Biochemical Journal

Research article

Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells

Penelope BUXTON, Xiang-Ming ZHANG, Bong WALSH, Absorn SRIRATANA, Irina SCHENBERG, Elizabeth MANICKAM, Tony ROWE

Abstract

Members of the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) superfamily [syntaxins, VAMPs (vesicle-associated membrane proteins) and SNAP25 (synaptosome-associated protein-25)-related proteins] are required for intracellular membrane-fusion events in eukaryotes. In neurons, assembly of SNARE core complexes comprising the presynaptic membrane-associated SNAREs syntaxin 1 and SNAP25, and the vesicle-associated SNARE VAMP2, is necessary for synaptic vesicle exocytosis. Several accessory factors have been described that associate with the synaptic SNAREs and modulate core complex assembly or mediate Ca2+ regulation. One such factor, Snapin, has been reported to be a brain-specific protein that interacts with SNAP25, and regulates association of the putative Ca2+-sensor synaptotagmin with the synaptic SNARE complex [Ilardi, Mochida and Sheng (1999) Nat. Neurosci. 2, 119–124]. Here we demonstrate that Snapin is expressed ubiquitously in neuronal and non-neuronal cells. Furthermore, using protein–protein-interaction assays we show that Snapin interacts with SNAP23, the widely expressed homologue of SNAP25, and that the predicted C-terminal helical domain of Snapin contains the SNAP23-binding site. Subcellular localization experiments revealed that Snapin is a soluble protein that exists in both cytosolic and peripheral membrane-bound pools in adipocytes. Moreover, association of Snapin with the plasma membrane was detected in cells overexpressing a Snapin–green fluorescent protein fusion protein. Finally, we show that Snapin is able to form a ternary complex with SNAP23 and syntaxin 4, suggesting that it is a component of non-neuronal SNARE complexes. An important implication of our results is that Snapin is likely to perform a general role in SNARE-mediated vesicle fusion events in non-neuronal cells in addition to its participation in Ca2+-regulated neurosecretion.

  • exocytosis
  • soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor (SNARE)
  • synaptosome-associated protein-23 (SNAP23)
  • syntaxin
  • trafficking
  • vesicle

Footnotes

  • 1 These authors contributed equally to this work.

  • Abbreviations used: GST, glutathione S-transferase; SNAP25, synaptosome-associated protein-25; SNAP23, synaptosome-associated protein-23; SNARE, soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor; Syn, syntaxin; VAMP, vesicle-associated membrane protein; PNS, post-nuclear supernatant; GFP, green fluorescent protein.