Epsin 1 is a polyubiquitin-selective clathrin-associated sorting protein.

Epsin 1 engages several core components of the endocytic clathrin coat, yet the precise mode of operation of the protein remains controversial. The occurrence of tandem ubiquitin-interacting motifs (UIMs) suggests that epsin could recognize a ubiquitin internalization tag, but the association of epsin with clathrin-coat components or monoubiquitin is reported to be mutually exclusive. Here, we show that endogenous epsin 1 is clearly an integral component of clathrin coats forming at the cell surface and is essentially absent from caveolin-1-containing structures under normal conditions. The UIM region of epsin 1 associates directly with polyubiquitin chains but has extremely poor affinity for monoubiquitin. Polyubiquitin binding is retained when epsin synchronously associates with phosphoinositides, the AP-2 adaptor complex and clathrin. The enrichment of epsin within clathrin-coated vesicles purified from different tissue sources varies and correlates with sorting of multiubiquitinated cargo, and in cultured cells, polyubiquitin, rather than non-conjugable monoubiquitin, promotes rapid internalization. As epsin interacts with eps15, which also contains a UIM region that binds to polyubiquitin, epsin and eps15 appear to be central components of the vertebrate poly/multiubiquitin-sorting endocytic clathrin machinery.

Dual engagement regulation of protein interactions with the AP-2 adaptor alpha appendage.

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.

A novel AP-2 adaptor interaction motif initially identified in the long-splice isoform of synaptojanin 1, SJ170.

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded alpha-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.

Disabled-2 exhibits the properties of a cargo-selective endocytic clathrin adaptor.

Clathrin-coated pits at the cell surface select material for transportation into the cell interior. A major mode of cargo selection at the bud site is via the micro 2 subunit of the AP-2 adaptor complex, which recognizes tyrosine-based internalization signals. Other internalization motifs and signals, including phosphorylation and ubiquitylation, also tag certain proteins for incorporation into a coated vesicle, but the mechanism of selection is unclear. Disabled-2 (Dab2) recognizes the FXNPXY internalization motif in LDL-receptor family members via an N-terminal phosphotyrosine-binding (PTB) domain. Here, we show that in addition to binding AP-2, Dab2 also binds directly to phosphoinositides and to clathrin, assembling triskelia into regular polyhedral coats. The FXNPXY motif and phosphoinositides contact different regions of the PTB domain, but can stably anchor Dab2 to the membrane surface, while the distal AP-2 and clathrin-binding determinants regulate clathrin lattice assembly. We propose that Dab2 is a typical member of a growing family of cargo-specific adaptor proteins, including beta-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.