Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells
Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells
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DOI：
10.1042/bj20030427
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发表时间：
2003-10-15
影响因子：
4.1
通讯作者：
Rowe, T
中科院分区：
文献类型：
--
作者：
Buxton, P;Zhang, XM;Rowe, T
SNARE（可溶性N-乙基马来酰亚胺敏感性融合蛋白附着蛋白受体）超家族成员[突触融合蛋白、VAMP（囊泡相关膜蛋白）和SNAP 25（突触体相关蛋白-25）相关蛋白]是真核生物细胞内膜融合事件所必需的。在神经元中，包含突触前膜相关SNARE突触融合蛋白I和SNAP 25以及囊泡相关SNARE VAMP 2的SNARE核心复合物的组装对于突触囊泡胞吐是必需的。已经描述了与突触SNARE相关并调节核心复合物组装或介导Ca 2+调节的几种辅助因子。据报道，一种这样的因子Snapin是脑特异性蛋白，其与SNAP 25相互作用，并调节推定的Ca 2 +-传感器突触结合蛋白与突触SNARE复合物的缔合[Ilardi，Mochida和Sheng（1999）Nat.Neurosci.2004，1999 - 1999]。2，119-124]。在这里，我们证明了Snapin在神经元和非神经元细胞中普遍表达。此外，使用蛋白质-蛋白质相互作用测定，我们表明，Snapin与SNAP 23，广泛表达的同源SNAP 25，预测的C-末端螺旋结构域的Snapin包含SNAP 23结合位点。亚细胞定位实验表明，Snapin是一种可溶性蛋白质，存在于脂肪细胞的细胞溶质和外周膜结合池。此外，协会的Snapin与质膜中检测到过表达的Snapin绿色荧光蛋白融合蛋白的细胞。最后，我们表明，Snapin是能够形成一个三元复合物与SNAP 23和syntaxin 4，这表明它是一个组件的非神经元陷阱复合物。我们的研究结果的一个重要意义是，Snapin是可能执行的SNARE介导的囊泡融合事件在非神经元细胞中的一般作用，除了其参与钙调节的神经分泌。
Members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) superfamily [syntaxins, VAMPs (vesicle-associated membrane proteins) and SNAP25 (synaptosome-associated protein-25)-related proteins] are required for intracellular membrane-fusion events in eukaryotes. In neurons, assembly of SNARE core complexes comprising the presynaptic membrane-associated SNAREs syntaxin I and SNAP25, and the vesicle-associated SNARE VAMP2, is necessary for synaptic vesicle exocytosis. Several accessory factors have been described that associate with the synaptic SNAREs and modulate core complex assembly or mediate Ca2+ regulation. One such factor, Snapin, has been reported to be a brain-specific protein that interacts with SNAP25, and regulates association of the putative Ca2+-sensor synaptotagmin with the synaptic SNARE complex [Ilardi, Mochida and Sheng (1999) Nat. Neurosci. 2, 119-124]. Here we demonstrate that Snapin is expressed ubiquitously in neuronal and non-neuronal cells. Furthermore, using protein-protein-interaction assays we show that Snapin interacts with SNAP23, the widely expressed homologue of SNAP25, and that the predicted C-terminal helical domain of Snapin contains the SNAP23-binding site. Subcellular localization experiments revealed that Snapin is a soluble protein that exists in both cytosolic and peripheral membrane-bound pools in adipocytes. Moreover, association of Snapin with the plasma membrane was detected in cells overexpressing a Snapin-green fluorescent protein fusion protein. Finally, we show that Snapin is able to form a ternary complex with SNAP23 and syntaxin 4, suggesting that it is a component of non-neuronal SNARE complexes. An important implication of our results is that Snapin is likely to perform a general role in SNARE-mediated vesicle fusion events in non-neuronal cells in addition to its participation in Ca2+-regulated neurosecretion.