FIGURE 8.

The import Hxk2-Kap60-Kap95 complex formation. GST pulldown assays of the effect of Gsp1 on the Hxk2-Kap60 (a) and Hxk2-Kap95 (b) interaction stability. GST-Hxk2, GST-Gsp1, and GST-Kap60 fusion proteins were purified on glutathione-Sepharose columns and incubated with thrombin to isolate Hxk2, Gsp1, and Kap60 proteins, respectively. The Gsp1 protein was loaded with GTP to generate Gsp1(GTP), or with GDP to generate Gsp1(GDP). The Hxk2 protein was incubated with purified GST-Kap60 or GST-Kap95 bound to glutathione-Sepharose beads in the presence of Gsp1(GTP) and Gsp1(GDP) in the absence or presence of Kap60 purified protein (lanes 5–7). The beads were washed extensively. Co-precipitated proteins were resolved by 12% SDS-PAGE and visualized on a Western blot with polyclonal anti-Hxk2 antibody.