Purification of Overexpressed Drg1 Protein

Overexpressed Drg1 was purified as described previously^{19 (link),21 (link)}. Essentially, frozen cells were thawed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, supplemented with 1 mM DTT and 1× complete protease inhibitor cocktail (Roche)) and disrupted by vigorous shaking in the presence of 0.6 mm glass beads using a Merckenschlager beadmill. Crude extracts were incubated for 90 min at 4 °C with GSH-agarose beads (Sigma-Aldrich) for affinity purification of GST-tagged Drg1 variants. After consecutive buffer washing steps (3× with lysis buffer plus 1 mM EDTA and 1 mM DTT and 1× with elution buffer plus 1 mM DTT), the protein was eluted in buffers specific for the respective experiment. Elution was performed by separating Drg1 from the GST-tag via Prescission protease treatment (Amersham) overnight at 4 °C on a rotator. Protein concentration was determined using the Bradford assay (Bio-Rad).