Adenovirus-mediated expression and functional analyses of SNARE proteins in salivery gland cells

SNARE蛋白被广泛认为参与细胞内囊泡运输的对接和融合过程。VAMP-2、syntaxin-4和SNAP-23可能是SNARE蛋白参与非神经元胞吐的候选蛋白。因此，我们通过将这些蛋白作为绿色荧光蛋白（GFP）和flag标记的融合蛋白在各种细胞（包括人腮腺上皮细胞系HSY细胞）中表达，研究了它们的定位、蛋白相互作用和细胞内运输。GFP-VAMP-2在高尔基区表达强烈，在质膜上表达弱。虽然GFP- snap -23似乎在细胞质中普遍表达，但当皂苷处理去除可溶性GFP- snap -23时，在质膜上可以清晰地看到GFP信号。GFP-syntaxin-4在质膜上检测不到，但在异常大的未知囊泡上强烈表达。没有跨膜结构域的GFP-syntaxin-4仍然不完全溶解，以聚集体的形式存在。当syntaxin-4和munc18c共表达时，syntaxin-4至少部分转移到质膜上。munc18c的共表达明显抑制了syntaxin-4和VAMP-2及其跨膜结构域之间的蛋白相互作用。这些结果表明，munc18c通过阻止syntaxin-4与其他蛋白质（包括SNARE蛋白）的过早相互作用，在将syntaxin-4运输到适当的目的地方面起着重要作用。

SNARE proteins are widely accepted to be involved in the docking and fusion process of intracellular vesicle trafficking. VAMP-2, syntaxin-4, and SNAP-23 are the plausible candidates of SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization, protein-protein interaction, and intracellular trafficking of these proteins by expressing them as green fluorescent protein (GFP)- and FLAG-tagged fusion proteins in various cells, including HSY cells, a human parotid epithelial cell line. GFP-VAMP-2 was expressed strongly in the Golgi area and weakly on the plasma membrane. Although GFP-SNAP-23 seemed to be expressed universally in the cytosol, the GFP signal was clearly seen on the plasma membrane, when soluble GFP-SNAP-23 was removed by treatment with saponin. GFP-syntaxin-4 was undetectable on the plasma membrane but was strongly expressed on unusually large unidentified vesicles. GFP-syntaxin-4 without its transmembrane domain was still incompletely soluble and observed as aggregates. When syntaxin-4 and munc18c were coexpressed, syntaxin-4 was translocated at least in part to the plasma membrane. The protein-protein interaction between syntaxin-4 and VAMP-2 with their transmembrane domains was markedly inhibited by coexpression of munc18c. These results suggest that munc18c plays an important role for the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.