P446L-importin-β inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules

Abstract

The P446L mutant Drosophila importin-β (P446L-imp-β) has been reported to prohibit - in dominant negative fashion - nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-β we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-β. A NE also forms over importin-β-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-β as efficiently recruits NE vesicles as normal importin-β. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-β appears to be related to icreased - as compared to normal importin-β - microtubule (MT) binding ability of P446L-imp-β. While wild-type importin-β is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-β cannot be removed from the MTs by RanGTP. P446L-imp-β, like normal importin-β, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-β on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.