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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.