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

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Abstract
Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargos 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 ortholog, FCHo1. These adaptors bind cargo via a C-terminal ì-homology domain (ìHD); however, few cargos exhibiting muniscin-dependent endocytosis have been identified, and the sorting sequence recognized by the μHD is unknown. To reveal Syp1 cargo-sorting motifs, we performed a phage display screen and used biochemical methods to demonstrate that the Syp1 μHD 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 cargos 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 clathrin-independent endocytic pathway that requires the Rho1 GTPase. These findings elucidate the mechanism of Syp1 cargo recognition and its role in trafficking.

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