Supplementary Figure 6: Hsc70ΔC competes for Hsc70 binding to cages and inhibits cage disassembly by Hsc70 at low pH.

Reduced disassembly activity and scattering increases with Hsc70ΔC vs. Hsc70 could be due to weaker binding of Hsc70ΔC to cages, rather than reduced self-association. If so then adding excess Hsc70ΔC to reactions with Hsc70 shouldn't inhibit disassembly by the latter since Hsc70ΔC shouldn't effectively compete with Hsc70 for cage binding. Conversely, if Hsc70ΔC and Hsc70 bind cages with similar affinity, but Hsc70ΔC doesn't disassemble as effectively once bound, then Hsc70ΔC should compete with and inhibit Hsc70 disassembly. To test this we carried out disassembly at pH 6.3 and 1 μM Hsc70, conditions under which disassembly is slow and depends on Hsc70 self-association. A. Cage disassembly reactions carried out at pH 6.3 with 1 μM Hsc70 and with 0 (black; average of 5 replicates), 3 μM (red; average of 8 replicates with trace thickness showing +/- se), or 9 μM (blue; average of 8 replicates) Hsc70ΔCterm. Hsc70ΔCterm at 3- and 9x excess over Hsc70 is observed to reduce disassembly rates by ~2x and ~4x, respectively. B. SDS PAGE analysis of pellet fractions of 1mM Hsc70, 0.23 μM Auxilin, 0.15 μM (lanes 1-6) or 0 μM (lanes 7-12) cages (concentrations of cages expressed as [CHC]) and with Hsc70ΔC at 1 (lanes 2, 8), 2 (lanes 3, 9), 4 (lanes 4, 10), 8 (lanes 5, 11) or 16 (lanes 6, 12) μM. Hsc70ΔC reduces the amount of Hsc70 that pellets with the cages. This experiment was independently performed 2x with similar results.