Wild-type rat clathrin heavy chain and a mutant truncated at residue 1637 (and hence lacking the Hsc70-binding motif Q1638LMLT) were expressed in insect cells and purified as described 16 (link). Rat clathrin light chain a1 (LCa) containing the mutations D203E (to restore the epitope recognized by the antibody CVC.6 24 (link)) and C218S (to remove one of the two cysteines in the light-chain sequence) was expressed in E. coli and purified as described 16 (link). Cys187 of LCa was reacted with an excess of a maleimide functionalized fluorophore (Alexa Fluor 488 or DyLight 649). The free dye was removed by ultrafiltration and the labeled LCa was purified by anion exchange chromatography. To reconstitute fluorescent clathrin, heavy-chain trimers were incubated with labeled LCa at a molar ratio of 1:2.4 for 40 min at room temperature. UV-visible absorption spectroscopy showed that >90% of heavy chains were occupied with a labeled LCa.
The C-terminal fragment of bovine auxilin (residues 547–910) containing the clathrin-binding and J-domain functions was expressed in E. coli and purified as described 16 (link).
Bovine Hsc70 with an N-terminal His-tag was expressed in E. coli strain BL21-DE3 using a pProEX HTc vector. The C-terminus of Hsc70 was modified for site-specific labeling by adding a glycine and a cysteine residue (Supplementary Figure 5). Affinity purification was followed by TEV protease cleavage to remove the His-tag. Undigested fusion protein was removed by binding to Co beads. The C-terminal cysteine residue of Hsc70 was labelled with an excess of Alexa Fluor 568-C5-maleimide. Labeled Hsc70 was purified by gel filtration. The labeling yield determined using UV-visible absorption spectroscopy was ~97%. Hsc70 without C-terminal cysteine did not incorporate an appreciable amount of label. Labeled Hsc70–ATP was monomeric and exhibited normal uncoating activity.