Research ArticleCELL BIOLOGY

Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles

See allHide authors and affiliations

Science Advances  22 Jul 2020:
Vol. 6, no. 30, eaba8381
DOI: 10.1126/sciadv.aba8381
  • Fig. 1 Overview of AP2 and clathrin structures.

    (A) Schematic representation of AP2 and clathrin polypeptide chains with marked domains. A length ruler (in amino acid residues) is given at the top. Note that the FLAP AP2 construct lacks α hinge and appendage regions, while the Core AP2 construct lacks α and β2 hinge and appendage regions. (B) Cartoon representation of the structures of AP2 (left) and clathrin (right). Subunits are color-coded as in (A) and known functional sites and domains are marked. The boxed panel shows a zoomed-out view of a clathrin triskelion with neighboring triskelia in white.

  • Fig. 2 Conformational activation and membrane interactions of AP2.

    (A) Comparison of single-particle structure of the cytosolic closed form (top) with the tomography-resolved open form of AP2 on the membrane in the presence of YxxΦ cargo (bottom). EM maps (semitransparent gray) are fitted with corresponding ribbon models of AP2 color-coded by chain. Arrowheads indicate membrane leaflets. Close-ups show details of membrane contacts with α and μ2 subunits and the lack of contact with β2, with PtdIns(4,5)P2 sites and cargo peptides marked. The μ2 basic loop includes K167, Y168, R169, and R170, while the μ2 basic patch includes K350, K367, and R368. (B) Surfaces of the atomic models for cytosolic and membrane-recruited AP2 in (A) are shown to illustrate the rearrangement between cytosolic and membrane-bound forms. The arrow indicates the major movement of Cμ2 out of the bowl of AP2. (C) Conformation and membrane interactions of AP2 recruited to the membrane via YxxΦ or YxxΦ/[ED]xxxL[LI] cargo signals within assembled clathrin coats. Additional tilting of α in respect to the membrane is apparent when [ED]xxxL[LI] cargo is present (compare top and bottom panels in the left column). The close-up demonstrates new membrane contacts formed by β2 and μ2 subunits, and additional density in the cargo binding pocket when [ED]xxxL[LI] cargo is present. The panel above the close-up shows YxxΦ/[ED]xxxL[LI]-bound AP2 viewed from the membrane side with peptide-binding sites marked. See also fig. S2E for close-up views of the cargo-binding sites.

  • Fig. 3 The structure of clathrin on coated membranes.

    EM maps (gray surface) of clathrin legs bounding hexagon and pentagon (see fig. S4 for details). The right column shows the EM map (sharpened to reveal high-resolution features) overlaid with a fitted ribbon model for the clathrin hub. N-terminal portions of CHC are clearly defined. Density corresponding to the AP2 β2 appendage indicates higher occupancy in hexagon than in pentagon maps (see also fig. S6). The dashed box indicates the region shown in Fig. 5.

  • Fig. 4 Experimental model of a coated bud formed on the YxxΦ cargo–containing membrane.

    The model of a representative bud was produced by placing densities for AP2 core (light green), AP2 β2 appendage (dark green), and clathrin legs (gold with NTDs colored orange) at positions and orientations determined by subtomogram averaging and classification (for β2 appendage) for that bud. AP2 core, β2 appendage, and clathrin densities were simulated on corresponding pseudoatomic models. The membrane was defined by segmenting the tomogram. (Top) The left panel shows the exterior of the bud. The front half of the clathrin cage is removed in the middle panel, revealing the random AP2 distribution on the membrane. The right panel shows a cross section through the entire bud. (Bottom) Close-up and cut-open views at the hexagon boxed in the top left panel.

  • Fig. 5 Interactions between clathrin and the AP2 β2 appendage domain.

    (A) Positions of NTD and the β2 appendage with respect to the clathrin lattice. Overlay of EM map of leg bounding hexagon enriched in β2 appendage (transparent gray; see fig. S6 for details) with fitted protein ribbon models. The β2 appendage makes contact with sites on three CHC: two in the ankle segment (CHC repeats 1 and 2) and one in the NTD. (B) Close-up of NTD and β2 appendage region indicating the binding sites of low-density lipoprotein receptor adaptor ARH and Eps15. ARH and Eps15 peptides are pink, positioned as in PDB 2g30 (23) and 2iv9 (24), together with a simulated 15-Å-resolution isosurface (transparent pink). Binding sites for clathrin boxes in the NTD are marked. (C) As in (B), showing the binding position of DNAJ auxilin domain in blue, as described in PDB 1xi5 (25).

Supplementary Materials

  • Supplementary Materials

    Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles

    Oleksiy Kovtun, Veronica Kane Dickson, Bernard T. Kelly, David J. Owen, John A. G. Briggs

    Download Supplement

    This PDF file includes:

    • Figs. S1 to S7
    • Tables S1 to S3

    Files in this Data Supplement:

Stay Connected to Science Advances

Navigate This Article