Figure 5.

Structure-based mutants verified the allosteric mechanism of NES release. (A) A pulldown assay showed that the C-terminal tail of Ran is required for RanBP1-mediated dissociation of NES from CRM1 and RanGTP. Immobilized GST-PKI (60 μg) was incubated with CRM1 (68 μg) and RanGTP (48 μg), unbound CRM1 and Ran washed away and then the beads were incubated without (lanes 1 and 3) or with (lanes 2 and 4) RanBP1 (130 μg). Bound and unbound fractions were analysed by SDS–PAGE. Lanes 1 and 2, full-length Ran; lanes 3 and 4, a C-terminal deletion mutant of Ran (residues 1–211). As the band of RanBP1 overlapped with that of GST-PKI, RanBP1 in the bound fraction was detected by western blotting; the other proteins were detected by Coomassie staining. (B–D) Mutational analysis demonstrated that the conserved hydrophobic residues in HEAT9 loop are important for loading and unloading of NES-cargo. (B) Pulldown assays to analyse the effects of mutating hydrophobic residues of HEAT9 loop on the binding of yeast CRM1 to PKI (NES-cargo) in the absence (lanes 1–4) or presence (lanes 5–8) of RanGTP. Immobilized GST-PKI (64 μg) was incubated with CRM1 (76 μg) without (lanes 1–4) or with (lanes 5–8) RanGTP (78 μg). Bound and unbound proteins were analysed by SDS–PAGE and Coomassie staining. SDS–PAGE of input proteins shows that the same amount of CRM1 (either wild type or mutant) was used in each experiment. The mutation(s) designed to disrupt the hydrophobic interactions between HEAT9 loop and the inner surface of HEAT repeats 11 and 12 strengthened the binding of CRM1 to PKI (NES-cargo) in the absence of RanGTP. (C, D) The mutations of the hydrophobic residues of HEAT9 loop also substantially reduced the rate of RanBP1-accelerated NES release. Preincubated yeast CRM1-CFP:YFP-NES (PKI residues 38–47):RanGTP mixture (0.4 μM CRM1 (wild type (red) or V441D (green) or V441A/L442A (orange) or V443D (magenta))-CFP, 2.0 μM YFP-NES, 6.0 μM RanGTP) was rapidly mixed with buffer alone (blue), or with 8 μM RanBP1.