The Na+ pump Ena1 is a yeast epsin-specific cargo requiring its ubiquitylation and phosphorylation sites for internalization

Abstract

It is well known that in addition to its classical role in protein turnover, ubiquitylation 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 (Ent1 and Ent2 in yeast)-specific cargo and that its internalization requires K¹⁰⁹⁰, which likely undergoes Art3-dependent ubiquitylation. In addition, an Ena1 serine and threonine (ST)-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 ubiquitylation. Furthermore, epsin-mediated internalization of Ena1 required a specific spatial organization of the ST patch with respect to K¹⁰⁹⁰ within the cytoplasmic tail of the pump. We hypothesize that ubiquitylation and phosphorylation of Ena1 are required for epsin-mediated internalization.