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PubMed2002

Karyopherins in nuclear pore biogenesis: a role for Kap121p in the assembly of Nup53p into nuclear pore complexes.

The mechanisms that govern the assembly of nuclear pore complexes (NPCs) remain largely unknown. Here, we have established a role for karyopherins in this process. We show that the yeast karyopherin Kap121p functions in the targeting and assembly of the nucleoporin Nup53p into NPCs by recognizing a nuclear localization signal (NLS) in Nup53p. This karyopherin-mediated function can also be performed by the Kap95p-Kap60p complex if the Kap121p-binding domain of Nup53p is replaced by a classical NLS, suggesting a more general role for karyopherins in NPC assembly. At the NPC, neighboring nucleoporins bind to two regions in Nup53p. One nucleoporin, Nup170p, associates with a region of Nup53p that overlaps with the Kap121p binding site and we show that they compete for binding to Nup53p. We propose that once targeted to the NPC, dissociation of the Kap121p-Nup53p complex is driven by the interaction of Nup53p with Nup170p. At the NPC, Nup53p exists in two separate complexes, one of which is capable of interacting with Kap121p and another that is bound to Nup170p. We propose that fluctuations between these two states drive the binding and release of Kap121p from Nup53p, thus facilitating Kap121p's movement through the NPC.

Binding Sites↗
PubMed2002

The permeability barrier of nuclear pore complexes appears to operate via hydrophobic exclusion.

Nuclear pore complexes (NPCs) restrict the nucleocytoplasmic flux of most macromolecules, but permit facilitated passage of nuclear transport receptors and their cargo complexes. We found that a simple hydrophobic interaction column can mimic the selectivity of NPCs surprisingly well and that nuclear transport receptors appear to be the most hydrophobic soluble proteins. This suggests that surface hydrophobicity represents a major sorting criterion of NPCs. The rate of NPC passage of cargo-receptor complexes is, however, not dominated just by properties of the receptors. We found that large cargo domains drastically hinder NPC passage and require more than one receptor molecule for rapid translocation. This argues against a rigid translocation channel and instead suggests that NPC passage involves a partitioning of the entire translocating species into a hydrophobic phase, whereby the receptor:cargo ratio determines the solubility in that permeability barrier. Finally, we show that interfering with hydrophobic interactions causes a reversible collapse of the permeability barrier of NPCs, which is consistent with the assumption that the barrier is formed by phenylalanine-rich nucleoporin repeats that attract each other through hydrophobic interactions.

Amino Acid Motifs↗
PubMed1993

The nuclear pore complex and nucleocytoplasmic transport.

In the past year we have seen two new three-dimensional views of the nuclear pore complex, the discovery of a nuclear pore complex protein that contains zinc fingers, the purification of nuclear localization signal recognition proteins, and the discovery that hsc70 participates in nuclear import. Other highlights include the finding that certain proteins associated with heterogeneous nuclear RNA shuttle between the nucleus and cytoplasm, and that a nucleolar nuclear localization signal binding protein, which also shuttles, is located on intranuclear tracks.

Animals↗
PubMed2000

Recombinant Nup153 incorporates in vivo into Xenopus oocyte nuclear pore complexes.

Nup153 is a molecular constituent of the nuclear basket of the nuclear pore complex (NPC) that plays a critical role in nuclear export of RNAs and proteins. In an effort to map this nucleoporin more precisely within the nuclear basket we have developed an experimental approach for localizing Nup153 expressed and incorporated in vivo into Xenopus oocyte NPCs. This approach involves the microinjection into the cytoplasm of Xenopus oocytes of in vitro synthesized mRNA from a vector encoding an epitope-tagged cDNA. Here we present results obtained by Western blots, fluorescence microscopy, and immuno-electron microscopy, which clearly document that the heterologous protein is properly expressed, targeted, and incorporated into preexisting Xenopus NPCs. This new approach for localizing nucleoporins within the structure of the NPC overcomes limitations of previous techniques and allows for greater specificity and resolution than have been possible with previous methods.

Animals↗
PubMed1995

Structural analysis of the p62 complex, an assembly of O-linked glycoproteins that localizes near the central gated channel of the nuclear pore complex.

