(A) Size exclusion chromatography (SEC) of 20 μM Imp9 alone (black), 60 μM RanGTP alone (dark grey), Imp9 +H2A-H2B 1:1 (20 μM; blue), and Imp9 +H2A-H2B 1:1 (20 μM) with Ran at 0.5 (10 μM; green), 1 (20 μM; red), 2 (40 μM; light grey) or 3 (60 μM; grey). The buffer was 20 mM HEPES pH 7.4, 200 mM sodium chloride, 2 mM magnesium acetate, 2 mM TCEP and 8% (v/v) glycerol. Column was Superdex S200 Increase 10/300. Imp9 alone elutes at 13.6 mL, while the 1:1 Imp9•H2A-H2B complex elutes at 13.5 mL. We see the formation of a 1:1:1 RanGTP•Imp9•H2A-H2B complex. Addition of an equimolar amount of RanGTP causes the Imp9•H2A-H2B peak to shift from 13.5 mL to 13.4 mL. Continued addition of RanGTP beyond a 1:1:1 mixture, results in the appearance of free RanGTP that elutes at 17.1 mL. Comparison to a Ran only control (60 μM; dark grey) shows that the Imp9•H2A-H2B•Ran complex has a 1:1:1 stoichiometry. Quantitatively, the free RanGTP peak is absent in the 1:1:1 sample, is one-third of the control in a 1:1:2 sample, and two-thirds of the control in a 1:1:3 sample. (B) SEC of 1:1 Imp9 +H2A-H2B (70 μM; blue) and 1:1:1 Imp9, H2A-H2B, and Ran (70 μM; red). The buffer was 20 mM HEPES pH 7.4, 200 mM NaCl, 2 mM magnesium acetate, 2 mM TCEP and 8% (v/v) glycerol. Column was Superdex S200 Increase 10/300. Peak fractions (Fractions 1–13) were analyzed on 15% SDS-PAGE stained with Coomassie blue. As in A), Imp9•H2A-H2B elutes at 13.5 mL and the peak eluting at 13.4 mL contains 1:1:1 RanGTP•Imp9•H2A-H2B. Analysis of peak fractions by SDS-PAGE shows the presence of Imp9, H2A-H2B, and Ran in the peak. Each protein stains in proportion to that seen in the input lane, consistent with the formation of a 1:1:1 complex. Also, in the SEC there is no free H2A-H2B or free Ran.