Takuma Taishin, Arakawa Toshiya, Okayama Miki, Mizoguchi Itaru, Tanimura Akihiko, Tajima Yoshifumi
Department of Oral Biochemistry, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-0293.
J Biochem. 2002 Nov;132(5):729-35. doi: 10.1093/oxfordjournals.jbchem.a003280.
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 plausible candidate 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 ex-pressed 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 unidentified unusually large 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 on coexpression of munc18c. These results suggest that munc18c plays an important role in the trafficking of syntaxin-4 to its proper destination by preventing premature interactions with other proteins, including SNARE proteins.
SNARE蛋白广泛参与细胞内囊泡运输的对接和融合过程，这一点已被广泛接受。VAMP - 2、 syntaxin - 4和SNAP - 23是参与非神经元胞吐作用的潜在SNARE蛋白候选者。因此，我们通过在包括人腮腺上皮细胞系HSY细胞在内的各种细胞中表达绿色荧光蛋白（GFP）和FLAG标记的融合蛋白，来研究这些蛋白的定位、蛋白质 - 蛋白质相互作用以及细胞内运输。GFP - VAMP - 2在高尔基体区域强烈表达，在质膜上表达较弱。尽管GFP - SNAP - 23似乎在细胞质中普遍表达，但在用皂苷处理去除可溶性GFP - SNAP - 23后，质膜上能清楚地看到GFP信号。GFP - syntaxin - 4在质膜上无法检测到，但在未识别的异常大的囊泡上强烈表达。没有跨膜结构域的GFP - syntaxin - 4仍然不完全可溶，并观察到聚集现象。当syntaxin - 4和munc18c共表达时，syntaxin - 4至少部分转运到质膜。munc18c共表达时，syntaxin - 4和VAMP - 2与其跨膜结构域之间的蛋白质 - 蛋白质相互作用受到显著抑制。这些结果表明，munc18c通过防止与包括SNARE蛋白在内的其他蛋白质过早相互作用，在syntaxin - 4运输到其正确目的地的过程中发挥重要作用。
