Abstract
Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P 2 (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.
Highlights
Clathrin-mediated endocytosis (CME) is a vital multistep process in all eukaryotes in which plasma membrane cargo proteins are first concentrated into a patch by clathrin adaptors, which are themselves clustered by interaction with a polymeric clathrin scaffold
The patch of membrane is deformed toward the cytoplasm and undergoes scission from the parent membrane to form a clathrin-coated vesicle (CCV), which subsequently delivers its cargo into the endocytic system [reviewed in [1, 2]]
We applied subtomogram averaging to determine the structure of adaptor protein 2 (AP2) to ~9 Å resolution
Summary
Clathrin-mediated endocytosis (CME) is a vital multistep process in all eukaryotes in which plasma membrane cargo proteins are first concentrated into a patch by clathrin adaptors, which are themselves clustered by interaction with a polymeric clathrin scaffold. In contrast to the sample lacking clathrin, buds and vesicles were formed with both AP2 and clathrin coating their membranes In vesicles and buds with both [ED]xxxL[LI] and Yxx cargoes, the dimeric form of AP2 is absent, likely because the N-terminal helix of 2 has moved to contact the membrane and can no longer contribute to dimerization interface.
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