SummaryThe AP2 adaptor complex (α, β2, σ2, and μ2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P2-containing membranes and transmembrane protein cargo. In the “locked” cytosolic form, AP2's binding sites for the two endocytic motifs, YxxΦ on the C-terminal domain of μ2 (C-μ2) and [ED]xxxL[LI] on σ2, are blocked by parts of β2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-μ2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured μ2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P2- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P2/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P2-containing membranes.

SummaryMost transmembrane proteins are selected as transport vesicle cargo through the recognition of short, linear amino acid motifs in their cytoplasmic portions by vesicle coat proteins. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by clathrin adaptors. The AP2 adaptor complex (subunits α,β2,μ2,σ2) recognises both major endocytic motifs: YxxΦ motifs 1 and [DE]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 2. The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing PtdIns4,5P2, but do not affect binding to YxxΦ motifs via μ2. In the ‘inactive’ AP2 core structure 3, both motif binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 N-terminus is displaced and becomes disordered; however, in this structure the YxxΦ binding site on μ2 remains blocked.

Clathrin-mediated endocytosis (CME) is vital for the internalization of most cell-surface proteins. In CME, plasma membrane-binding clathrin adaptors recruit and polymerize clathrin to form clathrin-coated 'pits' into which cargo is sorted. AP2 is the most abundant adaptor, and is pivotal to CME. By determining a new structure of AP2 that includes the clathrin-binding β2-hinge and developing an AP2-dependent budding assay, we reveal the existence of an autoinhibitory mechanism that prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide PtdIns(4,5)P 2 and cargo relieves this autoinhibition, so triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism constitutes an unsuspected layer of regulation that couples AP2's membrane recruitment to its key functions of cargo and clathrin binding.Clathrin adaptors provide an essential physical bridge connecting clathrin, which itself lacks membrane binding activity (1), to the membrane and to embedded transmembrane protein cargo. A central player in CME is the AP2 (Assembly Polypeptide 2) complex, (Figs 1A, S1), which both coordinates CCP formation and binds the many cargo proteins that contain 'acidic dileucine' and Yxxφ endocytic motifs (φ denotes a bulky hydrophobic residue) through its membrane proximal core (2, 3). Cargo binding is activated by a large-scale conformational change from the 'locked' or 'inactive' cytosolic form to an 'open' or 'active' form driven by localization to membranes containing the plasma membrane phosphoinositide PtdIns(4,5)P 2 (4, 5). The C-terminal 'appendages' of the α and β2 subunits bind other clathrin adaptors as well as CCV (clathrin-coated vesicle) assembly and disassembly accessory factors (3,(6)(7)(8) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts from the β2-trunk binds the N-terminal beta-propeller of the clathrin heavy chain using a canonical clathrin box motif (LLNLD; Fig 1A,B (9)). The β2 appendage domain also binds clathrin, albeit weakly, but both interactions are necessary for robust clathrin binding (10).A version of AP2 comprising full-length β2, μ2 and σ2 subunits, and the α-trunk domain, (FLβ.AP2) (Fig 1B)(11) was expressed in E.coli, avoiding contamination with other CCV components inherent to purification from brain tissue (12, 13). Despite most FLβ.AP2 possessing an intact β2 subunit (Fig 1C-E), it bound clathrin very poorly in pulldowns when immobilized on either glutathione sepharose beads ( Fig 1C) or via its N-terminal His6 tag (similarly positioned to the β2 PtdIns(4,5)P 2 binding site Fig1B (4, 5).) to liposomes containing the nickel-attached lipid NiNTA-DGS ( Fig 1E): in both cases the FLβ.AP2 will be in its locked cytosolic conformation (4). FLβ.AP2 also failed to stimulate clathrin cage assembly efficiently at physiological pH ( Fig 1D). In contrast, the isolated β2 hingeappendage ('GST-β2-h+app', Fig S1) bound clathrin efficiently ( Fig 1C) and stimu...

SummaryThe size of endocytic clathrin-coated vesicles (CCVs) is remarkably uniform, suggesting that it is optimized to achieve the appropriate levels of cargo and lipid internalization. The three most abundant proteins in mammalian endocytic CCVs are clathrin and the two cargo-selecting, clathrin adaptors, CALM and AP2. Here we demonstrate that depletion of CALM causes a substantial increase in the ratio of “open” clathrin-coated pits (CCPs) to “necked”/“closed” CCVs and a doubling of CCP/CCV diameter, whereas AP2 depletion has opposite effects. Depletion of either adaptor, however, significantly inhibits endocytosis of transferrin and epidermal growth factor. The phenotypic effects of CALM depletion can be rescued by re-expression of wild-type CALM, but not with CALM that lacks a functional N-terminal, membrane-inserting, curvature-sensing/driving amphipathic helix, the existence and properties of which are demonstrated. CALM is thus a major factor in controlling CCV size and maturation and hence in determining the rates of endocytic cargo uptake.