Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer
Intracellular interaction between syntaxin and Munc 18-1 revealed by fluorescence resonance energy transfer
复制标题
DOI：
10.1080/09687860500224892
复制
发表时间：
2005-01
影响因子：
--
通讯作者：
Adinarayanareddy Yerrapureddy;T. Korte;S. Hollmann;M. Nordhoff;G. Ahnert-Hilger;A. Herrmann;M. Veit
中科院分区：
文献类型：
--
作者：
Adinarayanareddy Yerrapureddy;T. Korte;S. Hollmann;M. Nordhoff;G. Ahnert-Hilger;A. Herrmann;M. Veit
神经分泌通过由SNAP-25、小突触泡蛋白和突触融合蛋白组成的可溶性N-乙基马来酰亚胺敏感性融合蛋白附着蛋白受体（SNARE）复合物的组装来催化。已知Munc 18-1在体外与突触融合蛋白结合。这种相互作用阻止了SNARE复合物的组装，但也可能影响蛋白质的细胞内靶向。我们已经将syntaxin和Munc 18融合到黄色荧光蛋白（YFP）或青色荧光蛋白（CFP）上，并在CHO和MDCK细胞中表达了这些构建体。我们已经研究了他们的本地化与共聚焦显微镜和可能的蛋白质-蛋白质相互作用与荧光共振能量转移（FRET）。YFP-突触融合蛋白定位于细胞内膜。CFP-Munc 18存在于细胞质中，如对于缺乏膜靶向结构域的蛋白质所预期的。然而，当共表达突触融合蛋白时，Munc 18被重定向到内膜，但仅观察到蛋白质向质膜的有限运输。通过FRET，可以证明Munc 18和syntaxin之间的相互作用，使用两种方法，敏化受体荧光和受体光漂白。已知在体外抑制与Munc 18结合的突触融合蛋白（L165 A，E166 A）中的突变阻止蛋白质的共定位以及FRET信号。因此，Munc 18和突触融合蛋白之间的蛋白质-蛋白质相互作用发生在细胞内膜上，这是必需的，但不足以将两种蛋白质定量转运到质膜。
Neurosecretion is catalyzed by assembly of a soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)-complex composed of SNAP-25, synaptobrevin and syntaxin. Munc 18-1 is known to bind to syntaxin in vitro. This interaction prevents assembly of the SNARE-complex, but might also affect intracellular targeting of the proteins. We have fused syntaxin and Munc 18 to the yellow- (YFP) or cyan-fluorescence-protein (CFP) and expressed the constructs in CHO- and MDCK-cells. We have studied their localization with confocal microscopy and a possible protein-protein interaction with fluorescence-resonance energy transfer (FRET). YFP-syntaxin localizes to intracellular membranes. CFP-Munc 18 is present in the cytoplasm as expected for a protein lacking membrane targeting domains. However, Munc 18 is redirected to internal membranes when syntaxin is coexpressed, but only limited transport of the proteins to the plasma membrane was observed. An interaction between Munc 18 and syntaxin could be demonstrated by FRET using two methods, sensitized acceptor fluorescence and acceptor photobleaching. A mutation in syntaxin (L165A, E166A), which is known to inhibit binding to Munc 18 in vitro, prevents colocalization of the proteins and also the FRET signal. Thus, a protein-protein interaction between Munc 18 and syntaxin occurs on intracellular membranes, which is required but not sufficient for quantitative transport of both proteins to the plasma membrane.