Molecular Recognition by Clathrin Adaptors

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Molecular Recognition by Clathrin Adaptors The clathrin coat plays a ubiquitous and fundamental role in endocytosis and in endosomal sorting within the eukaryotic cell. Clathrin forms a cage that surrounds cargo-bearing vesicles, but clathrin itself does not directly bind to cargo. Cargo is sorted into clathrin-coated vesicles by adaptor proteins that physically bridge cargo and clathrin. The best-known general purpose adaptors are the heterotetrameric adaptor protein complexes (AP complexes) and the multimodular GGA adaptor proteins. The overall goals of this project are 1) to identify the binding sites for cargo on adaptor proteins and measure their affinities quantitatively; 2) to determine the crystal structures of complexes between adaptors and soluble cargo fragments; and 3) to relate structure and function using mutational analysis. The subunits of the AP complexes and the GGAs define conserved structural domains that are also found in other endocytic proteins. In the past year, we collaborated with Beverly Wendland (JHU) to define the structures of the domains of the endocytic protein Syp1. Syp1 is proposed to be a founding member of the newly-named muniscin family of endocytic adaptors that is conserved from yeast to humans. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding mu homology domains (muHD). In vitro and in vivo assays in the Wendland laboratory confirmed membrane tubulation activity for muniscin EFC/F-BAR domains. The mHD domain has conserved interactions with the endocytic adaptor/scaffold Ede1/eps15, which influences muniscin localization. A transmembrane protein previously implicated in polarized Rho1 signaling was identified as a cargo of the yeast adaptor protein. We propose that muniscins directly link cargo selection with vesicle formation, thereby regulating receptors that stimulate cell polarization pathways.