Figure 3

Xenopus importin beta is an authentic negative regulator of nuclear pore assembly and is reversed by RanGTP. Pore-free BAPTA nuclear intermediates, which have fused nuclear membranes but contain no nuclear pores (left panel), when diluted into fresh cytosol (+ buffer), incorporate nuclear pores. The addition of His-tagged human importin beta (+h-β-Tag) or Xenopus untagged importin beta (+X-β) prevented nuclear pore assembly. Addition of RanQ69L-GTP with His-tagged human importin beta (+h-β-Tag +Ran) could not reverse the beta block to pore assembly, as previously observed [9]. However, addition of RanQ69L-GTP with untagged Xenopus importin beta (+X-β +Ran) did reverse the beta block to pore assembly. Nuclear pores were detected by the monoclonal antibody mAb414, which recognizes FG nucleoporins (FG Nups). Where indicated, importin beta was added at 20 μM and RanQ69L-GTP at 30 μM. The bar represents 10 microns. Black squares on the drawings at the right indicate FG-staining nuclear pores.