组装；然而，尽管 SN1 片段与 syntaxin 的结合产生稳定的 2:2（SN1:syntaxin）复合物，但 SN2 与 syntaxin 形成的复合物则主要是无序的。突触小泡相关膜蛋白 25 单独不能与 syntaxin 结合，但在 SN1 或 SN2 片段存在的情况下会与 syntaxin 结合；然而，突触小泡相关膜蛋白 25 与 syntaxin 的复合物仍然是无序的。总的来说，这些数据表明，突触小泡相关膜蛋白 25 和 syntaxin 不会在没有 SNAP25 的情况下组装，并且 SNAP25 的 SN2 片段是最后进入 SNARE 复合物的。
