Fig. 7. XMog1 and RanBP1 together promote GDP release from Ran and loading with GTP. (A) Binding between Ran (68 nM) and GST-RanBP1 (2.6 µM) in Mg²⁺-buffer with addition of GTPS or GDP. Incubations were carried out with or without addition of 6 µM XMog1. A western blot of the precipitates was developed using antibodies to Ran and XMog1. (B) [³H]GTP release from Ran induced by XMog1 is inhibited by RanBP1. (C) [³H]GDP release from Ran is promoted by XMog1 and RanBP1. In both (A) and (B), Ran (50 pmol) preloaded with [³H]-labelled guanine nucleotide was incubated in a total volume of 50 µl with the specified amount of XMog1 alone (open symbols) or with the addition of XMog1 and 18 pmol RanBP1 (closed symbols) in the absence of free nucleotide, as in Fig. 6. The amount of radioactivity remaining was counted and the amount released expressed as a percentage of the total. (D) [³H]GTP loading onto Ran in the presence of XMog1 and RanBP1. 50 pmol Ran-GDP was incubated with no additions (open squares), 300 pmol XMog1 alone (closed squares), 18 pmol RanBP1 alone (open triangles) or XMog1 and RanBP1 together (closed triangles) in a total volume of 50 µ1. [³H]GTP was added at 1.2 µM and GDP was present at 700 µM. Incubation was carried out for the times shown before determining the amount of radioactivity bound to Ran. Each time point was performed in duplicate.