AP2 controls clathrin polymerization with a membrane-activated switch

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Science  25 Jul 2014:
Vol. 345, Issue 6195, pp. 459-463
DOI: 10.1126/science.1254836

A membrane-activated switch to bind clathrin

Clathrin-mediated endocytosis—the process by which cells take up nutrients and signals within clathrin-coated vesicles—is very well understood. Kelly et al. reveal an unanticipated layer of regulation in this process. The proteins AP2 and clathrin are the major constituents of endocytic clathrin-coated vesicles. AP2 and clathrin stick together through a clathrin-binding motif in AP2. The authors now show that AP2's clathrin-binding motif is normally buried within the core of the AP2 protein. AP2 only ejects its clathrin-binding motif and recruits clathrin if it is associated with the correct cell membrane and an endocytic cargo.

Science, this issue p. 459


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. Assembly polypeptide 2 (AP2) is the most abundant adaptor and is pivotal to CME. Here, we determined a structure of AP2 that includes the clathrin-binding β2 hinge and developed an AP2-dependent budding assay. Our findings suggest that an autoinhibitory mechanism prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate and cargo relieves this autoinhibition, triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism can couple 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 clathrin-mediated endocytosis (CME) is the AP2 (assembly polypeptide 2) complex (Fig. 1A and fig. S1), which both coordinates clathrin-coated pit (CCP) formation and binds the many cargo proteins that contain acidic dileucine and Yxxϕ endocytic motifs (Y denotes Tyr; x, any amino acid; and ϕ, 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 phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] (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, 68). The flexible hinge separating the β2 appendage from the β2 trunk binds the N-terminal β-propeller of the clathrin heavy chain by using a canonical clathrin box motif [LLNLD; L, Leu; N, Asn; D, Asp (Fig. 1, A and B) (9)]. The β2 appendage domain also binds clathrin, albeit weakly, but both interactions are necessary for robust clathrin binding (10).

Fig. 1 AP2 can bind clathrin only when it is attached to a PtdIns(4,5)P2- and cargo-containing membrane.

(A) AP2 schematic, color-coded by subunit: α, blue; β2, green; N-μ2, dark magenta; C-μ2, pale magenta; σ, cyan. The unstructured α and β2 hinge and appendage subdomains, together with the core of the complex, are indicated. Parts shown in gray are not present in FLβ.AP2. Schematics of constructs are shown in fig. S1. (B) FLβ.AP2 showing the positions of the clathrin box and purification tags. (C) Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS-PAGE) of glutathione-sepharose pulldowns using GST-FLβ.AP2 or GST-β2-h+app. Supernatant (s) and pellet (p). The band marked with an asterisk results from proteolysis of β2. MW, molecular weight. (D) Coomassie-stained SDS-PAGE of clathrin cage assembly assays using 2.5 μM clathrin and 1.5 μM adaptors (as indicated), overnight at 21°C, centrifuged to separate clathrin cages (p) from supernatants (s). (E) Coomassie-stained SDS-PAGE of liposome pulldown assays. Liposomes were sequentially incubated with adaptors and clathrin, then centrifuged to separate unbound material (s) from the liposome pellet (p).

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 Escherichia 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. 1, C to E), it bound clathrin very poorly in pulldowns when immobilized either on glutathione sepharose beads (Fig. 1C) or via its N-terminal His6 tag [similarly positioned to the β2 PtdIns(4,5)P2 binding site (Fig. 1B) (4, 5)] to liposomes containing the nickel-attached nitrilotriacetic acid–dioleoylgycerosuccinyl (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 hinge-appendage [glutathione S-transferase (GST)-β2-h+app (fig. S1)] bound clathrin efficiently (Fig. 1C) and stimulated cage assembly (Fig. 1D). We next compared clathrin recruitment to synthetic liposomes composed of dioleoylphosphatidylcholine and dioleoylphosphatidylethanolamine supplemented either with NiNTA-DGS or with a mixture of PtdIns(4,5)P2 and a lipid-linked YxxΦ endocytic motif (5, 11, 14). β2-h+app fused to His6-tagged epsin N-terminal homology (ENTH) domain (His6-ENTH-β2-h+app), which can bind NiNTA-DGS or PtdIns(4,5)P2, recruited clathrin efficiently to both types of liposomes. In contrast, FLβ.AP2 recruited clathrin only when bound to PtdIns(4,5)P2- and YxxΦ-containing liposomes (Fig. 1E). Thus, no additional proteins are required to prevent clathrin binding to AP2 in solution, consistent with immunoprecipitation data (15). We conclude that the clathrin-binding activity of AP2 is autoinhibited in the cytosol to restrict inappropriate clathrin recruitment and that only upon encountering its physiological membrane ligands [PtdIns(4,5)P2 and cargo] can AP2 recruit clathrin efficiently. Previous reports that AP2 purified from brain could bind and polymerize clathrin (12) were likely due to other contaminating clathrin adaptors, such as AP180 (13).

