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

SNARE蛋白通常被认为参与细胞内膜的对接和融合过程。VAMP-2、syntaxin-4和SNAP-23是SNARE蛋白中最有可能用于非神经元胞吐的候选蛋白。因此，我们通过在各种细胞（包括培养的唾液细胞（HSY细胞）、COS-7细胞和3 T3-L1脂肪细胞）中表达作为绿色荧光蛋白（GFP）标记的融合蛋白来检查这些蛋白的定位和细胞内运输。在HSY细胞和COS-7细胞中，GFP标记的VAMP-2在高尔基体区域强烈表达。全长SNAP-23与GFP的普遍表达在细胞质中，除了细胞核。Syntaxin-4定位于未鉴定的大囊泡上。当GFP-SNAP-23和突触融合蛋白-4在这些细胞中共表达时，SNAP-23移动到GFP-突触融合蛋白-4定位的囊泡，表明SNAP-23的定位至少部分由突触融合蛋白-4决定。当这些蛋白质在3 T3-L1脂肪细胞中表达时，SNAP-23和VAMP-2部分定位于质膜上，尽管syntaxin-4仍然位于囊泡上。不含跨膜结构域的突触融合蛋白4不可溶，但在COS-7细胞和3 T3-L1脂肪细胞中均显示为聚集体。这些结果表明，SNARE蛋白的细胞内运输与其他SNARE蛋白或未鉴定的因子调节。

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.