Figure 8.

RanQ69L reverses the full-length importin β block to membrane fusion, but not the block to NPC assembly. Nuclear reconstitution reactions containing chromatin, cytosol, and membranes were supplemented at t = 0 with 30% volume buffer, full-length importin β (20 μM), full-length importin β (20 μM) plus RanQ69L (25 μM), or RanQ69L (25 μM) alone. The reactions containing importin β, or importin β plus RanQ69L, failed to incorporate FG nucleoporins as detected with Oregon Green-mAb414. FG nucleoporins incorporated into nuclear envelopes in reactions supplemented with buffer or RanQ69L. The RanQ69L reactions also showed FG-containing structures forming outside of the nucleus, presumed annulate lamellae. Additionally, the nuclei in the RanQ69L reactions contained areas of the nuclear envelope devoid of FG nucleoporin staining. These corresponded to outer nuclear membrane bubbles, which appear as gaps in the mAb414 stain (this figure) or as bubbles when DHCC membrane dye is used (our unpublished data). Bar, 10 μm.