Figure 4.

Altering importin beta by addition of a His-tag renders importin beta insensitive to RanGTP specifically in its block to nuclear pore assembly. A. Pore-free BAPTA intermediates rescued in the presence of cytosol plus His-tagged Xenopus importin beta were not able to assemble nuclear pores (+Tag-X-β). When RanQ69L-GTP was added along with His-tagged Xenopus importin beta, the block to pore assembly could not be reversed (+Tag-X-β +Ran). Where indicated, importin beta was added at 10 μM and RanQ69L-GTP at 50 μM. The bar represents 10 microns. B. Pore-free BAPTA nuclear intermediates rescued in the presence of cytosol and untagged human or Xenopus importin beta were not able to assemble nuclear pores (+X-β or +h-β). The inhibitory concentration of 10 μM used here was determined to be the approximate minimum concentration for pore assembly inhibition in a separate experiment (data not shown). When RanQ69L-GTP was added along with untagged human importin beta, the block to pore assembly was partially reversed (+h-β +Ran). The Xenopus importin beta block was fully reversed (+X-β +Ran). To better visualize the FG-nucleoporin stain, a section of the images (white dashed box) was enlarged by 3X (right most panel). Where indicated, importin beta was added at 10 μM and RanQ69L-GTP at 50 μM. The bar represents 10 microns.