We were unable to crystallize FLβ.AP2, so we determined the structure of a form of AP2 (βhingeHis6.AP2) whose β2 (residues 1 to 650) includes the clathrin box–containing hinge but not the β2 appendage. The structure closely resembles that of the locked conformation of the AP2 core, but additional protein difference electron density was visible in the center of the AP2 core (fig. S2). Crystallographic analysis of AP2s truncated at Leu636 and Gln619 of β2 suggested that this electron density corresponded to the region of β2 between these points (11). A series of mutant AP2s with single methionine substitutions throughout the hinge region was created, the mutants crystallized as selenomethionyl derivatives, and their structures solved (fig. S3). This allowed us to assign unambiguously this density to residues 618 to 634 of β2 (including the clathrin box at residues 631 to 635) and refine the structure (Fig. 2, A and B; fig. S2; and movies S1 and S2). There are two regions of contact: a β sheet interaction between β2 residues 618 to 624 and α trunk residues 490 to 493 and the packing of the clathrin box itself and the short helix immediately preceding it with residues of C-μ2 and the β2 trunk (Fig. 2C and fig. S4). In this position, the LLNLD clathrin box is inaccessible, explaining FLβ.AP2’s lack of clathrin binding (Fig. 1), because deletion of only the LLNLD clathrin motif in GST-β2-h+app abolishes clathrin recruitment (fig. S5). The β2 618 to 636 region is well conserved across a wide range of species (fig. S6) and between β2 and β1 (the equivalent subunit of the AP1 adaptor). This suggests that, in AP1, clathrin binding will be similarly regulated by membrane attachment, albeit stimulated mainly by binding to guanosine 5′-triphosphate (GTP)–bound Arf1 (16, 17). Interfering with the interactions that trap the clathrin box in the AP2 core should release the hinge, allowing increased clathrin recruitment. Indeed, deletion of C-μ2 (termed FLβ.AP2.ΔCμ2), disrupting key interactions with β2 625 to 635 (that includes the clathrin motif; fig. S4), had a profound effect, resulting in efficient clathrin binding and cage assembly in solution (Fig. 2D and fig. S7). Deletion of the hinge residues 617 to 624, removing the largely backbone-mediated interaction with 490 to 493 of the α subunit (fig. S4), resulted in a modest but significant increase in AP2-mediated clathrin polymerization in solution at physiological pH [25% ± 3.1% clathrin polymerized (mean ± SEM, three experiments) versus 9.9% ± 3.8%; difference tested by Student’s t test, P = 0.037 (Fig. 2E)].

Fig. 2 The AP2 β2 subunit LLNLD clathrin binding motif is buried in the center of the core.

Overall (A) and closeup (B) views of the structure of βhingeHis6.AP2. The residues of the hinge resolved in the structure are shown in green as a stick representation. The AP2 core is depicted in a surface representation, colored as in Fig. 1. The residues of the buried hinge are indicated in (B), with electron density shown as mesh (2mFo – DFc map, contoured at 0.34 e Å−3). Q, Gln. Also shown are the positions of the selenium (Se) sites found in the bowl for each of the methionine (M) mutants indicated, showing good agreement with the positions of the corresponding wild-type residues that were mutated. Individual log-likelihood gradient maps are shown in fig. S3. (C) Ligplot+ (25) diagram showing interactions between buried hinge residues (in pale green) with residues of α (blue), μ2 (magenta), and β2 (dark green). Red fans indicate hydrophobic interactions; dashed green lines indicate hydrogen bonds. The residues of the clathrin-binding motif are boxed. See also fig. S4. A, Ala; C, Cys; E, Glu; G, Gly; H, His; I, Ile; K, Lys; P, Pro; S, Ser; and V, Val. (D) Clathrin cage assembly assays. (D) is identical to Fig. 1D (2.5 μM clathrin, 1.5 μM adaptors) with the addition of the FLβ.AP2.ΔCμ2 lane. (E) Assays performed as in (D) but at 28°C and with 2 μM clathrin and 4 μM adaptors as shown.

