Transient Fcho1/2⋅Eps15/R⋅AP-2 Nanoclusters Prime the AP-2 Clathrin Adaptor for Cargo Binding

Abstract

SummaryClathrin-coated vesicles form by rapid assembly of discrete coat constituents into a cargo-sorting lattice. How the sequential phases of coat construction are choreographed is unclear, but transient protein-protein interactions mediated by short interaction motifs are pivotal. We show that arrayed Asp-Pro-Phe (DPF) motifs within the early-arriving endocytic pioneers Eps15/R are differentially decoded by other endocytic pioneers Fcho1/2 and AP-2. The structure of an Eps15/R⋅Fcho1 μ-homology domain complex reveals a spacing-dependent DPF triad, bound in a mechanistically distinct way from the mode of single DPF binding to AP-2. Using cells lacking FCHO1/2 and with Eps15 sequestered from the plasma membrane, we establish that without these two endocytic pioneers, AP-2 assemblies are fleeting and endocytosis stalls. Thus, distinct DPF-based codes within the unstructured Eps15/R C terminus direct the assembly of temporary Fcho1/2⋅Eps15/R⋅AP-2 ternary complexes to facilitate conformational activation of AP-2 by the Fcho1/2 interdomain linker to promote AP-2 cargo engagement.

Eps15R and clathrin regulate EphB2‐mediated cell repulsion

“…The 725 LDPF in Eps15R is slightly different in that when mutated it loses clathrin binding more readily than AP2 association, which is an interesting observation. It is established that the spacing of DPF motifs is critical for determining binding specificity for both FCHo2 and AP2 . How this motif spacing is deciphered and decoded by the interaction partner is not known.…”

Structural determinants controlling 14-3-3 recruitment to the endocytic adaptor Numb and dissociation of the Numb·α-adaptin complex

“…As the key component of the AP2 complex, α-appendage acts by recruiting a large number of accessory/regulatory proteins into CCPs/CCVs (6)(7)(8). During the past decades, two conserved target binding sites have been identified on α-appendage, namely, the "top" site that recognizes the DPF/FxDxF/FxxFxxL motifs and the "side" site that binds to the WVxF/W motif (7,9,40).…”

“…The α (also called α-adaptin) and β2 subunits mediate the binding to the target membrane and the recruitment of clathrin, respectively, whereas the µ2 and σ2 subunits recognize the Yxxφ (where x represents any amino acid and φ indicates a hydrophobic residue hereafter) and di-leucine (diLeu) motifs on cargo proteins, respectively (4,5). The appendage domain of α-adaptin (referred to as α-appendage hereafter) is also responsible for recruiting a large number of accessory/regulatory proteins, including Eps15, by binding to DPF motifs within these otherwise very different proteins (6)(7)(8). Two conserved target binding sites have been identified on α-appendage, namely, the "top" site that recognizes the DPF/FxDxF/FxxFxxL motifs and the "side" site that binds to the WVxF/W motif (6,7,9).…”

Conformational regulation of AP1 and AP2 clathrin adaptor complexes

“…Eps15, in turn, binds to the AP2 ears . Thus, these interactions may position the APA close to AP2 which would increase the efficiency of its activation (Figure B) . To understand how Eps15 engages binding partners using DPF motifs, structures of μHDs from FCHo1 and SGIP in complex with fragments of Eps15 were solved .…”