Mixed and non-cognate SNARE complexes - Characterization of assembly and biophysical properties

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein re ceptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have rec ently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes wi th components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1, When the Q-SNAREs syntaxin 2, 3, and 4, and the R -SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes co ntained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fu sion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy s howed that major conformational changes occur during assembly, which are as sociated with induction of structure from unstructured monomers, Furthermor e, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations . We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggest ing that specificity of membrane fusion in intracellular membrane traffic i s not due to intrinsic specificity of SNARE pairing.