Fig. 1. Clathrin polymerization. (A) Four triskelia together form one edge of a clathrin polyhedron. The completed edge (circled) is composed of four leg segments: two anti-parallel distal leg segments (lavender and cyan) and two anti-parallel proximal leg segments (yellow and orange). (B) This predicted arrangement of triskelia in one edge of the clathrin polyhedron, viewed from the side, shows two adjacent proximal leg segments (yellow, orange) coming from a vertex downward and crossing at an angle on the outer surface of the basket. Two crossing distal leg segments (lavender, cyan) lie underneath, with the globular N-terminal domain protruding down toward the inside of the basket. Based on the 21 Å structure of a clathrin basket (Smith et al., 1998). (C) A cross-section view of one edge of the clathrin polyhedron with two proximal leg segments on top (yellow, orange) and two distal leg segments beneath (lavender, cyan) indicates the center-to-center distance separation between leg segments. Based on the 21 Å structure of a clathrin basket (Smith et al., 1998).

Fig. 3. Sedimentation velocity assays. Polyhistidine-tagged constructs were expressed and purified and their interaction assessed using sedimentation velocity by analytical ultracentrifugation in a Beckman XL-I at 40K r.p.m., 4°C. SedFit was used for a continuous c(S) distribution analysis of the data. For each fit, the root mean square deviation (r.m.s.d.) for the fit of the curves to the data, the number of data points (n), sum of squared residuals (SSR) and frictional ratio (F, indicating shape deviations from a sphere) were noted. The confidence level was 0.95. (A) Proximal leg segment at pH 6.2 is composed of a single species with sedimentation coefficient S = 2.3 (r.m.s.d. = 0.006503, n = 8372, SSR = 0.354059, F = 1.8). This species was confirmed as a monomer by equilibrium sedimentation (Figure 4), and components of trimeric constructs with S = 2.3 were thus assigned as monomeric species in plots (B–D). (B) Hub at pH 7.9 is composed of three species in solution: the first two are at S = 2.3 and S = 3.9 and correspond to monomer (35.2%) and trimer (64.8%), respectively, and the third is a minor larger species at S = 6.1 (r.m.s.d. = 0.007666, n = 14198, SSR = 0.834386, F = 1.6). (C) Proximal-Ii chimera at pH 7.9 is composed of two species in solution, at S = 2.4 and S = 4.7 (r.m.s.d. = 0.013468, n = 16365, SSR = 2.968339, F = 1.3), which correspond to monomer (46.8%) and trimer (53.2%), respectively. (D) Distal-Ii chimera at pH 7.9 is composed of two species in solution, at S = 2.5 and S = 4.6 (r.m.s.d. = 0.0.008542, n = 18212, SSR = 1.328910, F = 1.2), which correspond to monomer (49.5%) and trimer (50.5%), respectively.

Fig. 5. Chimeric trimerized clathrin proximal or distal leg segments assemble. (A) Chimeric constructs distal-Ii and proximal-Ii were created with clathrin distal or proximal leg segments, respectively, attached to the trimerization domain of the invariant chain protein. The bar diagrams indicate the protein domains of each donor protein from which chimeras were constructed and the domains in the resulting chimeras. Numbers indicate the residues flanking protein domains and the junctions where fragments were recombined. (B) Hub (blue) and purified chimeric proteins proximal-Ii (brown) and distal-Ii (lavender) at pH 7.9 were induced to assemble by a drop in pH 6.7 through the addition of 1/25 volume of 1 M MES pH 6.7, and turbidity (change in OD at 320 nm) was monitored for 5 min. Disassembly was induced by increasing the pH with an addition of 1/25 volume of 1 M TRIS pH 9 and monitoring OD₃₂₀ for an additional 2 min as turbidity returned to baseline levels, demonstrating reversibility of the assembly process. Monomeric proximal leg segment (green) and trimeric MBP-Ii (cyan) served as controls (overlapping lines near the baseline). (C) The chimeras had a similar overall extent of assembly to purified clathrin Hub in the pH 6.7 turbidity assay. Samples were induced to assemble as above and the extent of assembly (OD₃₂₀ after 5 min) was recorded. Extent of assembly was normalized to average hub assembly over several preparations and averaged for this plot.

