The R-SNARE motif of tomosyn forms SNARE core complexes with syntaxin 1 and SNAP-25 and down-regulates exocytosis.
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.
Hatsuzawa
Kiyotaka
K
Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg, D-37077 Göttingen, Germany.
Lang
Thorsten
T
Fasshauer
Dirk
D
Bruns
Dieter
D
Jahn
Reinhard
R
D020816:Amino Acid Motifs; D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D015536:Down-Regulation; D005089:Exocytosis / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D017434:Protein Structure, Tertiary; D050683:R-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D000954:Antigens, Surface; D002352:Carrier Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D009479:Neuropeptides; D050683:R-SNARE Proteins; C494208:SNAP25 protein, human; D050600:SNARE Proteins; C494212:STX1A protein, human; C494210:Snap25 protein, rat; C494213:Stx1a protein, rat; C484769:Stxbp5 protein, rat; D050825:Synaptosomal-Associated Protein 25; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins