(A) Effects of wild-type and E1-Ran·GDP on
nuclear protein import in digitonin-permeabilized buffalo rat liver
cells. Assays were carried out at 21°C as described in MATERIALS AND
METHODS on permeabilized cells supplemented with 1.4 mg/ml of
Xenopus fraction A (which contains karyopherin α and
β), 1.5 μg/ml of purified human nuclear import factor p10, the
indicated concentrations of wild-type Ran·GDP (▪) or E1-Ran·GDP
(○), 5 μg/ml of NLS-tagged, rhodamine-labeled human
serum albumin, and 1 mM GTP. Protein import was measured as mean
nuclear fluorescence in arbitrary units. (B) Inhibition of nuclear
import by E1-Ran. Assays were performed at 21°C, in the presence of
1.45 mg/ml Xenopus fraction A, 1.5 μg/ml p10, 50
μg/ml wild-type Ran·GDP, 1 mM GTP, and the indicated concentrations
of additional wild-type Ran·GDP (▪) or E1-Ran·GDP (○).