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Publisher: National Academy of Sciences
Languages: English
Types: Article
Subjects: Biological Sciences, QH
An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from\ud the vesicle coat by the molecular chaperone, Hsc70 and the J-domain-containing protein,\ud auxilin, in an ATP-dependent process. We present the first detailed mechanistic analysis\ud of clathrin disassembly catalysed by Hsc70 and auxilin, using loss of perpendicular light\ud scattering to monitor the process. We report that a single auxilin per clathrin triskelion is\ud required for maximal rate of disassembly, that ATP is hydrolysed at the same rate that\ud disassembly occurs and that three ATP molecules are hydrolysed per clathrin triskelion\ud released. Stopped-flow measurements revealed a lag phase in which the scattering\ud intensity increased owing to association of Hsc70 with clathrin cages followed by serial\ud rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped flow data to\ud several physically plausible mechanisms showed the best fit to a model in which\ud sequential hydrolysis of three separate ATP molecules is required for the eventual\ud release of a triskelion from the clathrin-auxilin cage.
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