The structure suggests a mechanism by which clathrin binding is triggered by AP2’s membrane recruitment in the cell. Aligning the open and locked conformations on residues 480 to 510 of the α subunit [a translation-libration-screw group used in refinement (5) that juxtaposes the buried hinge fragment] revealed that the membrane and cargo-bound open conformation is incompatible with the autoinhibitory sequestration of the hinge (Fig. 3 and figs. S4 and S8): The β2 trunk now blocks the point of entry of the hinge into the bowl, and the relocation of C-μ2 removes one face of the pocket in which the helical β2 hinge segment rests (Fig. 2C and fig. S4). Thus, transition of AP2 from the locked to the open conformation, triggered by association with the plasma membrane, stimulates clathrin binding by releasing the clathrin box–containing β2 hinge from the center of the core (5).

Fig. 3 The open, activated form of AP2 is not compatible with the β2 hinge binding back into the core.

Release of the clathrin-binding motif stimulated by conformational change. (Top) Locked AP2 core, in solution or transiently bound to the plasma membrane via PtdIns(4,5)P2 (left), and open AP2 stably attached to the membrane via multiple PtdIns(4,5)P2s and cargo. (Bottom) Views of the hinge binding site in each conformational state; in the open state (right), the hinge residues from the locked state βhingeHis6.AP2 structure are superposed onto the open structure and shown in gray.

To address whether cargo binding is absolutely required to stimulate clathrin recruitment, we prepared synthetic liposomes supplemented either with PtdIns(4,5)P2 alone or with a mixture of PtdIns(4,5)P2 and a lipid-linked Yxxϕ motif and mixed these with FLβ.AP2 and clathrin, both at plausible cellular concentrations of 0.4 μM (18) (Fig. 4A). As expected, FLβ.AP2 bound more tightly to the cargo-containing liposomes (Fig. 4A) (5, 14). However, the ratio of clathrin to AP2 present in the cargo-containing liposome pellets was significantly greater (26.5% ± 5.3%) than in the PtdIns(4,5)P2-only liposomes (5.2% ± 1.9%; means ± SEM, four experiments; difference tested by Student’s t test, P = 0.0094). This finding suggests that PtdIns(4,5)P2- and cargo-bound FLβ.AP2 is able to recruit clathrin more efficiently than FLβ.AP2 bound only via PtdIns(4,5)P2, consistent with PtdIns(4,5)P2 driving the conformational change in AP2 that is then stabilized by cargo binding (5, 19).

Fig. 4 AP2 and clathrin are sufficient to generate clathrin-coated buds on membranes.

(A) Clathrin recruitment to liposomes. Synthetic liposomes supplemented with PtdIns(4,5)P2, or with PtdIns(4,5)P2 and TGN38 peptide (as indicated), were incubated with adaptor and clathrin as shown (both at 0.4 μM); supernatants (s) and pellets (p) were then separated and analyzed by gel electrophoresis. (B) Liposomes supplemented with PtdIns(4,5)P2 and lipid-linked TGN38 internalization were incubated sequentially with FLβ.AP2 and clathrin and analyzed by negative stain EM, showing clathrin-coated membrane buds (arrowheads). (C) Mean number of buds formed per μm2 of membrane, estimated for various combinations of lipid and adaptor. Note that no buds were found on PtdIns(4,5)P2 liposomes with or without TGN38 incubated sequentially with the AP2 core and clathrin or with clathrin only (fig. S9). Error bars indicate SEM, three experiments. (D and E) Liposomes supplemented with PtdIns(4,5)P2 and lipid-linked TGN38 internalization incubated sequentially with FLβ.AP2 and clathrin and analyzed by ultrathin sectioning. Arrowheads indicate examples of clathrin-coated structures; arrows indicate coated buds where the connection to the liposome is visible. (E) shows an enlarged image of a bud showing the “neck.” (F) Examples of liposomes supplemented with the lipids indicated [PtdIns(4,5)P2 or NiNTA-DGS] incubated sequentially with adaptors (as indicated) and clathrin, examined by negative stain EM. Some buds produced by FLβ.AP2 on PtdIns(4,5)P2-only liposomes seemed less invaginated than those shown here; this can be seen more clearly in ultrathin sections (fig. S10).

