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

Abstract

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell’s plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo–electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P2 (phosphatidylinositol 4,5-bisphosphate)–containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 β2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

Architecture and mechanism of metazoan retromer:SNX3 tubular coat assembly

“…Subtomogram alignment and averaging were done as previously described in 6 using MATLAB (MathWorks) functions adapted from the TOM 56 , AV3 57 . As in 52 we used a modified wedge mask representing the amplitudes of the determined CTF and applied exposure filters at each tilt 58,59 . Table S2 contains a summary of data processing parameters.…”

“…Image pre-processing and tomogram reconstruction were performed essentially as described in 52 . The IMOD v. 4.10.3 package 53 was used to align frames in raw movies and correct for detector gain and pixel defects.…”

Evolving models for assembling and shaping clathrin-coated pits

“…The recent cryo-EM-based structural studies of native CCVs and membrane-bound AP2/clathrin buds have also provided some insight into curvature generation. Interestingly, AP2 complexes are not uniformly distributed on the clathrin coat but are depleted from curvature-essential pentagons (Kovtun et al, 2020;Paraan et al, 2020). Consistent with this, the flat-tocurved transition of clathrin coats has been reported to be marked by a decrease in the AP2/clathrin ratio (Bucher et al, 2018).…”

“…Instead of the expected β2-hinge-NTD interactions, both cryo-EM structures revealed that one β2-appendage domain cross-links two adjacent clathrin NTDs, bringing the NTD into contact with its neighboring clathrin ankle region, presumably to promote clathrin cage assembly (Paraan et al, 2020). Consistent with their predominant role in the recruitment of overexpressed β2 adaptins to CCPs in vivo (Edeling et al, 2006), the β2-appendage binding sites (Y815 and Y888) seem to interact more consistently with two sites on the clathrin ankle and the Royle-box site on the NTD (Kovtun et al, 2020;Paraan et al, 2020). Interestingly, these two sites on the β2 appendage are also binding sites for other EAPs and adaptors; Y815 in the sandwich domain is required for binding AP180/CALM (phosphatidylinositol binding clathrin assembly protein), Eps15, and autosomal recessive hypercholesterolemia, whereas Y888 in the platform domain is required for binding β-arrestins and epsin (Edeling et al, 2006;Schmid et al, 2006).…”

“…Recent structural studies have also questioned the role of clathrin-box motifs and their binding site in mediating AP2clathrin interactions. Two new cryo-EM studies have determined the structures of AP2 and clathrin-coated buds formed on PI(4,5)P 2 -and cargo-containing membranes (Kovtun et al, 2020) and of native CCVs purified from bovine brains (Paraan et al, 2020). Surprisingly, the β2-hinge could not be detected in either structure, and no evidence for its binding to the NTD binding sites could be found, suggesting that these interactions are weak and transient, variable, or both.…”