Figure 6.

Impβ^45-462 multimerises and binds FG repeats more strongly than full-length importin β. (A) Nickel-Sepharose beads coated with the His-tagged FG/FxFG repeat domain of Nsp1p were incubated with an excess of untagged Impβ (lanes 1, 4, 7), untagged Impβ^45-452 (lanes 2, 5, 8) or a mixture of both proteins at physiological salt conditions. Bound NTRs and bait proteins were sequentially eluted and analysed by SDS–PAGE and Coomassie staining. Loaded material corresponds to 0.75% of input material or 3% of the eluted material. In comparison to the single incubations, binding of Impβ was drastically diminished in the presence of Impβ^45-452, whereas binding of the Impβ mutant itself was virtually unaffected. This indicates that Impβ^45-452 has an increased affinity for FG repeat domains. (B) Impβ^45-452 was mixed with plasmid DNA and GDP as markers for void volume and total volume, respectively, diluted to the indicated concentrations and subjected to analytical gel filtration. Absorbance profiles acquired at 280 nm were normalised to the maximal optical density. Note that the observed peak positions and shapes greatly depend on the protein concentration of the applied sample. Higher order oligomeric forms are observed at higher protein concentrations. The gradual decrease of apparent molecular weight with higher dilutions indicates dynamic association/dissociation equilibria between multiple association states.