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Reconstitution of clathrin-coated bud and vesicle formation with minimal components

Nature Cell Biology volume 14, pages 634–639 (2012)Cite this article

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Abstract

During the process of clathrin-mediated endocytosis an essentially planar area of membrane has to undergo a gross deformation to form a spherical bud. Three ways have been recognized by which membranes can be induced to transform themselves locally from a planar state to one of high curvature: a change in lipid distribution between the leaflets, insertion of a protein into one leaflet and formation of a protein scaffold over the surface1. Such a scaffold is spontaneously generated by clathrin2,3,4,5. Conjectures that the attachment of clathrin was the cause of the change in curvature were challenged on theoretical grounds6, and also by the discovery of a number of clathrin-associated proteins with the capacity to induce membrane curvature7,8,9. We have now developed a cell-free system that has enabled us to demonstrate that clathrin polymerization alone is sufficient to generate spherical buds in a membrane. This process is reversible, as shown by the reassimilation of the buds into the planar membrane when the intra-clathrin contacts are dissociated by the chaperone Hsc70. We further show that the final step in the formation of coated vesicles ensues when clathrin-coated buds are released through the action of dynamin.

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Figure 1: Specific association of H6-ΔENTH-epsin144–575 and clathrin with Ni2+-NTA-DOGS-containing liposomes.
Figure 2: Protein-anchor-dependent clathrin bud formation.
Figure 3: AP180- and epsin-dependent recruitment of clathrin to liposomes.
Figure 4: Effect of Hsc70 and auxilin 1 on clathrin lattice structure and stability of membrane buds.
Figure 5: Generation of clathrin-coated vesicles by dynamin-catalysed scission.

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Acknowledgements

We thank H. Böning, C. Lemke and H. Ungewickell for expert technical assistance. Special thanks go to W. B. Gratzer and A. Ungewickell for their critical comments on this work. The German Research Foundation (DFG) supported early stages of this study.

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Authors and Affiliations

  1. Department of Cell Biology, Centre of Anatomy, Hannover Medical School, Carl-Neuberg Str. 1, D-30625 Hannover, Germany

    Philip N. Dannhauser & Ernst J. Ungewickell

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  1. Philip N. Dannhauser
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E.J.U. and P.N.D. designed the study, conducted the experiments, interpreted the results and wrote the manuscript.

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Correspondence to Ernst J. Ungewickell.

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Dannhauser, P., Ungewickell, E. Reconstitution of clathrin-coated bud and vesicle formation with minimal components. Nat Cell Biol 14, 634–639 (2012). https://doi.org/10.1038/ncb2478

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