SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways. Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known. We now describe the subunit structure, assembly, and regulation of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity 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 pattern 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 membrane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, we demonstrate that the quaternary complex containing sec22b is not an in vitro entity only, but is a bona fide species in living cells.

This study establishes a role for luminal Ca2+ in ER/Golgi transport organelles and elucidates an effector mechanism involving the EF-hand protein ALG-2 and regulation of COPII coat retention.

The endoplasmic reticulum/Golgi SNARE rbet1 cycles between the endoplasmic reticulum and Golgi and is essential for cargo transport in the secretory pathway. Although the quaternary SNARE complex containing rbet1 is known to function in membrane fusion, the structural role of rbet1 is unclear. Furthermore, the structural determinants for rbet1 targeting and its cyclical itinerary have not been investigated. We utilized protein interaction assays to demonstrate that the rbet1 SNARE motif plays a structural role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly of a conserved salt bridge between rbet1 and sec22b. We also examined the potential role of the rbet1 SNARE motif and SNARE interactions in rbet1 localization and dynamics. We found that, in contrast to what has been observed for syntaxin 5, the rbet1 SNARE motif was essential for proper targeting. To test whether SNARE interactions were important for the targeting function of the SNARE motif, we used charge repulsion mutations at the conserved salt bridge position that rendered rbet1 defective for binary, ternary, and quaternary SNARE interactions. We found that heteromeric SNARE interactions are not required at any step in rbet1 targeting or dynamics. Furthermore, the heteromeric state of the SNARE motif does not influence its interaction with the COPI coat or efficient recruitment onto transport vesicles. We conclude that protein targeting is a completely independent function of the rbet1 SNARE motif, which is capable of distinct classes of protein interactions.

No abstract