Figure 6.

Kap114p, Kap121p, and Kap95p bind directly to the H2A and H2B NLSs. (a) Reticulocyte lysate containing ³⁵S-methionine–labeled Kap114p, Kap95p, Kap121p, or Kap108p was incubated with Sepharose-bound GST (GST), GST-H2A amino acids 1–46 (GST-H2A), or GST-H2B amino acids 1–52 (GST-H2B). The bound material was separated by SDS-PAGE. Kaps were visualized by fluorography of the gels (top four panels). The reticulocyte lysate input (2% of reaction) is also shown. The GST and GST-fusion proteins for a representative experiment, visualized by Coomassie blue staining, are shown (bottom). (b) 1 μg of purified, bacterially expressed Kap114p, Kap121p, or Kap95p was incubated with Sepharose-bound GST (GST), GST-H2A amino acids 1–46 (GST-H2A), or GST-H2B amino acids 1–52 (GST-H2B). Where indicated, the Kap was preincubated with 20 μM human RanGTP Q69L. The bound material was separated by SDS-PAGE and visualized by Coomassie blue staining. Positions of GST-fusion proteins are indicated; additional lower bands are probably degradation products.