Deubiquitinating enzymes Ubp2 and Ubp15 regulate endocytosis by limiting ubiquitination and degradation of ARTs.

Endocytic down-regulation of cell-surface proteins is a fundamental cellular process for cell survival and adaptation to environmental stimuli. Ubiquitination of cargo proteins serves as the sorting signal for downstream trafficking and relies on the arrestin-related trafficking adaptor (ART)-Rsp5 ubiquitin ligase adaptor network in yeast. Hence proper regulation of the abundance and activity of these ligase-adaptor complexes is critical for main-tenance of optimal plasma membrane protein composition. Here we report that the stability of ARTs is regulated by the deubiquitinating enzymes (DUBs) Ubp2 and Ubp15. By counteracting the E3 ubiquitin ligase Rsp5, Ubp2 and Ubp15 prevent hyperubiquitination and proteasomal degradation of ARTs. Specifically, we show that loss of both Ubp2 and Ubp15 results in a defect in Hxt6 endocytosis associated with Art4 instability. Our results uncover a novel function for DUBs in the endocytic pathway by which Ubp2 and Ubp15 positively regulate the ART-Rsp5 network.