Article: article from journal or magazin.
The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis.
Journal of Biological Chemistry
Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfolding-refolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor alpha-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.
Amino Acid Motifs, Animals, Antigens, Surface/metabolism, Carrier Proteins/chemistry, Carrier Proteins/genetics, Cell Membrane/metabolism, Down-Regulation, Exocytosis/physiology, Gene Expression, Humans, Membrane Proteins/chemistry, Membrane Proteins/metabolism, Nerve Tissue Proteins/metabolism, Neuropeptides/chemistry, Neuropeptides/genetics, PC12 Cells, Protein Structure, Tertiary, R-SNARE Proteins, Rats, SNARE Proteins, Synaptosomal-Associated Protein 25, Syntaxin 1, Vesicular Transport Proteins
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