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. BLOC-1 (biogenesis of lysosome-related organelles complex-1) is critical for melanosome biogenesis and has also been implicated in neurological function and disease. BLOC-1 is not a clathrin adaptor, but functions in melanosome biogenesis in concert with the clathrin adaptor complex AP-3. We found that BLOC-1 is an elongated complex that contains one copy each of the eight subunits pallidin, Cappuccino, dysbindin, Snapin, Muted, BLOS1, BLOS2, and BLOS3. The complex appears as a linear chain of eight globular domains, ∼300 Ålong and ∼30 Åin diameter. The individual domains are flexibly connected such that the linear chain undergoes bending by as much as 45. Two stable subcomplexes were defined, pallidin-Cappuccino-BLOS1 and dysbindin-Snapin-BLOS2. Both subcomplexes are 1:1:1 heterotrimers that form extended structures as indicated by their hydrodynamic properties. The two subcomplexes appear to constitute flexible units within the larger BLOC-1 chain, an arrangement conducive to simultaneous interactions with multiple BLOC-1 partners in the course of tubular endosome biogenesis and sorting.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2013
Total Cost
$274,703
Indirect Cost
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