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Abstract. Selective transport of proteins is a major mechanism by which biochemical differences are maintained between the cytoplasm and nucleus. To begin to investigate the molecular mechanism of nuclear transport, we used an in vitro transport system composed of a Xenopus egg extract, rat liver nuclei, and a fluorescently labeled nuclear protein, nucleoplasmin. With this system, we screened for inhibitors of transport. We found that the lectin, wheat germ agglutinin (WGA), completely inhibits the nuclear transport of fluorescently labeled nucleoplasmin. No other lectin tested affected nuclear transport. The inhibition by WGA was not seen when N-acetylglucosamine was present and was reversible by subsequent addition of sugar. When rat liver nuclei that had been incubated with ferritin-labeled WGA were examined by electron microscopy, multiple molecules of WGA were found bound to the cytoplasmic face of each nuclear pore. Gel electrophoresis and nitrocellulose transfer identified one major and several minor nuclear protein bands as binding t25I-labeled WGA. The most abundant protein of these, a 63-65-kD glycoprotein, is a candidate for the inhibitory site of action of WGA on nuclear protein transport. WGA is the first identified inhibitor of nuclear protein transport and interacts directly with the nuclear pore.

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Nuclear pores span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm, from individual proteins and RNAs to entire viral genomes. By far the largest subunit of the nuclear pore is the Nup107-160 complex, which consists of nine proteins and is critical for nuclear pore assembly. At mitosis, the Nup107-160 complex localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107-160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107-160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division.Nup107-160 complex ͉ MEL-28 ͉ Nup133 ͉ mitosis E ssential for cell survival, nuclear pore complexes are large multiprotein assemblages, Ϸ30 times the size of the ribosome. Structurally, nuclear pores are comprised of three major domains inserted in the nuclear membranes. These domains include a massive central scaffold, cytoplasmic filaments, and a nuclear basket (1). Nuclear pores consist of multiple copies of Ϸ30 different proteins termed nucleoporins (Nups) (2). A third of these contain phenylalanine-glycine (FG) repeat domains, believed to be key sites for interaction with transport receptors (3).During vertebrate mitosis, the nuclear pore disassembles into approximately a dozen subunits, concurrent with the breakdown of the nuclear envelope. Most diffuse throughout the mitotic cytoplasm, playing no role in mitotic progression identified to date. However, a small number of nuclear pore proteins, including the Nup107-160 complex, localize to regions of the mitotic kinetochore and͞or spindle, pointing toward a function in mitotic chromosome segregation (4-15). We now know that, in vitro, the Nup107-160 complex is required for spindle assembly (15).Nuclear reassembly, which begins in late anaphase and continues through telophase, occurs at the chromatin periphery. During this time, the nuclear pore subunits reassemble, stepwise, into pore complexes within the double nuclear membrane. The Nup107-160 complex, by far the largest of the pore subunits, has been shown to play a critical role in nuclear pore assembly. The Nup107-160 complex consists to date of nine proteins (Fig. 1C: Nup160, Nup133, Nup107, Nup96, Nup8...

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

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 ...

Importin beta, once thought to be exclusively a nuclear transport receptor, is emerging as a global regulator of diverse cellular functions. Importin beta acts positively in multiple interphase roles: in nuclear import, as a chaperone for highly charged nuclear proteins, and as a potential motor adaptor for movement along microtubules. In contrast, importin beta plays a negative regulatory role in mitotic spindle assembly, centrosome dynamics, nuclear membrane formation, and nuclear pore assembly. In most of these, importin beta is counteracted by its regulator, Ran-GTP. In light of this, the recent discovery of Ran's involvement in spindle checkpoint control suggested a potential new arena for importin beta action, although it is also possible that one of importin beta's relatives, the karyopherin family of proteins, manages this checkpoint. Lastly, importin beta plays a role in transducing damage signals from the axons of injured neurons back to the cell body.

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.