PMC

The three-step sequential mechanism for clathrin cage disassembly that fitted the kinetic data most accurately when five alternate kinetic mechanisms were tested (Schemes S3–S7). Each step consists of an association event between clathrin.auxilin (C) and Hsc70.ATP (T) with rate constant k_a, followed by hydrolysis of ATP to ADP with rate constant k_r. Once the three steps have taken place, the clathrin.auxilin cage disassembles with rate constant k_d.

The hydrolysis of ATP during clathrin cage disassembly. The amount of P_i produced from ATP hydrolysis by Hsc70 during clathrin cage disassembly was monitored. Clathrin (0.33 μM triskelia), 0.35 μM auxilin, and 8 μM Hsc70 were mixed together. The reaction was initiated by addition of ATP (50 μM), and at specific time points samples were removed, quenched, and assayed for P_i content with malachite green solution. The data are fitted to a single exponential superimposed on a linear function (Eq. S2).

Stopped-flow measurements of light scattering to examine the early stages of clathrin cage disassembly. Clathrin cages (0.09 μM triskelia) premixed with 0.1 μM auxilin were mixed with Hsc70 (concentrations in μM shown on graph) and 500 μM ATP, and perpendicular light scattering was measured using stopped-flow techniques. The closely spaced raw data was reduced to a frequency of 3.3 s^-1 (closed circles) and fitted (dashed lines) to a system of first-order ordinary differential equations corresponding to the reaction mechanism shown in , using the software DYNAFIT (, ). Data corresponding to 0 and 0.25 μM Hsc70 were omitted from the fit because of their low information content, as was data collected after 12 s.

The effect of [Hsc70] or [auxilin] on clathrin cage disassembly. (A) Representative light-scattering curves containing clathrin cages (0.09 μM triskelia), auxilin (0.3 μM), and ATP (500 μM) with disassembly initiated by various concentrations of Hsc70 (0.15–4 μM). (D) Representative disassembly curves with clathrin cages (0.09 μM triskelia) premixed with varying [auxilin] (0.022–0.36 μM) and 500 μM ATP, and initiated by addition of 2 μM Hsc70. (B and E) Symbol t_1/2 on the vertical axis denotes the average time taken for disassembly of 50% of clathrin cages obtained from raw data traces as in A and D, respectively. Data points and error bars, respectively, are mean ± standard error from replicated measurements (n≥4). (C and F) Average amplitude (total amount of disassembly) at the end of each assay from curves obtained as in A and D, respectively. Data points and error bars, respectively, are mean ± standard error from replicated measurements (n≥4). The smooth curves in panels B, C, and F represent a fit to the hyperbolic saturation function (Eq. S1), which serves as an empirical description of the data.

A real-time in vitro assay for clathrin cage disassembly and correlation with electron microscopy images of clathrin cages. (A) Representative trace of the right-angle light-scattering assay for clathrin cage disassembly. Clathrin cages (0.09 μM triskelia) were premixed with 0.1 μM auxilin, and after 60 s, cage disassembly was initiated by addition of 1 μM Hsc70 and 500 μM ATP. (B) Average results for three different disassembly assays monitored both by light scattering as in A and compared with electron microscopy images as in C–E. The single points represent the average number of cages counted per image, initiated with 0.1 μM (triangles), 0.2 μM (circles), or 0.5 μM (squares) Hsc70. Data are mean ± SD. The single lines represent the light-scattering results obtained under the same conditions. (C–E) Representative transmission electron micrographs of negatively stained grids prepared at 0, 60, and 180 s during a disassembly assay containing clathrin cages (0.09 μM triskelia), 0.1 μM auxilin, 500 μM ATP, and initiated with 0.2 μM Hsc70. The scale bar in the bottom left-hand corner of each image represents 0.2 μm.

An illustration of the serial Hsc70 binding and ATP hydrolysis model for the disassembly of clathrin cages highlighting the sequence of events on a single triskelion. 1: Auxilin has a high affinity for triskelion legs forming part of a clathrin cage and binds at a stoichiometry of one per triskelion. 2: An Hsc70∶ATP complex binds to the clathrin∶auxilin complex. 3: The interaction between auxilin’s J domain and Hsc70 stimulates ATP hydrolysis and induces a conformational change in Hsc70, increasing its affinity for clathrin. 4: Auxilin repositions, and a second Hsc70∶ATP is recruited to the clathrin∶auxilin complex. 5: The second Hsc70 interacts with auxilin’s J domain, ATP is hydrolyzed, and the resulting Hsc70∶ADP complex binds tightly to clathrin. Following hydrolysis, the auxilin again repositions, and a third Hsc70∶ATP binds to the clathrin∶auxilin complex. 6: Hydrolysis of the third ATP results in a weak interaction between the triskelion(Hsc70∶ADP)₃ complex and the cage. The rate-limiting step is the disassembly of triskelia leading to cage collapse. 7: The Hsc70∶ADP complex has a high affinity for the released triskelia and remains bound, whereas the affinity of triskelia(Hsc70∶ADP)₃ for auxilin is low; the previously bound auxilin is free now to rebind the cage in a catalytic manner ().