The Na+ pump Ena1 is a yeast Epsin-specific cargo requiring its ubiquitination/phosphorylation sites for internalization

Abstract

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 units are identical, how do different elements of the sorting machinery recognize their specific cargoes? Our results indicate that the yeast Na+ pump Ena1 is an epsin-specific cargo and that its internalization required Lys1090, which likely undergoes Art3-dependent ubiquitination. In addition, an Ena1 Ser/Thr-rich patch, proposed to be targeted for phosphorylation by casein kinases, was also required for its uptake. Interestingly, our data suggest that this phosphorylation was not needed for cargo ubiquitination. Further, epsin-mediated internalization of Ena1 required a specific spatial organization of the Ser/Thr-rich patch with respect to Lys1090 within the cytoplasmic tail of the pump. We hypothesize that ubiquitination and phosphorylation of Ena1 are required for epsin-mediated internalization.