Fig. 6. GTP-binding and hydrolysis activity of two Mfn proteins. (A) FLAG-tagged Mfn2 or Mfn1 was expressed in HeLa cells and their PNS were solubilized by digitonin. The lysates were incubated with GTP-agarose beads on ice for 60 minutes, or at 30°C, for the indicated times. The bound proteins and PNS used (5%) were analyzed by immunoblotting using anti-FLAG antibodies. (B) FLAG-tagged Mfn2, Mfn2^K109T, Mfn1, or Mfn1^K88T was incubated with GTP-agarose at 30°C for 1 hour and bound proteins were analyzed by immunoblotting using anti-FLAG antibodies. (C) His₆-tagged Mfn2 and Mfn1 proteins were expressed in E. coli and purified as described. The purified proteins were analyzed by SDS-PAGE followed by Coomassie Brilliant blue staining. (D,E) His-tagged Mfn2 or Mfn1 was incubated with [-³²P]GTP at 37°C for the indicated times, and analyzed by thin layer chromatography followed by digital autoradiography as described, using bovine serum albumin as a control. The positions of GDP, GTP, and the origin are shown by an arrow, filled arrowhead, and open arrowhead, respectively. GTP and GDP radioactivity was quantified using an image analyzer (E).