Stable SNARE complex prior to evoked synaptic vesicle fusion revealed by fluorescence resonance energy transfer

Although it is clear that soluble N-ethylmaleimide-sensitive factor (NSF) a ttachment protein receptor (SNARE) complex plays an essential role in synap tic vesicle fusion, the dynamics of SNARE assembly during vesicle fusion re main to be determined. In this report, we employ fluorescence resonance ene rgy transfer technique to study the formation of SNARE complexes. Donor/acc eptor pair variants of green fluorescent protein (GFP), cyan fluorescent pr otein (CFP), and yellow fluorescent protein (YFP) are fused with the N term ini of SNAP-25 and synaptobrevin, respectively. lit vitro assembly of SNARE core complex in the presence of syntaxin shows strong fluorescence resonan ce energy transfer (FRET) between the CFP-SNAP-25 and YFP-synaptobrevin. Un der the same conditions, CFP fused to the C terminus of SNAP-25, and YFP- s ynaptobrevin have no FRET, Adenovirus-mediated gene transfer is used to exp ress the fusion proteins in PC12 cells and cultured rat cerebellar granule cells. Strong FRET is associated with neurite membranes and vesicular struc tures in PC12 cells co expressing CFP-SNAP-25 and YFP-synaptobrevin, In cul tured rat cerebellar granule cells, FRET between CFP-SNAP-25 and YFP-synapt obrevin is mostly associated with sites presumed to be synaptic junctions, Neurosecretion in PC12 cells initiated by KCl depolarization leads to an in crease in the extent of FRET. These results demonstrate that significant am ounts of stable SNARE complex exist prior to evoked synaptic vesicle fusion and that the assembly of SNARE complex occurs during vesicle docking/primi ng stage. Moreover, it demonstrates that FRET can be used as an effective t ool for investigating dynamic SNARE interactions during synaptic vesicle fu sion.