Trafficking of green fluorescent protein-tagged SNARE proteins in HSY cells

Abstract

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.