Curvature of clathrin-coated pits driven by epsin

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Abstract

Clathrin-mediated endocytosis involves cargo selection and membrane budding into vesicles with the aid of a protein coat. Formation of invaginated pits on the plasma membrane and subsequent budding of vesicles is an energetically demanding process that involves the cooperation of clathrin with many different proteins. Here we investigate the role of the brain-enriched protein epsin 1 in this process. Epsin is targeted to areas of endocytosis by binding the membrane lipid phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2). We show here that epsin 1 directly modifies membrane curvature on binding to PtdIns(4,5)P2 in conjunction with clathrin polymerization. We have discovered that formation of an amphipathic α-helix in epsin is coupled to PtdIns(4,5)P2 binding. Mutation of residues on the hydrophobic region of this helix abolishes the ability to curve membranes. We propose that this helix is inserted into one leaflet of the lipid bilayer, inducing curvature. On lipid monolayers epsin alone is sufficient to facilitate the formation of clathrin-coated invaginations.

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Figure 1: Epsin 1 ENTH domain tubulates liposomes.
Figure 2: Structure of epsin ENTH bound to Ins(1,4,5)P3.
Figure 3: Binding of epsin ENTH and mutants to phosphoinositides.
Figure 4: Effect of full-length epsin and mutants on the distribution of the AP2 complex in COS-7 cells visualized by sequential imaging with confocal microscopy.
Figure 5: Mutations of L6 on helix 0 affect the ability of epsin to tubulate liposomes.
Figure 6: Clathrin recruitment to lipid monolayers by epsin.

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Acknowledgements

We thank O. Perisic and R. Williams for advice on protein–membrane interactions; B. Collins and staff at the ESRF for assistance with data collection; J. Butler for assistance with analytical ultracentrifugation; and J. Berriman for assistance with electron microscopy. We also thank B. Pearse, N. Unwin, M. Stowell, M. Goedert, S. Munro, P. Rosenthal and members of our laboratories for extensive discussions, and J. Gallop for a supply of dynamin. I.G.M. was supported by an MRC Postdoctoral Fellowship, B.J.P was supported by an EMBO Long Term Fellowship, and G.J.K.P. was supported by a Marie Curie Fellowship.

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Correspondence to Philip R. Evans or Harvey T. McMahon.

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Ford, M., Mills, I., Peter, B. et al. Curvature of clathrin-coated pits driven by epsin. Nature 419, 361–366 (2002) doi:10.1038/nature01020

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