The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis

J Biol Chem. 2003 Aug 15;278(33):31159-66. doi: 10.1074/jbc.M305500200. Epub 2003 Jun 2.


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.

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Antigens, Surface / metabolism*
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism*
  • Cell Membrane / metabolism
  • Down-Regulation
  • Exocytosis / physiology*
  • Gene Expression
  • Humans
  • Membrane Proteins / chemistry
  • Membrane Proteins / metabolism*
  • Nerve Tissue Proteins / metabolism*
  • Neuropeptides / chemistry
  • Neuropeptides / genetics
  • Neuropeptides / metabolism*
  • PC12 Cells
  • Protein Structure, Tertiary
  • R-SNARE Proteins
  • Rats
  • SNARE Proteins
  • Synaptosomal-Associated Protein 25
  • Syntaxin 1
  • Vesicular Transport Proteins*


  • Antigens, Surface
  • Carrier Proteins
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neuropeptides
  • R-SNARE Proteins
  • SNAP25 protein, human
  • SNARE Proteins
  • STX1A protein, human
  • Snap25 protein, rat
  • Stx1a protein, rat
  • Stxbp5 protein, rat
  • Synaptosomal-Associated Protein 25
  • Syntaxin 1
  • Vesicular Transport Proteins