Synapsin is a novel Rab3 effector protein on small synaptic vesicles -: I.: Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals
Synapsin is a novel Rab3 effector protein on small synaptic vesicles -: I.: Identification and characterization of the synapsin I-Rab3 interactions in vitro and in intact nerve terminals
复制标题
DOI：
10.1074/jbc.m403293200
复制
发表时间：
2004-10-15
影响因子：
4.8
通讯作者：
Benfenati, F
中科院分区：
文献类型：
--
作者：
Giovedì, S;Vaccaro, P;Benfenati, F
突触蛋白是一类神经元特异性磷酸化蛋白，通过磷酸化依赖性结合突触囊泡和肌动蛋白细胞骨架来调节突触囊泡的胞吐作用。虽然上述观察结果强烈支持突触蛋白在突触囊泡储备池的组装和维持中的预对接作用，但最近的结果表明突触蛋白也可能参与胞吐的一些后期步骤。为了研究突触蛋白与神经末梢蛋白的其他相互作用，我们采用噬菌体展示文库技术来选择与突触蛋白I具有高亲和力结合的肽序列。针对肽YQYIETTR（syn 21）的抗体特异性识别Rab 3A，Rab 3A是一种突触囊泡特异性小G蛋白，参与胞吐的多个步骤。突触蛋白I和Rab 3A之间的相互作用，证实了纯化的突触囊泡上的光亲和标记实验和突触蛋白I和Rab 3A在完整的神经末梢之间的化学交联复合物的形成。突触蛋白I可以有效地共沉淀突触体提取物固定化重组Rab 3A在GTP依赖的方式。使用纯化的蛋白质在体外结合试验证实了Rab 3A-GTP的突触蛋白I的结合偏好，并揭示了突触蛋白I的COOH-末端区域和Rab 3A效应域是与Rab 3A发生相互作用所必需的。这些数据表明，突触蛋白I是一种新的Rab 3相互作用的突触囊泡，并建议突触蛋白Rab 3的相互作用可能参与调节突触囊泡内的神经末梢运输。
Synapsins, a family of neuron-specific phosphoproteins, have been demonstrated to regulate the availability of synaptic vesicles for exocytosis by binding to both synaptic vesicles and the actin cytoskeleton in a phosphorylation-dependent manner. Although the above-mentioned observations strongly support a pre-docking role of the synapsins in the assembly and maintenance of a reserve pool of synaptic vesicles, recent results suggest that the synapsins may also be involved in some later step of exocytosis. In order to investigate additional interactions of the synapsins with nerve terminal proteins, we have employed phage display library technology to select peptide sequences binding with high affinity to synapsin I. Antibodies raised against the peptide YQYIETSMQ (syn21) specifically recognized Rab3A, a synaptic vesicle-specific small G protein implicated in multiple steps of exocytosis. The interaction between synapsin I and Rab3A was confirmed by photoaffinity labeling experiments on purified synaptic vesicles and by the formation of a chemically cross-linked complex between synapsin I and Rab3A in intact nerve terminals. Synapsin I could be effectively co-precipitated from synaptosomal extracts by immobilized recombinant Rab3A in a GTP-dependent fashion. In vitro binding assays using purified proteins confirmed the binding preference of synapsin I for Rab3A-GTP and revealed that the COOH-terminal regions of synapsin I and the Rab3A effector domain are required for the interaction with Rab3A to occur. The data indicate that synapsin I is a novel Rab3 interactor on synaptic vesicles and suggest that the synapsin-Rab3 interaction may participate in the regulation of synaptic vesicle trafficking within the nerve terminals.