Differential interaction of tomosyn with syntaxin and SNAP25 depends on domains in the WD40 β-propeller core and determines its inhibitory activity

J Biol Chem. 2014 Jun 13;289(24):17087-99. doi: 10.1074/jbc.M113.515296. Epub 2014 Apr 29.

Abstract

Neuronal exocytosis depends on efficient formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and is regulated by tomosyn, a SNARE-binding protein. To gain new information about tomosyn's activity, we characterized its mobility and organization on the plasma membrane (PM) in relation to other SNARE proteins and inhibition of exocytosis. By using direct stochastic optical reconstruction microscopy (dSTORM), we found tomosyn to be organized in small clusters adjacent to syntaxin clusters. In addition, we show that tomosyn is present in both syntaxin-tomosyn complexes and syntaxin-SNAP25-tomosyn complexes. Tomosyn mutants that lack residues 537-578 or 897-917 from its β-propeller core diffused faster on the PM and exhibited reduced binding to SNAP25, suggesting that these mutants shift the equilibrium between tomosyn-syntaxin-SNAP25 complexes on the PM to tomosyn-syntaxin complexes. As these deletion mutants impose less inhibition on exocytosis, we suggest that tomosyn inhibition is mediated via tomosyn-syntaxin-SNAP25 complexes and not tomosyn-syntaxin complexes. These findings characterize, for the first time, tomosyn's dynamics at the PM and its relation to its inhibition of exocytosis.

Keywords: Exocytosis; FRAP; Membrane Fusion; Microscopy; Neurobiology; SNARE Proteins; Tomosyn.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Binding Sites
  • Cell Membrane / metabolism
  • Exocytosis
  • Gene Deletion
  • HEK293 Cells
  • Humans
  • Mice
  • Nerve Tissue Proteins / chemistry
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • PC12 Cells
  • Protein Binding
  • Protein Transport
  • R-SNARE Proteins / chemistry
  • R-SNARE Proteins / genetics
  • R-SNARE Proteins / metabolism*
  • Rats
  • Synaptosomal-Associated Protein 25 / chemistry
  • Synaptosomal-Associated Protein 25 / genetics
  • Synaptosomal-Associated Protein 25 / metabolism*
  • Syntaxin 1 / chemistry
  • Syntaxin 1 / genetics
  • Syntaxin 1 / metabolism*

Substances

  • Nerve Tissue Proteins
  • R-SNARE Proteins
  • Snap25 protein, rat
  • Stx1a protein, rat
  • Stxbp5 protein, rat
  • Synaptosomal-Associated Protein 25
  • Syntaxin 1