Subunit structure of a mammalian ER/Golgi SNARE complex

SNAP receptor (SNARE) complexes bridge opposing membranes to promote membra ne fusion within the secretory and endosomal pathways. Because only the exo cytic SNARE complexes have been characterized in detail, the structural fea tures shared by SNARE complexes from different fusion steps are not known. We now describe the subunit structure, assembly, and regulation of a quater nary SNARE complex, which appears to mediate an early step in endoplasmic r eticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinit y binding site for sec22b, an R-SNARE. The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly. The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pat tern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membr ane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others ass emble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, we demonstrate that the quaternary complex co ntaining sec22b is not an in vitro entity only, but is a bona fide species in living cells.