Assembly of a eukaryotic nucleus involves three distinct events: membrane recruitment, fusion to form a double nuclear membrane, and nuclear pore complex (NPC) assembly. We report that importin ␤ negatively regulates two of these events, membrane fusion and NPC assembly. When excess importin ␤ is added to a full Xenopus nuclear reconstitution reaction, vesicles are recruited to chromatin but their fusion is blocked. The importin ␤ down-regulation of membrane fusion is Ran-GTP reversible. Indeed, excess RanGTP (RanQ69L) alone stimulates excessive membrane fusion, leading to intranuclear membrane tubules and cytoplasmic annulate lamellae-like structures. We propose that a precise balance of importin ␤ to Ran is required to create a correct double nuclear membrane and simultaneously to repress undesirable fusion events. Interestingly, truncated importin ␤ 45-462 allows membrane fusion but produces nuclei lacking any NPCs. This reveals distinct importin ␤-regulation of NPC assembly. Excess full-length importin ␤ and ␤ 45-462 act similarly when added to prefused nuclear intermediates, i.e., both block NPC assembly. The importin ␤ NPC block, which maps downstream of GTP␥S and BAPTA-sensitive steps in NPC assembly, is reversible by cytosol. Remarkably, it is not reversible by 25 M RanGTP, a concentration that easily reverses fusion inhibition. This report, using a full reconstitution system and natural chromatin substrates, significantly expands the repertoire of importin ␤. Its roles now encompass negative regulation of two of the major events of nuclear assembly: membrane fusion and NPC assembly. INTRODUCTIONIn cells from yeast to mammals, importin ␣ and ␤ act together to ferry classical nuclear localization signal (NLS)-bearing proteins into the nucleus (Gorlich and Kutay, 1999;Stoffler et al., 1999;Damelin and Silver, 2000;Rout et al., 2000;Conti and Izaurralde, 2001;Vasu and Forbes, 2001;Damelin et al., 2002;Weis, 2003). Once in the nucleus the small GTPase Ran binds to importin ␤, displacing importin ␣ and the NLS cargo, thus completing import. In the nucleus, Ran is kept in a GTP state by the constant action of its chromatinbound Ran-GEF, RCC1 (Melchior and Gerace, 1998;Macara, 2001;Dasso, 2002;Kalab et al., 2002;Schwoebel et al., 2002;Steggerda and Paschal, 2002). In contrast, RanGDP is the predominant form found in the cytoplasm due to the cytoplasmic localization of RanGAP.The horizons for importin ␣ and ␤ were unexpectedly broadened when they were found to play a very different role at mitosis. In metazoans, importin ␣ and ␤ are released to the cytosol by nuclear breakdown, where they act to inhibit proteins essential for mitotic spindle assembly. However, the inhibition of spindle assembly is reversed by Ran in the vicinity of mitotic chromosomes, where RanGTP continues to be produced by chromatin-bound RCC1 (Kalab et al., 1999;Gruss et al., 2001;Nachury et al., 2001;Wiese et al., 2001;Dasso, 2002). Thus, a spindle forms only around the mitotic (ER) chromosomes and not elsewhere in the cytoplasm.At the end ...

Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events. INTRODUCTIONDuring mitosis, the intracellular architecture of the higher eukaryotic cell undergoes constant and dramatic change. The chromosomes condense, the nuclear envelope breaks down, a mitotic spindle forms and separates the chromosomes, and finally a nuclear envelope is reassembled around each set of daughter chromosomes. The movement of the cell cycle from one stage of mitosis to the next involves the coordinated action of multiple kinases and phosphatases. Recently, however, new regulators have been identified that contribute not to the timing but to the spatial control of assembly of both the mitotic spindle and the nucleus: karyopherins and RanGTP.Karyopherins are most well known for their function in interphase as the transport receptors for nuclear import and export (reviewed in Tran and Wente, 2006;Stewart, 2007). Nuclear proteins larger than ϳ20 -40 kDa are actively imported, whereas snRNAs, tRNAs, and other cargoes are exported through the nuclear pores by karyopherins.Karyopherins consist of a large family of importins (import receptors) and exportins (export receptors; reviewed in Weis, 2003;Pemberton and Paschal, 2005). The first and most studied import receptor of the karyopherin family was importin beta (Adam and Adam, 1994;Chi et al., 1995;Gorlich et al., 1995;Imamoto et al., 1995;Radu et al., 1995;Floer et al., 1997). Importin beta mediates the nuclear import of many proteins through the use of a specific adaptor, such as importin alpha (reviewed in Cook et al., 2007). Importin alpha recognizes cargoes containing classical or bipartite nuclear localization signals (NLSs), which are composed largely of basic amino acids (reviewed ...

Background: Human importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue.

Nuclear import is a critical process for the cell: molecules are selectively permitted into the nuclear interior where the sheltered genome resides. The process of nuclear import can be biochemically studied in vitro using nuclei reconstituted from Xenopus egg extract components and Xenopus sperm chromatin. This in vitro system allows for the visualization of nuclear import by monitoring the accumulation of fluorescent nuclear import substrates in the reconstituted nuclei. A powerful aspect of the system is that "biochemically mutant" nuclei can be readily generated, either by immunodepletion of proteins from or addition of proteins to the reaction. This ability allows ascertainment of the role of specific proteins in nuclear import.