Specific interaction between SNAREs and ENTH domains of epsin-related proteins in TGN to endosome transport

Subbulakshmi Chidambaram, Nina Müllers, Katrin Wiederhold, Volker Haucke, and Gabriele Fischer von Mollard

SNARE proteins on transport vesicles and target membranes have important roles in vesicle targeting and fusion. Therefore, localization and activity of SNAREs has to be tightly controlled. Regulatory proteins bind to N-terminal domains of some SNAREs. Vti1b is a mammalian SNARE which functions in late endosomal fusion. To investigate the role of the N-terminus of vti1b we performed a yeast two-hybrid screen. The N-terminus of vti1b interacted specifically with the ENTH domain of enthoprotin/CLINT/epsinR. The interaction was confirmed using in vitro binding assays. This complex formation between a SNARE and an ENTH domain was conserved between mammals and yeast. Yeast Vti1p interacted with the ENTH domain of Ent3p. ENTH proteins are involved in the formation of clathrin coated vesicles. Both, epsinR and Ent3p bind adaptor proteins at the TGN. Vti1p is required for multiple transport steps in the endosomal system. Genetic interactions between VTI1 and ENT3 were investigated. Synthetic defects suggested that Vti1p and Ent3p cooperate in transport from the TGN to the prevacuolar endosome. Our experiments identified the first cytoplasmic protein binding to specific ENTH domains. These results point towards a novel function of the ENTH domain and a connection between proteins which function either in vesicle formation or in vesicle fusion.