Epsin N-terminal homology domains bind on opposite sides of two SNAREs

SNARE proteins are crucial components of the cellular membrane fusion machinery. Four SNARE motifs from three or four SNAREs on opposing membranes interact to form a four-helix-bundle, pulling the membranes together, thus facilitating fusion. The specificity of the fusion reaction requires a specific localization of SNAREs on different membranes. SNAREs are recruited to different subcellular membranes by cargo adaptors which specifically recognize their cargo. The N-terminal three-helix-bundle (Habc) of several SNARE proteins is recognized by a few Epsin N-terminal homology (ENTH) proteins. EpsinR is a cargo adaptor for the mammalian late endosomal SNAREs vti1b, syntaxin7 and syntaxin8 while the yeast homologue Ent3p binds to Vti1p, Pep12p and Syn8p. Here we describe three crystal structures of yeast proteins: The Habc domain of Vti1p, the ENTH domain of Ent3p, and their complex. The complex structure reveals that the Habc domain of Vti1p binds to the ENTH domain of Ent3p using an opposite surface compared to the corresponding mammalian structure of vti1b and EpsinR. Mutations based on the yeast complex disrupt the interaction in a yeast-two-hybrid system and in an in vitro pull-down assay while mutations based on the mammalian interface do not. Disruptive mutations affected the sorting of Vti1p in vivo as assayed by sucrose densitiy gradient centrifugation and size exclusion chromatography. Thus, the observed interface is responsible for Vti1p-Ent3p recognition in yeast.