Arpita Sen

It is well-known that in addition to its classical role in protein turnover, ubiquitination is required for a variety of membrane protein sorting events. However, and despite substantial progress in the field, a long-standing question remains:given that all ubiquitin (Ub) units are identical, how do different elements of the sorting machinery recognize their specific cargoes?Here we provide an answer to this question as we discovered a mechanism based on the coincidence detection of lysine ubiquitination and Ser/Thr phosphorylation for the endocytic adaptor epsin to mediate the internalization of the yeast Na⁺pump Ena1.Internalization of Ena1-GFP was abolished in double epsin knock-out inS. cerevisiaeand was rescued by re-introducing either one of the 2 yeast epsins, Ent1 or Ent2 in an UIM (Ub InteractingMotif)-dependent manner. Further, our results indicate that ubiquitination of its C-terminal Lys¹⁰⁹⁰is needed for internalization of Ena1 and requires the arrestin-related-trafficking adaptor, Art3.We determined that in addition to ubiquitination of K¹⁰⁹⁰, the presence of a Ser/Thr-rich patch (S¹⁰⁷⁶TST¹⁰⁷⁹) within Ena1 was also essential for its internalization. Our results suggest that this ST motif is targeted for phosphorylation by casein kinases. Nevertheless, phosphorylation of this S/T patch was not required for ubiquitination. Instead, ubiquitination of K¹⁰⁹⁰and phosphorylation of the ST motif were independently needed for epsin-mediated internalization of Ena1.We propose a model in which a dual detection mechanism is used by Ub-binding elements of the sorting machinery to differentiate among multiple Ub-cargoes.