A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

Abstract

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin-auxilin cage.clathrin-mediated endocytosis | kinetic mechanism | macromolecular assemblies | vesicle uncoating | mathematical model

Diversity of Clathrin Function: New Tricks for an Old Protein

“…ATP hydrolysis by Hsc70 bound in this region potentially could alter triskelion pucker and put strain on the lattice to cause the destabilization suggested by in silico models. The issue of how many ATPs must be hydrolyzed to induce disassembly and the precise sequence of molecular interactions during uncoating is not resolved (Bocking et al 2011, Rothnie et al 2011. Three studies have also suggested a role for CLC in uncoating through independent influence on hsc70 and auxillin activity (DeLuca-Flaherty et al 1990, Ferreira et al 2012, Schmid et al 1984, though uncoating CHC17 can occur without CLC (Ungewickell et al 1995).…”

Structures and mechanisms of vesicle coat components and multisubunit tethering complexes

“…Recent structural [47,48] and biophysical studies [49] have further elucidated the role of auxilin in coat disassembly. Auxilin binds directly to both clathrin and the uncoating ATPase, Hsc70.…”

Clathrin Coat Disassembly by the Yeast Hsc70/Ssa1p and Auxilin/Swa2p Proteins Observed by Single-particle Burst Analysis Spectroscopy

“…Clathrin coat disassembly has been reported as the appearance of clathrin triskelia in the supernatant fraction of a high speed centrifugation of fractionated brain homogenates, although baskets remain in the pellet (9,23). Disassembly of neuronal clathrin coats has also been observed by measuring changes in average light scattering as a function of Hsc70 ATPase activity (24,25). Recent work has suggested a cooperative disassembly model based on total internal reflection fluorescence microscopy measurements with tethered neuronal clathrin baskets reconstituted from purified components (20).…”