Figure 5.

Cyclin A-GFP/Cdk2^(K33R) import is not dependent on Rch1/importin-β but is blocked by the IBB. (A) Permeabilized HeLa cells were incubated with cyclin A-GFP/Cdk2^K33R, cyclin E-GFP/Cdk2^K33R, or cyclin B15E-GFP. Control levels of nuclear import with exogenous Rch1, importin-β, and Ran GDP after 30 min of incubation at 22°C were set to 100%. Import was measured in parallel experiments, without exogenous Rch1, without importin-β, with 60 μM RanQ69L, or under standard control conditions but with incubation on ice instead of at 22°C. For each experimental condition, the average amount of each substrate imported into 100 nuclei was calculated and the mean of at least two independent experiments is shown. (B) Permeabilized HeLa cells were incubated in import buffer containing Rch1, importin-β, Ran GDP, and an energy-regenerating mixture, with TRITC-labeled nucleoplasmin (top), cyclin A-GFP/Cdk2^K33R (middle), or Texas Red-labeled M9 domain (bottom) in the absence (left) or presence (right) of IBB (15 μM final concentration). In the experiments using TRITC-labeled M9 domain (a3 and b3), the protein was incubated under standard nuclear import assay conditions but with 300 nM transportin instead of Rch1 and importin-β in the reaction mixture. After 30 min of incubation at 22°C, nuclear import was visualized by confocal fluorescence microscopy. Transportin was unable to stimulate cyclin A-GFP/Cdk2^K33R import (not shown).