Promoting t-SNARE binary complex assembly and membrane fusion by the exocyst protein Sec3

Membrane fusion is the final step of intracellular trafficking, which is essential to eukaryotic cells. Exocyst is an octameric protein complex that mediates the initial contact between secretory vesicles and the plasma membrane. The final fusion event is mediated by SNAREs (Soluble N-ethylmaleimide Sensitive Factor Attachment Protein Receptor), proteins residing on both the secretory vesicles and the plasma membrane. The assembly of the SNARE complex drives membrane fusion. SNARE mediated fusion at the plasma membrane in yeast begins with the formation of a binary t SNARE complex composed of Sso1/2 and Sec9 followed by its binding to the v SNARE protein Snc1/2. It was previously shown that SNARE assembly is disrupted in yeast exocyst mutant cells. However, the molecular mechanism by which the exocyst regulates SNAREs assembly is unknown. In this study, I have found that one of the exocyst subunits, Sec3, directly interacts with the t-SNARE protein Sso1/2. Pre-incubation of Sso2 with Sec3 promotes the interaction between Sso2 and Sec9. In the in vitro liposome mixing assay, Sec3 accelerates the reaction rate of fusion. Furthermore, FRET and BRET assays strongly suggest that Sec3 relieves the self-inhibitory conformation of Sso2. The structure of Sso2-Sec3 complex was solved at 2.2Å resolution. The crystal structure shows that the binding of Sec3 bends the N-terminus of Sso2 SNARE domain approximately 20°. The mutations disrupting Sso2-Sec3 interaction lead to growth defect and a block in secretion in yeast cells. I propose that the interaction between Sec3 and Sso2 activates Sso2 by relieving its auto-inhibition, thus facilitating its binding to Sec9. As such, Sec3 accelerates SNARE-mediated membrane fusion