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Molecular cloning and functional analysis of VAMP-2 binding proteins in parotid acinar cells.

腮腺腺泡细胞中 VAMP-2 结合蛋白的分子克隆和功能分析。

基本信息

批准号：
11671849
负责人：
TAKUMA Taishin
金额：
$ 2.24万
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：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11671849/
关键词：
SNARE hypothesis VAMP-2 GFP SNAP-23 Exocytosis Parotid acinar cell

项目摘要

SNARE proteins are generally accepted to be involved in the docking and fusion process of intracellular membranes. VAMP-2, syntaxin-4, and SNAP-23 are most plausible candidates of SNARE proteins for non-neuronal exocytosis. Thus, we examined the localization and intracellular trafficking of these proteins by expressing as green fluorescent protein (GFP)-tagged fusion proteins in various cells, including cultured salivary cells (HSY cells), COS-7 cells, and 3T3-L1 adipocytes. In HSY cells and COS-7 cells, VAMP-2 tagged with GFP was strongly expressed in the Golgi area. The full length of SNAP-23 with GFP was universally expressed in the cytosol except the cell nuclei. Syntaxin-4 was localized on large vesicles unidentified. When GFP-SNAP-23 and syntaxin-4 were co-expressed in these cells, SNAP-23 moved to the vesicles where GFP-syntaxin-4 was localized, suggesting that localization of SNAP-23 is determined at least partly by syntaxin-4. When these proteins were expressed in 3T3-L1 adipocytes, SNAP-23 and VAMP-2 were partly localized on the plasma membrane, although syntaxin-4 was still on the vesicles. Syntaxin-4 without transmembrane domain was not soluble but revealed as aggregates in both COS-7 cells and 3T3-L1 adipocytes. These results suggest that intracellular trafficking of SNARE proteins are regulated with other SNARE proteins or factors unidentified.

SNARE蛋白被普遍认为参与了细胞内膜的对接和融合过程。VAMP-2、Synaxin-4和SNAP-23是最有可能用于非神经元胞吐的SNARE蛋白。因此，我们通过在不同的细胞中表达绿色荧光蛋白(GFP)标记的融合蛋白来检测这些蛋白的定位和细胞内转运，这些细胞包括培养的唾液细胞(HSY细胞)、COS-7细胞和3T3-L1脂肪细胞。在HSY细胞和COS-7细胞中，GFP标记的VAMP-2在高尔基体区有较强的表达。带有GFP的SNAP-23全长蛋白在胞浆中普遍表达，但在细胞核中不表达。Synaxin-4定位于未鉴定的大小泡上。当GFP-SNAP-23和Synaxin-4在这些细胞中共表达时，SNAP-23移动到GFP-Synaxin-4定位的小泡中，这表明SNAP-23的定位至少部分由Synaxin-4决定。当这些蛋白在3T3-L1脂肪细胞中表达时，SNAP-23和VAMP-2部分定位在质膜上，尽管Synaxin-4仍然位于小泡上。不含跨膜结构域的Synaxin-4在COS-7细胞和3T3-L1脂肪细胞中均呈聚集态，而不是可溶性的。这些结果表明，SNARE蛋白在细胞内的运输受到其他SNARE蛋白或未知因素的调控。