Last, we sought to determine whether AP2 alone is sufficient to initiate and drive clathrin-coated bud formation on appropriate membranes. When FLβ.AP2-loaded PtdIns(4,5)P2- and lipid-linked Yxxϕ cargo-supplemented liposomes were incubated with clathrin and the results examined by negative stain electron microscopy (EM) (Fig. 4B), we observed numerous clathrin-coated buds, of ~80-nm diameter. Ultrathin sectioning revealed that the buds encapsulated invaginated membrane or vesicles of 30- to 40-nm diameter, similar to CCVs isolated from brain (Fig. 4, D and E). Fewer buds were found on PtdIns(4,5)P2 liposomes incubated with FLβ.AP2 and clathrin (Fig. 4, C and F). We were unable to find buds on NiNTA liposomes similarly treated with FLβ.AP2 (Fig. 4, C and F), but some clathrin-coated buds were found on NiNTA liposomes incubated with clathrin and C-μ2–deleted FLβ.AP2 (FLβ.AP2.ΔCμ2), whereas His12-tagged GST-β2-h+app produced ample buds on NiNTA liposomes (Fig. 4, C and F). Thus, once activated by binding to its physiological ligands, AP2 is sufficient to drive clathrin-coated bud formation (at least in vitro); no other clathrin adaptors, including those currently described as driving membrane curvature, are required.

AP2 is the most abundant endocytic clathrin adaptor (20) and the first to be recruited to sites of CCP formation (2, 8). AP2 knockdown results in a ~12-fold reduction in CCP formation in HeLaM cells (21). Together with our findings that PtdIns(4,5)P2-activated and cargo-stabilized AP2 is sufficient to drive bud formation on liposomes, these data suggest that in vivo the dominant mechanism for endocytic CCP initiation will be the recruitment of AP2 to PtdIns(4,5)P2-enriched sites in its locked form with its clathrin binding autoinhibited, followed by transition to the open form if sufficient PtdIns(4,5)P2 is present (5, 19). AP2’s conformational change will expel the β2 hinge, allowing clathrin triskelia to bind. The presence of cargo will stabilize AP2’s open form and increase its dwell time on the membrane (5, 14, 19, 22), thus increasing the chances of it binding clathrin and forming a sufficiently stable nucleating structure. Once a small nucleus of AP2 and clathrin has formed, further AP2, clathrin, and other clathrin adaptors that bind the α appendage can then be recruited in random order to produce a CCP, which can ultimately be severed from the membrane (3, 23, 24).

Supplementary Materials

Materials and Methods

Figs. S1 to S11

Tables S1 and S2

References (2638)

Movies S1 and S2

References and Notes

  1. Materials and methods are available as supplementary materials on Science Online.
  2. Acknowledgments: We would like to thank the I02, I03, and I04-1 beamline staff at the Diamond Lightsource; A. McCoy and P. Evans for crystallographic advice; C. Oubridge for advice and assistance with SeMet mapping of the hinge residues; N. Bright, C. Savva, and S. Miller for advice and assistance with EM; C. Smith for reagents; and H. Böning and H. Ungewickell for expert technical assistance. D.J.O. and B.T.K. are supported by a Wellcome Trust Principal Research fellowship (090909/Z/09/Z). S.H. is supported by a grant of the German Science Foundation (SFB 635, TP A3). S.C.G. is supported by a Sir Henry Dale fellowship from the Wellcome Trust and the Royal Society (098406/Z/12/Z). CIMR is supported by a Wellcome Trust Strategic Award (079895). Coordinates have been deposited in the Protein Data Bank with PDB ID 4uqi.
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