Figure 3.

p10 mutants that cannot bind RanGDP do not support nuclear accumulation of FITC–RanGDP in digitonin-permeabilized cells. (A) Recombinant p10 proteins were dotted onto nitrocellulose, as described in the Materials and Methods, and the nitrocellulose was incubated with either Ran[α-³²P]GDP or Ran[α-³²P]GTP, washed, and exposed to film. (B) The ability of different p10 mutants to mediate the import of FITC–RanGDP (2 μM) in permeabilized BRL cells was assayed, as described in the legend to Fig. 2. Samples contained 0.5 mM GTP, 1 mM ATP plus a regenerating system, and 1 μM of the indicated p10 protein (dimer). Import was for 20 min at room temperature before washing and fixation.