Role of Importin-β in the Control of Nuclear Envelope Assembly by Ran

and determines the directionality of nucleocytoplasmic transport by controlling the stability of complexes formed between cargo proteins carrying specific tar-leases proteins required for mitotic spindle assembly from inhibitory complexes with importin-␤ [5–7]. Peking University Beijing 100871 Ran also functions in the assembly of the nuclear envelope (NE) at the end of mitosis when the compart-China 3 ETH Zü rich mentalization of the nucleus is reestablished [1, 2, 8]. In cell-free systems for NE assembly made from Xenopus Institut fü r Biochemie Universitä tsstrasse 16 laevis eggs, NE assembly requires the generation of Ran-GTP from Ran-GDP by the guanine nucleotide ex-8092 Zü rich Switzerland change factor RCC1, as well as GTP hydrolysis on Ran [1, 2]. Localized concentration of Ran is sufficient to induce NE assembly in Xenopus egg extracts in the absence of chromatin, since Sepharose beads coated Summary with Ran will assemble NE-like structures containing nuclear pore complexes (NPCs) around them, forming Compartmentalization of the genetic material into a pseudonuclei that actively import karyophilic proteins nucleus bounded by a nuclear envelope (NE) is the [2]. However, the mechanism by which Ran controls NE hallmark of a eukaryotic cell. The control of NE assem-assembly is unknown. bly is poorly understood, but in a cell-free system made To investigate the possible role of Ran-interacting from Xenopus eggs, NE assembly involves the small proteins such as importin-␤ in NE assembly, we de-GTPase Ran [1, 2]. In this system, Sepharose beads pleted Xenopus egg extracts using RanQ69L, a mutant coated with Ran induce the formation of functional defective in GTPase activity and therefore locked in the NEs in the absence of chromatin [2]. Here, we show GTP-bound form [9]. This procedure removed more than that importin-␤, an effector of Ran involved in nucleo-90% of importin-␤ from the extracts (Figure 1A), as well cytoplasmic transport and mitotic spindle assembly, as other Ran-GTP binding proteins [6]. Extracts depleted is required for NE assembly induced by Ran. Concen-of Ran binding proteins (⌬RanBP extracts) were defi-tration of importin-␤ on beads is sufficient to induce cient in the ability to promote membrane vesicle recruit-NE assembly in Xenopus egg extracts. The function ment and fusion to form continuous membranes around of importin-␤ in NE assembly is disrupted by a muta-Sepharose beads coated with Ran (Figure 1B). NE as-tion that decreases affinity for nucleoporins containing sembly activity in ⌬RanBP extracts was restored by the FxFG repeats. By contrast, a truncated …