Figure 1.

Nup159 promotes Dbp5 release of ADP. (A) Equilibrium binding assays show an increase in initial rate of Dbp5–¹⁴C-ADP binding: Dbp5 (1 μM) was incubated with increasing concentrations of ¹⁴C-ADP (0.5–150 μM) over the course of 24 h. Samples were removed at several time points and used in filter-binding assays. Background integrated density signals of ¹⁴C-ADP at each concentration were subtracted from the Dbp5-bound ADP signal. As listed in the table, initial velocities of binding (fraction ¹⁴C-ADP bound per minute) were calculated during the linear phase of the binding curve at each Dbp5:¹⁴C-ADP ratio tested. (B) Dbp5 does not release bound ADP after equilibration: After 24-h equilibration of 1 μM Dbp5 and 5 μM ¹⁴C-ADP:MgCl₂, excess unlabeled ATP:MgCl₂ or ADP:MgCl₂ (10 mM of each) was added, and filter-binding assays were conducted after an additional 24 h incubation. (C) Nup159-NTD does not promote release of the nonhydrolyzable ATP analog ADP-BeFx: After 26-h equilibration of 1 μM Dbp5 with 5 μM ¹⁴C-ADP-BeFx, either 1 μM Nup159NTD, 500 nM Gle1 with 200 nM IP₆, or 2 μM poly(A)₂₅ RNA was added, and filter-binding assays were performed at 24 h after addition. (D) Nup159-NTD specifically promotes Dbp5 release of bound ADP: After 24-h equilibration of 1 μM Dbp5 with 5 μM ¹⁴C-ADP:MgCl₂, either 1 μM Nup159-NTD, 500 nM Gle1 with 200 nM IP₆, or 2 μM RNA was added, and filter-binding assays were performed at 10 min and 24 h after addition. Two altered nup159 proteins (nup159^DD and nup159^VI) that do not bind Dbp5 in vitro were used as negative controls. (E) Increasing concentration of Nup159 promotes faster Dbp5 release of bound ¹⁴C-ADP. Dbp5 (1 μM) was incubated with 5 μM ¹⁴C-ADP for 24 h. Increasing concentrations of Nup159 were added and filter-binding assays were performed over the course of 2 h. All data points and bars represent at least three independent experiments with standard error bars.