Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

The mechanism for nuclear envelope （NE） assembly is not fully understood. Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS （nuclear localization sequence） proteins provide docking sites for the NE precursor membrane vesicles and nucleo- porins via importin-a and -β during NE assembly in Xenopus egg extracts. We show that along with the fast recruit- ment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-α binds the chromatin NLS proteins rapidly. Meanwhile, importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-α on the chromatin NLS pro- teins, importin-β targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran- GTP on the chromatin generated by RCC1, importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.