Identification and characterization of Snapin as a ubiquitously expressed SNARE-binding protein that interacts with SNAP23 in non-neuronal cells.
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.
Buxton
Penelope
P
Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
Zhang
Xiang-Ming
XM
Walsh
Bong
B
Sriratana
Absorn
A
Schenberg
Irina
I
Manickam
Elizabeth
E
Rowe
Tony
T
D041721:3T3-L1 Cells; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002522:Chlorocebus aethiops; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018613:Microscopy, Confocal; D011485:Protein Binding; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050600:SNARE Proteins; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C100493:SNAP23 protein, human; D050600:SNARE Proteins; C494212:STX1A protein, human; C107017:Snap23 protein, mouse; C498451:Snapin protein, mouse; C494214:Stx1a protein, mouse; D050827:Syntaxin 1; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins