IDENTIFICATION OF A MINIMAL CORE OF THE SYNAPTIC SNARE COMPLEX SUFFICIENT FOR REVERSIBLE ASSEMBLY AND DISASSEMBLY

Assembly of the three neuronal membrane proteins synaptobrevin, syntax in, and SNAP-25 is thought to be one of the key steps in mediating exo cytosis of synaptic vesicles. In vivo and in vitro, these proteins for m a tight complex. Assembly is associated with a large increase in alp ha-helical content, suggesting that major structural and conformationa l changes are associated with the assembly reaction. Limited proteolys is by trypsin, chymotrypsin, and proteinase K of the ternary complex f ormed from recombinant proteins lacking their membrane anchors reveale d a SDS-resistant minimal core, The components of this core complex we re purified and characterized by N-terminal sequencing and mass spectr ometry. They include a slightly shortened synaptobrevin fragment, C- a nd N-terminal fragments of SNAP-25, and a C-terminal fragment of synta xin that is slightly larger than the previously characterized H3 domai n. Recombinant proteins corresponding to these fragments are sufficien t for assembly and disassembly. In addition, each of the two SNAP-25 f ragments can individually form complexes with syntaxin and synaptobrev in, suggesting that they both contribute to the assembly of the SNARE complex. Upon complex assembly, a large increase in alpha-helical cont ent is observed along with a significantly increased melting temperatu re(T-m). Like the full-length complex, the minimal complex tends to fo rm an oligomeric species; global analysis of equilibrium ultracentrifu gation data suggests a monomer-trimer equilibrium exists. These conser ved biophysical properties may thus be of fundamental importance in th e mechanism of membrane fusion.