ENTH domain proteins are cargo adaptors for multiple SNARE proteins at the TGN endosome.

ABSTRACT

ENTH and ANTH domain proteins are involved in budding of clathrin-coated vesicles. SNAREs are fusogenic proteins that function in the targeting and fusion of transport vesicles. In mammalian and yeast cells, ENTH domain proteins (epsinR and Ent3p) interact with SNAREs of the vti1 family (Vti1b or Vti1p). This interaction indicates that ENTH proteins could function in cargo sorting, which prompted us to search for additional SNAREs as potential cargo for Ent3p and epsinR. We carried out specific yeast two-hybrid assays, which identified interactions between epsinR and the mammalian late endosomal SNAREs syntaxin 7 and syntaxin 8 as well as between Ent3p and the endosomal SNAREs Pep12p and Syn8p from yeast. Lack of Ent3p affected the trafficking of Pep12p. Ent3p binding to Pep12p required the FSD late endosomal sorting signal in Pep12p. Inactivation of the sorting signal had a similar effect to removal of Ent3p on Pep12p stability indicating that Ent3p acts as a cargo adaptor for Pep12p by binding to the sorting signal. As Vti1p, Pep12p and Syn8p participate in a SNARE complex whereas Vti1b, syntaxin 7 and syntaxin 8 are mammalian SNARE partners, we propose that ENTH domain proteins at the TGN-endosome are cargo adaptors for these endosomal SNAREs.