contributed equally to this work 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 m2 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 phosphotyrosinebinding (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-speci®c adaptor proteins, including b-arrestin, AP180, epsin, HIP1 and numb, which regulate clathrin-coat assembly at the plasma membrane by synchronizing cargo selection and lattice polymerization events.

Clathrin-mediated endocytosis is a major pathway for the internalization of macromolecules into the cytoplasm of eukaryotic cells. The principle coat components, clathrin and the AP-2 adaptor complex, assemble a polyhedral lattice at plasma membrane bud sites with the aid of several endocytic accessory proteins. Here, we show that huntingtin-interacting protein 1 (HIP1), a binding partner of huntingtin, copurifies with brain clathrin-coated vesicles and associates directly with both AP-2 and clathrin. The discrete interaction sequences within HIP1 that facilitate binding are analogous to motifs present in other accessory proteins, including AP180, amphiphysin, and epsin. Bound to a phosphoinositide-containing membrane surface via an epsin N-terminal homology (ENTH) domain, HIP1 associates with AP-2 to provide coincident clathrin-binding sites that together efficiently recruit clathrin to the bilayer. Our data implicate HIP1 in endocytosis, and the similar modular architecture and function of HIP1, epsin, and AP180 suggest a common role in lipid-regulated clathrin lattice biogenesis.

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 ␣-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.The major intracellular pool of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) 1 is found, at steady state, within the cytosolic leaflet of the plasma membrane. PtdIns(4,5)P 2 -specific probes, such as the phospholipase C-␦1 pleckstrin homology domain fused to the green fluorescent protein, reveal a highly selective distribution of this important regulatory phospholipid over the cell surface (1, 2). This same PtdIns(4,5)P 2 -binding pleckstrin homology domain prevents receptor-mediated endocytosis in vitro by interfering with clathrin-coated vesicle assembly (3) underscoring the inter-relationship between phosphoinositides and clathrin-dependent internalization. PtdIns(4,5)P 2 regulates coat assembly in part by physically interacting with several of the components of the endocytic clathrin machinery, including the heterotetrameric AP-2 adaptor complex (4 -6), AP180 (7-9), epsin (10 -13), HIP1 (11), Dab2 (12), and the ␤-arrestins (14). The activity of type I phosphatidylinositol 4-phosphate 5-kinases promotes AP-2-containing coat assembly upon membrane templates in vitro (15,16) where the generated PtdIns(4,5)P 2 appears to act as an organizing principle to promote proper placement of endocytic components at the bud site (9,(11)(12)(13)17). In fact, overexpression of phosphatidylinositol 4-phosphate 5-kinase ␤ promotes AP-2 association with the plasma membrane, increases the number of clathrin-coated pits, and potentiates transferrin uptake (18).At the n...