Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif

Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling of network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargoes at the cell surface and link them to the PM and clathrin coat. Muniscins are a conserved family of endocytic adaptors, including Syp1 in budding yeast and its mammalian orthologue, FCHo1. These adaptors bind cargo via a C-terminal mu-homology domain (mu HD); however, few cargoes exhibiting muniscin-dependent endocytosis have been identified, and the sorting sequence recognized by the mu HD is unknown. To reveal Syp1 cargo-sorting motifs, we performed a phage display screen and used biochemical methods to demonstrate that the Syp1 mu HD binds DxY motifs in the previously identified Syp1 cargo Mid2 and the v-SNARE Snc1. We also executed an unbiased visual screen, which identified the peptide transporter Ptr2 and the ammonium permease Mep3 as Syp1 cargoes containing DxY motifs. Finally, we determined that, in addition to regulating cargo entry through CME, Syp1 can promote internalization of Ptr2 through a recently identified clathrinin-dependent endocytic pathway that requires the Rho1 GTPase. These findings elucidate the mechanism of Syp1 cargo recognition and its role in trafficking.