Article: article from journal or magazin.
Tomosyn-1 is involved in a post-docking event required for pancreatic beta-cell exocytosis.
Journal of Cell Science
Although the assembly of a ternary complex between the SNARE proteins syntaxin-1, SNAP25 and VAMP2 is known to be crucial for insulin exocytosis, the mechanisms controlling this key event are poorly understood. We found that pancreatic beta-cells express different isoforms of tomosyn-1, a syntaxin-1-binding protein possessing a SNARE-like motif. Using atomic force microscopy we show that the SNARE-like domain of tomosyn-1 can form a complex with syntaxin-1 and SNAP25 but displays binding forces that are weaker than those observed for VAMP2 (237+/-13 versus 279+/-3 pN). In pancreatic beta-cells tomosyn-1 was found to be concentrated in cellular compartments enriched in insulin-containing secretory granules. Silencing of tomosyn-1 in the rat beta-cell line INS-1E by RNA interference did not affect the number of secretory granules docked at the plasma membrane but led to a reduction in stimulus-induced exocytosis. Replacement of endogenous tomosyn-1 with mouse tomosyn-1, which differs in the nucleotide sequence from its rat homologue and escapes silencing, restored a normal secretory rate. Taken together, our data suggest that tomosyn-1 is involved in a post-docking event that prepares secretory granules for fusion and is necessary to sustain exocytosis of pancreatic beta-cells in response to insulin secretagogues.
Animals, COS Cells, Calcium Signaling, Cells, Cultured, Cercopithecus aethiops, Down-Regulation/genetics, Exocytosis, Gene Silencing, Insulin/secretion, Insulin-Secreting Cells/cytology, Insulin-Secreting Cells/secretion, Mice, Nerve Tissue Proteins/genetics, Nerve Tissue Proteins/metabolism, Protein Binding, Protein Structure, Tertiary, Protein Transport, R-SNARE Proteins/genetics, R-SNARE Proteins/metabolism, RNA, Messenger/genetics, RNA, Messenger/metabolism, Rats, Secretory Vesicles/metabolism, Synaptosomal-Associated Protein 25/metabolism, Syntaxin 1/metabolism, Thermodynamics, Up-Regulation/genetics
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