GST-Mid1p, 6His-Vps4p or 6His-C-term Myo2p plasmids were grown in BL21 E. coli until an OD of 0.6-0.8, with protein production induced by adding 1 mM IPTG. Mid1p 578-799 was not found to be expressed under these conditions, and so was not used for further experiments. 
Bacterial pellets were produced by centrifugation with cells re-suspended in 20 ml re- suspension buffer with EDTA free protease inhibitors; for GST-Mid1p fusion proteins PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4, pH 7.4) was used, whereas for 6His-Vps4p, HEPES buffer (25 mM HEPES, 400 mM KCl and 10% (v/v) glycerol, pH 7.4) was used. Cells were then lysed by sonication, where a final concentration of 1 mg ml-1 lysozyme was added for cell wall digestion followed by sonication, with 0.1 % (v/v) Triton X-100 added prior to lysis. A clear lysate was produced by centrifugation. GST-tagged fusion proteins were purified using 1ml l-1 glutathione beads in PBS buffer, while 6His-tagged Vps4p or C-term Myo2p were purified using 500 μl l-1 Ni-NTA beads in HEPES buffer, either for 2 h or overnight at 4 ̊C. Mid1p was eluted from glutathione beads using Reduced glutathione buffer. 
Vps4p or C-term Myo2p were washed and eluted from Ni-NTA beads using HEPES buffers. Elution was carried out for 2 h at 4 ̊C. Samples of proteins from each step were subjected to SDS-PAGE to determine elution efficiency.