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.
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Acknowledgements
We thank W. Boll and I. Rapoport for help with protein preparations; E. Marino for maintaining the Imaging Resource used in this study; G. Danuser, A. van Oijen and D. Floyd for advice; and members of our laboratories for discussions. T.B. acknowledges a Cross-Disciplinary Fellowship of the Human Frontier Science Program Organization, and F.A. a Swiss National Science Foundation fellowship and partial support from US National Institutes of Health (NIH) grant GM-073165 (to G. Danuser, Harvard Medical School). This work was supported by NIH grants GM-075252 and U54 AI057159 (New England Regional Center of Excellence in Biodefense and Emerging Infectious Disease, Core Imaging Facility) to T.K. S.C.H. is an investigator in the Howard Hughes Medical Institute.
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T.B. and T.K. developed the single-molecule assay and T.B. performed experiments; T.B. and F.A. analyzed data; F.A. developed the kinetic model. T.B., S.C.H. and T.K. designed experiments. All authors discussed the results and contributed to the final manuscript.
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Böcking, T., Aguet, F., Harrison, S. et al. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18, 295–301 (2011). https://doi.org/10.1038/nsmb.1985
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DOI: https://doi.org/10.1038/nsmb.1985
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