National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)
R01 GM127548-01A1
United States
Citation
Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization. Authors: Edward A Partlow / Kevin S Cannon / Gunther Hollopeter / Richard W Baker / Abstract: Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage ...Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.
Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force -10, 4 second blot, 4 uL sample.
Details
Heparin added at a 2-fold excess 20 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT, 0.05% beta-OG
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Electron microscopy
Microscope
FEI TALOS ARCTICA
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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