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

腺病毒介导的唾液腺细胞中 SNARE 蛋白的表达和功能分析

基本信息

批准号：
13671946
负责人：
TAKUMA Taishin
金额：
$ 1.98万
依托单位：
Health Sciences University of Hokkaido
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671946/
关键词：
SNARE hypothesis VAMP-2 GFP SNAP-23 Exocytosis Parotid acinar cell アデノウイルス SNARE 唾液腺開口分泌

项目摘要

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.

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是SNARE蛋白质用于非神经元胞吐的合理候选者。因此，我们研究了这些蛋白质的定位，蛋白质-蛋白质相互作用，和细胞内运输通过表达它们作为绿色荧光蛋白（GFP）和FLAG标记的融合蛋白在各种细胞，包括HSY细胞，人腮腺上皮细胞系。GFP-VAMP-2在高尔基体区域表达较强，在质膜上表达较弱。虽然GFP-SNAP-23似乎在胞质溶胶中普遍表达，但当通过用皂苷处理去除可溶性GFP-SNAP-23时，在质膜上清楚地看到GFP信号。GFP-突触融合蛋白-4在质膜上检测不到，但在异常大的不明囊泡上强烈表达。没有其跨膜结构域的GFP-突触融合蛋白-4仍然不完全可溶，并且观察到聚集体。当syntaxin-4和munc 18 c共表达时，syntaxin-4至少部分被转运到质膜。munc 18 c的共表达显著抑制了syntaxin-4和VAMP-2跨膜结构域之间的蛋白质-蛋白质相互作用。这些结果表明，munc 18 c通过阻止与其他蛋白质（包括SNARE蛋白）的过早相互作用，在将syntaxin-4运输到其适当目的地方面发挥重要作用。