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ABSTRACT: Heat shock cognate protein-70 (Hsc70) supports remodeling of protein complexes, such as disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP-driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by cryo-EM, in the vicinity of each occupied site and that accumulation of local strains destabilizes the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.
Nature Structural & Molecular Biology 01/2011; 18(3):295-301. · 12.71 Impact Factor
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ABSTRACT: The chaperone Hsc70 drives the clathrin assembly-disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 A resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.
The EMBO Journal 02/2010; 29(3):655-65. · 9.20 Impact Factor
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ABSTRACT: The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.
Molecular biology of the cell 02/2008; 19(1):405-13. · 5.98 Impact Factor
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ABSTRACT: Molecular and Cellular Biology The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.
