Downregulation of the broad-specificity amino acid permease Agp1 mediated by the ubiquitin ligase Rsp5 and the arrestin-like protein Bul1 in yeast.

Most of plasma membrane transporters are downregulated by ubiquitination-dependent endocytosis to avoid the excess uptake of their substrates. In Saccharomyces cerevisiae, ubiquitination of transporters is mediated by the HECT-type ubiquitin ligase Rsp5. We report here a mechanism underlying the substrate-induced endocytosis of the broad-specificity amino acid permease Agp1. First, we found that Agp1 underwent ubiquitination and endocytosis in response to the addition of excess asparagine, which is a substrate of Agp1. Moreover, the substrate-induced internalization of Agp1 was dependent on the ubiquitination activity of Rsp5. Since Rsp5 requires α-arrestin family proteins as adaptors to bind with substrates, we next developed a method of genetic screening to identify adaptor proteins for Agp1 endocytosis. This screening and biochemical analysis revealed that Bul1, but not its paralogue Bul2, was essential for the substrate-induced endocytosis of Agp1. Our results support that the substrate-induced endocytosis of Agp1 requires Rsp5 and Bul1.