Fig. 2. Leg segments are monomeric by FPLC. Fragments representing the clathrin proximal leg and terminal–distal domain were expressed as polyhistidine-tagged recombinant proteins and analyzed by gel permeation chromatography. K_av versus molecular weight for sample and standards were plotted. Proximal leg segments are monomeric on a calibrated FPLC column. (A) Proximal leg fragment and calibration standards were independently chromatographed on a Superdex 200 HR 10/30 column in Bis–TRIS pH 6.2 and elution times were noted. Proximal leg segment K_av was calculated as 0.454, indicating a size for clathrin proximal leg segment consistent with a monomeric 52 kDa species. (B) Isolated proximal and distal leg segments are monomeric and do not form a complex when combined. Proximal and terminal-distal domains were chromatographed on a Superose 6 prep grade column in MES/TRIS pH 6.7 and elution times were compared with elution of Hub or leg segments at pH 7.3. K_av was calculated. Mixtures of proximal and distal domain segments eluted as two separate populations; no larger peak corresponding to a complex was observed. (C) Proximal leg segments bind light chains normally but do not dimerize in the presence of light chain. Proximal leg segments with and without purified light chains and Hub were chromatographed on a Superose 6 prep grade column in Bis–TRIS pH 6.2 and elution times were noted for calculation of K_av. While the mixture of proximal leg with light chain eluted as a complex, indicating proper folding of the recombinant protein fragments, the K_av of the complex is consistent with each complex containing only one light chain and one proximal leg. Thus the presence of light chain did not induce homodimerization of the proximal leg.

Fig. 4. Proximal leg segment is a monomer at pH 6.2 in an equilibrium sedimentation assay. Proximal leg was expressed and purified and its self-interaction was assessed by equilibrium sedimentation in a Beckman XL-I analytical ultracentrifuge at 40K r.p.m., 4°C, pH 6.2. WinNonLin was used to simultaneously fit data points from samples of 0.4 (left), 0.2 (center), and 0.1 mg/ml (right) at 7K (green), 10K (orange), and 14K (purple) to the lines shown in black. (Radial distance)²/2 (R^{^}2/2) is plotted against A280 (AU). A plot of the residuals (lower panel) indicates the systematic error in the fit between the lines and the raw data points. The fit gave values for parameter σ = 0.623 (0.547–0.696) where for the expected monomer σ = 0.667. The fit is consistent with a proximal leg segment fitted to an N-mer of N = 0.94, which represents monomer. Variance of fit was 4.6 × 10^–5 with 1234 degrees of freedom.

Fig. 6. Surface plasmon resonance detects interaction between proximal leg segments. Clathrin constructs were expressed, purified and tested for interactions using a BIAcore Processing Unit. Proximal leg segments, Hubs, or chimeras were immobilized to a CM5 chip. Mobile solutions of clathrin fragments or chimeras were flowed over the surface at pH 6.7 and changes in refractive index monitored. Lower curves represent lower concentrations of mobile species. (A) Immobilized proximal leg segment (5029 RU) interacts with proximal leg segments at 2.5, 5.0, 7.5, 10.0 and 12.5 µM. (B) Immobilized Hub (6144 RU) interacts with Hub at 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 µM. (C) Immobilized proximal-Ii chimera (5731 RU) interacts with proximal-Ii chimera at 1.0, 2.0, 2.5, 3.0, 4.0 and 5.0 µM. (D) Immobilized distal-Ii chimera (5115 RU) interacts with distal-Ii chimera at 0.25, 0.50, 0.75, 2.5 and 5.0 µM. (E) Immobilized Hub (6144 RU) interacts with distal-Ii chimera at 0.25, 0.50, 0.75, 2.5 and 5.0 µM.