Evaluation of Potential Binding Interactions Between the Sec1/Munc18 Family Protein Sly1 and Its Cognate SNAREs

Abstract

This Abstract contains text that is based closely on, or identical to, text found in my Junior Paper [Sarah Howells, SM family protein Sly1 Interaction with R-SNAREs in the Anterograde and Retrograde Trafficking Pathways from Endoplasmic Reticulum to Golgi Apparatus, Junior Independent Work Paper, Spring 2015]. Cell trafficking is a process by which cargo-carrying vesicles fuse with target membranes within the cell to release proteins into specific cellular compartments. The Sec1/Munc18 (SM) protein Sly1 chaperones trans-SNARE complex formation to promote vesicle fusion with target membranes. It is well established that Sly1 binds Qa- SNAREs in trafficking pathways from the endoplasmic reticulum (ER) to the Golgi apparatus. Yet the possibility of functionally important interactions between Sly1 and its cognate Qb-SNAREs, Qc-SNARES, and R-SNAREs has not been rigorously explored. Recently, our lab discovered that the SM protein Vps33 binds the R-SNARE Nyv1 in a surface groove homologous to that covered by a “lid” formed by a short helical region, α20, on Sly1. Mutations that destabilize α20 suppress deleterious mutations in trafficking proteins such as vesicle-bound Rab GTPases and some multisubunit tethering complexes, including Dsl1. Using gel filtration binding assays, we determined that wild-type Sly1 and lid mutant Sly1-20 do not bind the SNARE domains of the cognate Qb-SNARE Sec20, Qc-SNARE Use1, and R-SNAREs Sec22 and Ykt6. We also established that Sly1-20 binds the retrograde Qa-SNARE Ufe1, like wild-type Sly1, although Sly1-20 failed to form a ternary complex with the Ufe1 N-peptide and Ykt6. Future work should test ternary complex formation of cytoplasmic Qa-SNAREs and R-SNAREs with Sly1-20 and compete crystallization trials of Sly1-20 with SNAREs. Our results suggest that unlike Vps33, but like the SM protein Munc18, Sly1 initiates SNARE complex formation by interaction with its cognate Qa-SNAREs.