Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly

Abstract

Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

Figure 1. New Protein Interaction Partners for the α- and β-Appendage Domains of AP2 Adaptors

(A) Plot of the network of protein interactions in clathrin-mediated endocytosis. AP2 adaptors and clathrin have disproportionately large numbers of interactors and so are the hubs of this network. Dynamin is a “party” hub as it is shared between different networks but we have not included all its interactors. (B) Scheme of AP2 showing the overall domain architecture and the appendages where most of the protein interactors bind are located on flexible linkers called “hinges.” (C) Protein interactors of α- and β2 appendages from HeLa cells as determined by LC-MS/MS of Coomassie stained bands. Bolded proteins were not detected previously. The interaction of CVAK104 and CVAK90 were tested and confirmed by yeast-2-hybrid analysis. The numbers of peptides sequenced from each protein are given in brackets. Further mass spectrometry data from brain and liver samples, accession numbers, domain structures and details are given in Figures S1 and S2.

Figure 2. The β2-Appendage Has Top and Side Sites for Protein Interactions

(A) Surface residues that are conserved from yeast to man are coloured from maroon (highly conserved) through white to sky-blue (least conserved). The α-appendage is shown for comparison. Both have two conserved patches, a top site and a side site. (B) Mutants of top and side sites in β compared with mutants of α. GST wild-type and mutant appendages were used in pull-downs from brain extract. β-arrestin binds specifically to the top site of β2, while most other ligands appear to bind more tightly to the side site. (C) Coomassie gel analysis of protein interactors with α- and β2-appendages from brain extracts. The boxed areas show the most effective mutants at displacing ligands. Asterisks mark positions of co-purified chaperones.

Figure 3. High Avidity Interactions of Eps15 for Clustered Appendages, yet Low Affinity Interactions of Ligands with Isolated Appendages

(A) Surface plasmon resonance measurements of Eps15-MD binding α and β-appendages. Note that the protein binds, but does not come off even after extensive washing. (B) Affinities of β-arrestin P-long (see D) for wild-type β2-appendage and mutants measured by ITC. (C) Affinity measurement of protein domains with β2-appendages. (D) Table of peptides used, highlighting possible motifs, and a summary of affinities.

Figure 4. Eps15 Peptide Binds in a Tight Turn to the Side Site of the β2-Appendage

(A) Ribbon diagram showing peptide in yellow bound to side site of the β-appendage. (B and C) The two principle Phe residues of the peptide bind in a groove. A single turn of a helix can also be seen for this peptide. (D) Peptide displayed as a linear chain showing hydrogen-bonding potential (green lines) and hydrophobic interactions (grey lines). A cluster of hydrogen-bonds in the peptide is consistent with the α-helical conformation in this region. (E) Density map for the peptide contoured at 1.24 σ.

Figure 5. Peptide from the C-Terminus of β-Arrestin Folds as a Helix in a Groove on the Platform Sub-domain of the β2-Appendage

(A) Ribbon diagram showing peptide in purple bound to top site of the platform sub-domain of the β-appendage as an α-helix. (B and D) The peptide binds into a groove with interacting residues of the peptide lining one side of the helix. The top of the groove is generally positive and D₃ of the peptide is found here. F₆ and F₉ are in apolar environments and there is limited space for F₉. R₁₃ binds to the negatively charged patch of the groove. (C) Peptide displayed as a linear chain showing hydrogen-bonding potential (green lines) and hydrophobic interactions (grey lines). Hydrogen bonds within the α-helix of the peptide are not shown for clarity. The residues for which there is little density are dotted. (E) Mutagenesis of the Phe residues of β-arrestin DxxFxxFxxxR motif prevent the binding of AP2 complexes to GST-β-arrestin C1 in brain extracts. (F) Conservation of the interaction motif between arrestins and ARH and possible motifs from epsin and CVAK90.

Figure 6. Introducing the Clathrin Hub

(A) Displacement of β-appendage interactors with clathrin binding. A comparison of protein interactors with the β-appendage +/− the connecting linker to the core of the adaptor complex (the hinge domain) is made. Rat brain extract was used. The asterisks point to a reduced binding of Eps15 to the Y888V mutant only when clathrin is present. It can be noted that β-arrestin is not enriched in the pull-downs over total lysates (where 1% of the volume used for the pull-down is loaded) and given the high affinity of arrestins for the β-appendage we conclude that only a small percentage of the arrestin is in the “open” conformation necessary for β-appendage binding. Amphiphysin is also not enriched, but it has a much lower affinity for the β-appendage. (B) β1 appendage+hinge binding to clathrin results in reduced accessory protein interactions. Appendage and appendage+hinge proteins from β1 to β4 adaptins and their interactors are compared. We found no specific bands by mass spectrometry that interact with β3 and β4 in these experiments (apart from chaperones) See Figure S3 for β1–4 appendage homologies. (C) Clustered clathrin N-terminal domain interactions with accessory proteins. The GST-β-propeller domain has been bound to sepharose beads and thus we should especially detect proteins that can bind to clathrin via multiple interactions. (D) Clathrin binding of MDs from various accessory proteins except Eps15. GST-MDs on beads were incubated with rat brain extract and clathrin was detected by Western blotting.

Figure 7. Selection of Partners to Remain during Coated Pit Maturation; β-Arrestin but not Eps15 Co-Localise with AP2 Adaptors in CCPs

(A) GFP-βarrestin2 C-terminal domain co-localisation with AP2 adaptors while mutants are cytoplasmic. GFP-Eps15-MD is cytoplasmic. (B) Full length GFP-β-arrestin2 co-localisation with AP2 adaptor following stimulation of GPCR receptors with TRH for 15s. A mutant of β2-appendage interaction does not localise to coated pits.

Figure 8. Network Dynamics

(A) Network of protein interactions of AP2 appendages and clathrin. The α- and β2-appendage interaction data are from our mass spectrometry analysis and from the literature. The AP2 hub (yellow shaded area) is made up of 2-sub-hubs that increase the interactome repertoire (side arms) but for the majority of proteins they increase the interaction avidity (central circle). As the network matures and clathrin is recruited, many of the previous interactors become clathrin interactors (green shaded area). Of the remaining proteins we only know of a few that do not bind to clathrin, while others have simply not been tested. (B) Limiting the mobility of appendage domains by interactions of both domains with the same accessory protein. (C) The changing environment of adaptor protein complexes, A, in CCV formation, showing the gradual movement from simple affinity based interactions with accessory proteins, B, to avidity based interactions, once adaptors are recruited to the membrane. As the coated pit matures and clathrin, C, polymerises into a matrix then accessory proteins that cannot bind directly to clathrin are displaced to the edge where the pit is still growing. ATP hydrolysis is needed to depolymerise clathrin and re-prime the system.