SNARE proteins on transport vesicles and target membranes have important roles in vesicle targetingand fusion. Therefore, localization and activity of SNAREs have to be tightly controlled. Regulatory proteins bind to N-terminal domains of some SNAREs. vti1b is a mammalian SNARE that 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 epsin Nterminal homology (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 trans-Golgi network. 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 trans-Golgi network to the prevacuolar endosome. Our experiments identified the first cytoplasmic protein binding to specific ENTH domains. These results point toward a novel function of the ENTH domain and a connection between proteins that function either in vesicle formation or in vesicle fusion. SNARE 1 proteins on transport vesicles and target membranes mediate recognition and fusion between membranes by complex formation with other SNAREs via SNARE motifs (1). SNAREs can be subdivided into four different groups (R-, Qa-, Qb-, and Qc-SNAREs, with R and Q being the single-letter abbreviations of arginine and glutamine, respectively) according to similarities in their amino acid sequences. All well characterized SNARE complexes are composed of four different SNARE helices, one from each group. The conserved SNARE motif is located next to a C-terminal transmembrane anchor in most SNAREs. Many SNAREs contain N-terminal domains that fold independently and are highly divergent in their amino acid sequences. The N-terminal domains of SNAREs belonging to syntaxins or to Qa-SNAREs can bind proteins that regulate SNARE complex formation. Other binding partners recruit Qaor R-SNAREs into budding vesicles. The N-terminal domains of the R-SNAREs Sec22p (2) and Ykt6p (3) form a mixed ␣-helical/ ␤-sheet profilin-like fold. By contrast, the N-terminal domains of Qa-SNAREs consist of a three-helix bundle.We set out to identify interaction partners for Qb-SNAREs, which function in endosomal traffic. The yeast Qb-SNARE Vti1p is required for the following: (i) transport from the TGN to the prevacuolar endosome; (ii) traffic to the vacuole (equivalent to the mammalian lysosome); (iii) retrograde traffic to the cis-Golgi; and (iv) homotypic TGN fusion as part of four different SNARE complexes (4 -6). Mammalian cells contain two homologs of yeast Vti1p. vti1a and vti1b share 30% of their amino acid residues with each other as well as with V...

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