Nanobiology Institute, Yale University School of Medicine, New Haven, CT 06520.
Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06520.
Proc Natl Acad Sci U S A. 2023 Nov 7;120(45):e2311484120. doi: 10.1073/pnas.2311484120. Epub 2023 Oct 30.
The synaptic vesicle protein Synaptophysin (Syp) has long been known to form a complex with the Vesicle associated soluble N-ethylmaleimide sensitive fusion protein attachment receptor (v-SNARE) Vesicle associated membrane protein (VAMP), but a more specific molecular function or mechanism of action in exocytosis has been lacking because gene knockouts have minimal effects. Utilizing fully defined reconstitution and single-molecule measurements, we now report that Syp functions as a chaperone that determines the number of SNAREpins assembling between a ready-release vesicle and its target membrane bilayer. Specifically, Syp directs the assembly of 12 ± 1 SNAREpins under each docked vesicle, even in the face of an excess of SNARE proteins. The SNAREpins assemble in successive waves of 6 ± 1 and 5 ± 2 SNAREpins, respectively, tightly linked to oligomerization of and binding to the vesicle Ca sensor Synaptotagmin. Templating of 12 SNAREpins by Syp is likely the direct result of its hexamer structure and its binding of VAMP2 dimers, both of which we demonstrate in detergent extracts and lipid bilayers.
突触小泡蛋白 Synaptophysin（Syp）长期以来一直与囊泡相关可溶性 N-乙基马来酰亚胺敏感融合蛋白附着受体（v-SNARE）囊泡相关膜蛋白（VAMP）形成复合物，但由于基因敲除的影响极小，其在胞吐作用中的更具体的分子功能或作用机制一直缺乏。利用完全定义的重组和单分子测量，我们现在报告说 Syp 作为一种伴侣蛋白发挥作用，决定了在准备释放的囊泡与其靶双层膜之间组装的 SNAREpins 的数量。具体来说，即使存在过量的 SNARE 蛋白，Syp 也能指导每个停泊囊泡下 12±1 个 SNAREpins 的组装。SNAREpins 分别以 6±1 和 5±2 个 SNAREpins 的连续波组装，与囊泡钙传感器突触结合蛋白的寡聚化和结合紧密相关。Syp 对 12 个 SNAREpins 的模板化可能是其六聚体结构及其与 VAMP2 二聚体结合的直接结果，我们在去污剂提取物和脂质双层中都证明了这一点。
