Clathrin-coated vesicles transport selected integral membrane proteins from the cell surface and the trans-Golgi network to the endosomal system. Before fusing with their target the vesicles must be stripped of their coats. This process is effected by the chaperone protein hsp70c together with a 100K cofactor which we here identify as the coat protein auxilin. Auxilin binds with high affinity to assembled clathrin lattices and, in the presence of ATP, recruits hsp70c. Dissociation of the lattice does not depend as previously supposed on clathrin light chains or on the amino-terminal domain of the heavy chain. The presence of a J-domain at its carboxy terminus now defines auxilin as a member of the DnaJ protein family. In conjunction with hsp70, DnaJ proteins catalyse protein folding, protein transport across membranes and the selective disruption of protein-protein interactions. We show that deletion of the J-domain of auxilin results in the loss of cofactor activity.

We have established by peptide mapping and immunochemical analysis of purified clathrin assembly protein preparations from bovine brain, that the cluster of components of mol. wt 100-120 kd fall into four classes, which we term a, (, , are immunologically related and generate a series of common tryptic peptides. The same criteria reveal no such homologies between the a, (3G') and -y polypeptides. The so-called HA-II assembly protein group contains equimolar amounts of a and (3 class polypeptides, which are shown to interact with each other. In the HA-I group assembly protein complex ry and (3' class polypeptides form a stoichiometric complex. Immunofluorescence microscopy reveals that the HA-I complex is specifically associated with clathrin-coated membranes in the Golgi region of cultured cells, whereas the HA-II complex appears to be restricted to coated pits on the plasma membrane. The data lead to the tentative conclusion that the clathrin assembly proteins are involved in the recognition of the intracellular targets by uncoated vesicles.

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the ␣-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of ␣-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in ␣-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.

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. AP2 is the most abundant adaptor, and is pivotal to CME. By determining a new structure of AP2 that includes the clathrin-binding β2-hinge and developing an AP2-dependent budding assay, we reveal the existence of an autoinhibitory mechanism that prevents clathrin recruitment by cytosolic AP2. A large-scale conformational change driven by the plasma membrane phosphoinositide PtdIns(4,5)P 2 and cargo relieves this autoinhibition, so triggering clathrin recruitment and hence clathrin-coated bud formation. This molecular switching mechanism constitutes an unsuspected layer of regulation that couples 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 CME is the AP2 (Assembly Polypeptide 2) complex, (Figs 1A, S1), which both coordinates CCP formation and binds the many cargo proteins that contain 'acidic dileucine' and Yxxφ endocytic motifs (φ denotes 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 PtdIns(4,5)P 2 (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,(6)(7)(8) Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts from the β2-trunk binds the N-terminal beta-propeller of the clathrin heavy chain using a canonical clathrin box motif (LLNLD; Fig 1A,B (9)). The β2 appendage domain also binds clathrin, albeit weakly, but both interactions are necessary for robust clathrin binding (10).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 E.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 1C-E), it bound clathrin very poorly in pulldowns when immobilized on either glutathione sepharose beads ( Fig 1C) or via its N-terminal His6 tag (similarly positioned to the β2 PtdIns(4,5)P 2 binding site Fig1B (4, 5).) to liposomes containing the nickel-attached lipid 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 hingeappendage ('GST-β2-h+app', Fig S1) bound clathrin efficiently ( Fig 1C) and stimu...

Clathrin, a polypeptide of molecular weight (MW) 180,000, is the main constituent of the polygonal network that forms the coat of coated pits and vesicles; these vesicles play a part in intracellular transport between membranous organelles. This function involves specific recognition of target membranes as well as fusion and fission events that must be coordinated with the assembly, partial disassembly or reorganization of the clathrin coats. To understand these interactions on a molecular level, information about the structure of clathrin and the interactions of clathrin with itself and other proteins is required. Here we show that purified clathrin coats dissociate reversibly into triskelions, structures composed of three usually bent, rather flexible legs irradiated from a centre. We have determined the molecular weight of these triskelions and conclude that they contain trimers of clathrin together with about three light molecular weight polypeptide chains.

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Depending on conditions of extraction from the membrane, spectrin may be recovered either as a dimer or a tetramer. It is demonstrated that these species are linked by a simple equilibrium, and can be readily interconverted. At low temperature, however, either species is kinetically trapped, and no interconversion occurs over periods of up to several days. In the range 25 -40 "C, equilibrium is achieved on a time scale of hours to minutes. First-order and second-order rate constants and thermodynamic parameters for the equilibrium have been determined. The large activation energy indicates that conformational effects are rate-limiting. In the absence of other proteins, no associated states higher than the tetramer are formed. Neither phosphorylation of the spectrin with endogenous kinase, nor dephosphorylation with phosphatase have any effect on the dimer-tetramer equilibrium, neither do they promote formation of higher associated states. It is therefore improbable that phosphorylation-dependent shape changes in the erythrocyte are related to the association of spectrin per se, and are likely instead to be the direct result of a spectrin-actin interaction. The dimer-tetramer equilibrium is strongly affected by ionic strength, and at low salt concentrations, such as those used to extract spectrin from membranes, the dimer becomes strongly favoured. Thus the recovery of tetramer by extraction at low temperature implies that this is the basic unit present in the membrane. Calcium and magnesium ions cause further association to higher oligomers, though only at high concentrations (in the millimolar range) of the cation.Spectrin is the most abundant constituent of the mammalian erythrocyte membrane [l]. Together with actin, it appears to play a major role in stabilising the membrane and maintaining its discoid shape [2]. The protein contains two subunits, differing somewhat in molecular weight (220 000 and 240 000), the smaller of which undergoes phosphorylation in the presence of ATP. This process appears to mediate the interaction between spectrin and actin [3], which in turn probably controls the ATP-dependent shape changes to which the cell is subject.Spectrin can be recovered in a highly aggregated form, together with actin and some minor protein constituents, by extraction of the erythrocyte membrane with non-ionic detergent [3,4]. Exposure of the membranes to media of low ionic strength, on the other hand, leads to release of spectrin into the solution. Brief extraction at 37 "C liberates the spectrin predominantly as a species of molecular weight about 500000 containing therefore two subunits ' [5,6]; actin and traceamounts ofotherproteins are also present. By contrast, prolonged dialysis of the ghosts in the cold gives rise to spectrin mainly in tetrameric form [6,7). The dimer does not revert to tetramer on cooling.Ralston et al. [8] have reported, moreover, that the tetramer, extracted in low ionic strength in the cold, is dissociated irreversibly to dimer when incubated at 3 7 T , and thereby raised the ...