Structural Analysis of the Interaction between the Dsl1 Subunit Tip20 and the ER SNARE Sec20

Abstract

In intracellular trafficking, multi-subunit tethering complexes (MTCs) mediate the initial long-range attachment between a transport vesicle and its target organelle. The vesicle and target organelle membranes fuse following the assembly of SNARE (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor) proteins into complexes. Although it is known that MTCs regulate SNARE proteins in most trafficking pathways, the mechanisms by which they do so are largely unknown. This is partly due to the lack of structural information on the interactions between MTCs and SNAREs. Accordingly, to better understand the mechanisms that underlie MTC-mediated SNARE regulation, we have characterized the interaction between Sec20, a SNARE protein, and Tip20, a subunit of the Dsl1 complex that tethers vesicles from the Golgi apparatus to the endoplasmic reticulum (ER). Our lab previously solved the complete structure of the Dsl1 complex and showed that it accelerates the assembly of ER SNAREs in vitro. After testing in vitro properties of various orthologs of the Tip20/Sec20 complex, we obtained a well-behaved complex from proteins derived from Eremothecium gossypii. We report the 3.2 Å resolution crystal structure of the N-terminal domain of Sec20 bound to Tip20. The structure reveals that Sec20 binds domains C, D, and E of Tip20 in an orientation that positions its C-terminal SNARE motif to interact productively with other ER SNAREs. Our results suggest that Dsl1 likely serves as a molecular scaffold that binds and orients SNAREs for assembly.