Glucose remodels the post-translational modifications of the yeast arrestin-like protein Rod1 to promote glucose-induced transporter endocytosis.

Mammalian arrestins have a major role in the intracellular trafficking of seven-transmembrane (7TM) receptors. The fungal ambient pH signaling pathway involves an arrestin-related protein, PalF/Rim8, and the ESCRT (endosomal sorting complex required for transport) machinery. We found that in Saccharomyces cerevisiae, Rim8 binds to both the putative 7TM pH sensor Rim21 and the ESCRT-I subunit Vps23. We show that an SXP motif in Rim8 mediates binding to the Vps23 ubiquitin E2 variant (UEV) domain and that a monoubiquitinated residue near the SXP motif contributes to this interaction. We present evidence that Rim8 ubiquitination is dependent on the Rsp5 E3 ubiquitin ligase and triggered upon binding of Vps23 UEV to both the SXP motif and ubiquitin, thus suggesting a two-step binding mechanism. We further show that Rim8 coimmunoprecipitates with ESCRT-I subunits Vps23 and Vps28, supporting the idea that binding of Rim8 to Vps23 mediates the association of Rim8 with the ESCRT-I complex. Fluorescence microscopic analyses indicate that overexpressed Rim8 and Vps23 colocalize at cortical punctate structures, providing additional evidence of the interaction between these two proteins. Strikingly, our findings indicate that evolutionary conserved mechanisms control the recruitment of the ESCRT machinery to Pal/Rim proteins in fungi and retroviral Gag proteins in animal cells.Arrestins play an essential role in the regulation of seventransmembrane (7TM) receptors (31). Although initially identified on the basis of their ability to uncouple 7TM receptors from heterotrimeric G proteins in a process known as desensitization, arrestins were later found to serve as endocytic adaptors that recruit clathrin and the clathrin adaptor protein AP-2 and facilitate 7TM receptor internalization via clathrincoated vesicles (17,29,30).Until recently, arrestins were thought to be restricted to the animal kingdom. However, the identification of an arrestin-related protein in Aspergillus nidulans, PalF, demonstrated that members of this protein family are also present in fungi (22). PalF and its yeast homolog Rim8 are involved in the Pal/Rim signaling pathway, which mediates the ambient pH response and is activated in neutral-alkaline environments (41). As mammalian beta-arrestins, PalF, which contains arrestin N-terminal and C-terminal domains, binds to the cytoplasmic domain of a 7TM protein, the putative pH sensor PalH, and is ubiquitinated in a signal-and 7TM receptor-dependent manner (22).The PalF/Rim8 signaling function does not appear to be related to receptor desensitization, as there is no evidence of G protein involvement in the Pal/Rim pathway. However, evidence strongly suggests that these proteins, as mammalian ␤-arrestins, play a role as endocytic adaptors. Most of the components of the ESCRT (endosomal sorting complex required for transport) machinery involved in the MVB (multivesicular body) sorting pathway (24, 48), play an essential role in the Pal/Rim signaling pathway (4,7,12,15,20,28,43,45,62). According to the current m...