Interaction assays in yeast and cultured cells confirm known and identify novel partners of the synaptic vesicle protein synaptophysin
Interaction assays in yeast and cultured cells confirm known and identify novel partners of the synaptic vesicle protein synaptophysin
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DOI：
10.1016/j.neuroscience.2008.07.033
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发表时间：
2008-10-02
期刊：
影响因子：
3.3
通讯作者：
Leube, R. E.
中科院分区：
文献类型：
--
作者：
Felkl, M.;Leube, R. E.
突触素（SYP）是一种主要的蛋白质的神经递质含有囊泡跨越膜四次，并有助于突触囊泡周期的各个方面。使用分裂泛素酵母双杂交系统来表征膜结合的全长小鼠SYP的分子相互作用。以这种方式，已知的嗜同性SYP-SYP协会可以确认和异嗜性结合SYP的其他tetraspan囊泡膜蛋白的分泌载体相关的膜和突触回蛋白类型可以检测到的第一次。还观察到vSNARE小突触蛋白2和各种膜和膜相关蛋白的SYP结合。进行鼠视网膜的双标记免疫荧光显微术、共免疫沉淀实验和荧光蛋白标记的多肽之间的荧光能量共振转移（FRET）分析，以验证和进一步表征SYP与四面体囊泡膜蛋白分泌载体相关膜蛋白1和突触回蛋白3、与小突触蛋白2和新鉴定的结合伴侣磷脂酶D4的关联，stathmin样3、Rho家族GTP酶2和ADP-核糖基化因子相互作用蛋白2。据观察，SYP的羧基末端是与膜结合多肽结合所必需的，而与膜结合蛋白结合则是必需的。后者似乎是由磷酸化调节，因为src同源2-结构域显示连接到多个羧基末端磷酸酪氨酸残基的SYP。总之，SYP与不同的tetraspan囊泡膜蛋白的关联表明共享的功能和多种其他相互作用确定SYP作为膜平台的一部分，作为突触囊泡周期的各个步骤的促进剂。(C)2008年IBRO。由爱思唯尔有限公司出版。保留所有权利。
Synaptophysin (SYP) is a major protein of neurotransmitter-containing vesicles spanning the membrane four times and contributing to various aspects of the synaptic vesicle cycle. The split-ubiquitin yeast two-hybrid system was used to characterize molecular interactions of membrane-bound, full-length murine SYP. In this way, the known homophilic SYP-SYP association could be confirmed and heterophilic binding of SYP to other tetraspan vesicle membrane proteins of the secretory carrier-associated membrane- and synaptogyrin-type could be detected for the first time. SYP-binding was also observed for the vSNARE synaptobrevin2 and various membrane and membrane-associated proteins. Double labeling immunofluorescence microscopy of murine retina, co-immunoprecipitation experiments and fluorescence energy resonance transfer (FRET) analyses between fluorescent protein-tagged polypeptides were carried out to validate and further characterize the association of SYP with the tetraspan vesicle membrane proteins secretory carrier-associated membrane protein 1 and synaptogyrin3, with synaptobrevin2, and the newly identified binding partners phospholipase D4, stathmin-like3, Rho family GTPase2 and ADP-ribosylation factor interacting protein2. It was observed that the carboxyterminus of SYP is dispensable for association with integral membrane proteins while it is needed for binding to membrane-associated polypeptides. The latter appears to be regulated by phosphorylation, since src homology 2-domains were shown to attach to the multiple carboxyterminal phosphotyrosine residues of SYP. In conclusion, the association of SYP with different tetraspan vesicle membrane proteins suggests shared functions and the multiple other interactions identify SYP as part of a membrane platform acting as a facilitator of various steps of the synaptic vesicle cycle. (C) 2008 IBRO. Published by Elsevier Ltd. All rights reserved.