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. Author manuscript; available in PMC: 2011 May 14.
Published in final edited form as: Mol Cell. 2010 May 14;38(3):369–382. doi: 10.1016/j.molcel.2010.02.038

Figure 4. Importin-β and RanGTP regulate the ubiquitination of HURP and NuSAP.

Figure 4

A. Importin-β blocks the APC/C-dependent ubiquitination of HURP. 35S-HURP was incubated with APC/CCdh1, Ube2S, and UbcH10. As indicated, Emi1, importin-β, importin-α-CT, or importin-α/β were added. Reactions were analyzed by autoradiography. B. Importin-β inhibits HURP-ubiquitination in a dose-dependent manner. 35S-HURP was incubated with APC/CCdh1, increasing concentrations of RanQ69L, and importin-β, as indicated. C. Quantification of effects of RanGTP on HURP-degradation in mitotic extracts (red); HURP-ubiquitination by APC/CCdh1 (green) and HURP-dissociation from importin-β (blue). D. An intact cargo-binding domain is required for importin-β to act as APC/C-inhibitor. Importin-β and two mutants with reduced cargo affinity (importin-β3WA; importin-β462) were analyzed for their effect on ubiquitination of HURP by APC/C as described above. E. RanGTP allows HURP-ubiquitination in the presence of importin-β. 35S-HURP was incubated with APC/CCdh1, Ube2S, and UbcH10. Importin-β/α and GTP-bound RanQ69L were added as indicated. Reactions were analyzed by autoradiography. F. Importin-β blocks the APC/C-dependent ubiquitination of NuSAP. 35S-NuSAP was analyzed for APC/C-dependent ubiquitination as described above. G. RanGTP allows NuSAP-ubiquitination in the presence of importin-β. 35S-NuSAP was incubated with APC/CCdh1, Ube2S, and UbcH10. Importin-β/α and RanQ69L were added as indicated, and reactions were analyzed by autoradiography.