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Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating

Nature volume 432, pages 649–653 (2004)Cite this article

Abstract

Clathrin-coated pits invaginate from specific membrane compartments and pinch off as coated vesicles. These vesicles then uncoat rapidly once released. The Hsc70 molecular chaperone effects the uncoating reaction, and is guided to appropriate locations on clathrin lattices by the J-domain-containing co-chaperone molecule auxilin1,2,3,4. This raises the question of how a local event such as ATP hydrolysis by Hsc70 can catalyse a global disassembly. Here, we have used electron cryomicroscopy to determine 12-Å-resolution structures of in-vitro-assembled clathrin coats in association with a carboxy-terminal fragment of auxilin that contains both the clathrin-binding region and the J domain. We have located the auxilin fragment by computing differences between these structures and those lacking auxilin (described in an accompanying paper5). Auxilin binds within the clathrin lattice near contacts between an inward-projecting C-terminal helical tripod and the crossing of two ‘ankle’ segments; it also contacts the terminal domain of yet another clathrin ‘leg’. It therefore recruits Hsc70 to the neighbourhood of a set of critical interactions. Auxilin binding produces a local change in heavy-chain contacts, creating a detectable global distortion of the clathrin coat. We propose a mechanism by which local destabilization of the lattice promotes general uncoating.

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Figure 1: Binding of auxilin(547–910) to clathrin coats.
Figure 2: Three-dimensional image reconstruction of a clathrin D6 barrel with bound auxilin(547–910) at 12 Å resolution.
Figure 3: Changes in clathrin heavy-chain contacts produced by auxilin binding.
Figure 4: Fit of the auxilin J domain into the difference density.

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Acknowledgements

We are grateful to W. Boll and I. Rapoport for help in the purification of clathrin and adaptors, and to P. Sliz for advice on computational methods. This work was supported by grants from the NIH to T.K. and to D. De Rosier. N.G. and S.C.H. are investigators in the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Biophysics Graduate Program, Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Alexander Fotin

  2. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Yifan Cheng & Thomas Walz

  3. Howard Hughes Medical Institute and Department of Biochemistry, Rosenstiel Basic Medical Sciences Research Center, Brandeis University, 415 South Street, Waltham, Massachusetts, 02454, USA

    Nikolaus Grigorieff

  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, and Howard Hughes Medical Institute, 250 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Stephen C. Harrison

  5. Department of Cell Biology and the CBR Institute for Biomedical Research, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts, 02115, USA

    Tomas Kirchhausen

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  1. Alexander Fotin
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Correspondence to Tomas Kirchhausen.

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Fotin, A., Cheng, Y., Grigorieff, N. et al. Structure of an auxilin-bound clathrin coat and its implications for the mechanism of uncoating. Nature 432, 649–653 (2004). https://doi.org/10.1038/nature03078

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