The Template Model of SNARE Assembly: An Investigation of the SM Vps33 and R-SNARE Nyv1 Interface

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins function in membrane fusion during vesicle trafficking. The assembly of SNAREs into four-helix bundles bridges the two membranes and provides the driving force for fusion. Sec1/Munc18 (SM) proteins help regulate SNARE bundle assembly but how exactly they do so is not well understood. A recently determined crystal structure demonstrated the SM protein Vps33 simultaneously bound to its R-SNARE Nyv1 and Qa-SNARE Vam3. This structure supports the hypothesis of the “SM template model” that suggests SM proteins align the SNARE motifs and form a “half-zippered” conformation of the R- and Qa-SNAREs. This thesis examined the interaction between the SM protein Vps33 and the R-SNARE Nyv1 to identify relative Nyv1 residue contributions to Vps33 binding. Alanine-scanning mutagenesis was performed across the entire ordered region of the Nyv1 SNARE motif. Small-scale pulldown binding assays were then used to examine how the Nyv1 mutations impacted interactions with Vps33. We identified regions of the Nyv1 SNARE motif that are less critical for Vps33 binding, like the zero-layer residue (R180) and distal C-terminal end of the motif, and hypothesize that they provide more flexibility for SNARE bundle assembly. On the other hand, residues of Nyv1 (187-191) found to be critical for Vps33 binding were hypothesized to be important for Vps33:R-SNARE selectivity. Together, these findings strengthen the hypothesis of SM proteins functioning as templates by suggesting that Vps33 recognizes specific residues within the Nyv1 SNARE motif while still enabling flexibility for SNARE bundle assembly.