Measuring GTPase Activity of Mitofusins

The GTP hydrolysis activity assay was carried out as previously described [41 (link), 50 (link), 51 (link)]. The free r-phosphate (Pi) release of GTP was measured by the colorimetric ATPase/GTPase activity assay kit (Sigma-Aldrich). 293T cells transiently co-transfected with Mfn1-Myc or Mfn2-Myc and either empty vector, MIEF1-V5, or MIEF2-V5 for 20–22 h were washed twice with phosphate-free buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl) and lysed in phosphate-free lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, protease inhibitor cocktail complete EDTA-free, Roche Diagnostics) on ice for 1 h. After centrifugation, the supernatants were subjected to immunoprecipitation (IP) with 40 μl of anti-Myc tag agarose bead (Abcam) for 2h at room temperature. After washing with lysis buffer 3 times and twice with 0.5 M Tris-HCl pH 7.5, beads were re-suspended in assay buffer provided with the kit and subjected to GTPase activity assay according to the manufacturer’s protocol. The absorbance at 620 nm was read for all samples. Anti-Myc tag agarose beads incubated with the cell lysate prepared from 293T cells transfected with empty vector were used as the mock control in the GTPase activity assay. To evaluate the quantities of input immunopurified proteins for GTPase activity assay, the proteins binding with anti-Myc tag agarose beads were subjected to Western blot analysis.