Supplementary Figure 5: Binding of large numbers of Hsc70s per CHC in pH 6–stabilized cages requires ATP, auxilin and an Hsc70-binding site in the CHC tails.

Plot of the # of Hsc70s per CHC pelleting with cages in absence of ATP, auxilin or an Hsc70 binding site (FLAG-tag cages; error is +se for 2 analyses of one experiment). Residual Hsc70 binding to cages in which the Hsc70 binding site is replaced with a FLAG-tag appears to be primarily due to Hsc70 binding weakly to other sites on the tails, as we found that complete deletion of the tails reduced binding (as assessed by the amount of 70 that pellets with cages) even more (to a level not meaningfully different than seen in the absence of cages; data not shown, available on request) than does altering the Hsc70 binding site by mutation. The nature of the 'non-specific' sites at which 70's bind to such cages is unknown, but such binding is consistent with the known promiscuous binding specificity of Hsc70 and the conclusion (Bocking, T., Aguet, F., Harrison, S.C. & Kirchhausen, T. Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating. Nat Struct Mol Biol 18, 295-301 (2011)), that, even with WT cages, approximately half of the Hsc70 molecules that associate with cages bind at 'non-specific' sites that are ineffective for disassembly.