Analysis of SM-SNARE Interactions in Golgi-to-ER Retrograde Vesicular Transport

Abstract

SNAREs form membrane bridging complexes to enable fusion. For this to occur, a SNARE must selectively assemble in proper alignment with its three cognate SNAREs to form a four-helix bundle. Sec1-Munc18 (SM) proteins are thought to facilitate SNARE complex formation, though the precise mechanism remains a mystery. In order to gain insight into how SM proteins and SNAREs interact, in vitro binding and SNARE assembly assays were performed using the SM protein Sly1 and its cognate Golgi-to-ER retrograde SNAREs. Our results reveal that the Qa-SNARE, Ufe1, has a propensity to form inactive oligomers mediated by asymmetric associations between the SNARE domain of one monomer and the Habc domain of another. Binding of Sly1 rescues Ufe1 back to a monomeric form. SNARE assembly assays further reveal that the Sly1:Ufe1 interaction precedes SNARE complex formation and that Sly1 acts as both a chaperone and a catalyst in this assembly reaction. There is a dynamic equilibrium present between oligomerized Ufe1, monomeric Ufe1 bound to Sly1, and Ufe1 in the SNARE bundle with the Sly1:Ufe1 complex being the kinetic product and the SNARE bundle being the thermodynamic product; Sly1 was not observed to bind to the SNARE bundle, suggesting it disengages with Ufe1 shortly before SNARE assembly is complete. Biochemical analysis of Sly1 surface properties suggest that the SM protein can engage with SNAREs at the SNARE domains if a regulatory helix, 20, is displaced. Dislodging this helix appears to require another agent/factor. Overall, this work offers new insights 7 into Sly1’s role in Golgi-to-ER retrograde trafficking and points to mechanisms that may be more broadly applicable to other SM-SNARE interactions.