The p62 complex is an oligomeric assembly of O-linked glycoproteins of the nuclear pore complex that interacts with cytosolic transport factors and is part of the machinery for nuclear protein import. In this study we have purified the p62 complex from rat liver nuclear envelopes and analyzed its structure and composition. The p62 complex consists of four distinct polypeptides (p62, p58, p54, and p45) and has a mass of approximately 234 kDa, calculated from its hydrodynamic properties and supported by chemical cross-linking and scanning transmission electron microscopy. These data suggest that the p62 complex contains one copy of each constituent polypeptide. Analysis of preparations of the p62 complex by electron microscopy using rotary metal shadowing and negative staining revealed donut-shaped particles with a diameter of approximately 15 nm. Immunogold electron microscopy of isolated rat liver nuclear envelopes demonstrated that p62 occurs on both the nucleoplasmic and cytoplasmic sides of the pore complex near the central gated channel involved in active transport of proteins and RNAs. The properties and localization of the p62 complex suggest that it may be involved in binding transport ligands near the center of the nuclear pore complex and in subsequently transferring them to the gated transport channel.

Animals↗
PubMed1989

Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

Animals↗
PubMed2003

Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating.

The nuclear pore complex (NPC) is a permeable sieve that can dilate to facilitate the bidirectional translocation of a wide size range of receptor-cargo complexes. The binding of receptors to FG nucleoporin docking sites triggers channel gating by an unknown mechanism. Previously, we used deoxyglucose and chilling treatments to implicate Nup170p and Nup188p in the control of NPC sieving in Saccharomyces cerevisiae. Here, we report that aliphatic alcohols increase the permeability of wild-type and nup170Delta NPCs. In conjunction with increases in permeability, aliphatic alcohols, deoxyglucose, and chilling trigger the reversible dissociation of several nucleoporins from nup170Delta NPCs. These results are consistent with the hypothesis that NPC gating occurs when molecular latches composed of FG repeats and structural nucleoporins dissociate.

Alcohols↗
PubMed2001

Kinetic analysis of translocation through nuclear pore complexes.

The mechanism of facilitated translocation through nuclear pore complexes (NPCs) is only poorly understood. Here, we present a kinetic analysis of the process using various model substrates. We find that the translocation capacity of NPCs is unexpectedly high, with a single NPC allowing a mass flow of nearly 100 MDa/s and rates in the order of 10(3) translocation events per second. Our data further indicate that high affinity interactions between the translocation substrate and NPC components are dispensable for translocation. We propose a 'selective phase model' that could explain how NPCs function as a permeability barrier for inert molecules and yet become selectively permeable for nuclear transport receptors and receptor-cargo complexes.

Active Transport, Cell Nucleus↗
PubMed2003

Sec13 shuttles between the nucleus and the cytoplasm and stably interacts with Nup96 at the nuclear pore complex.

Sec13 is a constituent of the endoplasmic reticulum and the nuclear pore complex (NPC). At the endoplasmic reticulum, Sec13 is involved in the biogenesis of COPII-coated vesicles, whereas at the NPC its function is unknown. We show here, by yeast two-hybrid screenings and biochemical assays, that a region at the amino terminus of the human nuclear pore complex protein Nup96 interacts with the WD (Trp-Asp) repeat region of human Sec13. By using immunofluorescence and confocal and immunoelectron microscopy, we found that in interphase, Sec13 and Nup96 are localized at both sides of the NPC in addition to other intracellular sites. In mitosis, Sec13 was found dispersed throughout the cell, whereas a pool of Nup96 colocalized with the spindle apparatus. Photobleaching experiments showed that Sec13 shuttles between intranuclear sites and the cytoplasm, and a fraction of Sec13 is stably associated with NPCs. Cotransfection of Sec13 and the Sec13 binding site of Nup96 decreased the mobile pool of Sec13, demonstrating the interaction of Sec13 and Nup96 in vivo. Targeting studies showed that Sec13 is actively transported into the nucleus and contains a nuclear localization signal. These results indicate that Sec13 stably interacts with Nup96 at the NPC during interphase and that the shuttling of Sec13 between the nucleus and the cytoplasm may couple and regulate functions between these two compartments.

Active Transport, Cell Nucleus↗
PubMed1993

Sequential O-glycosylation of nuclear pore complex protein gp62 in vitro.

Gp62 is a nuclear pore complex glycoprotein of vertebrates containing multiple O-linked N-acetylglucosamine monosaccharides. We have recently shown (Cordes et al., Eur. J. Cell Biol. 55, 31-47 (1991)) that gp62 of mouse and Xenopus laevis is predominantly glycosylated in the amino-terminal half of the molecule. Here we describe in vitro glycosylation in rabbit reticulocyte lysate of gp62 and of individual segments of this protein expressed as recombinant polypeptides in E. coli. Competition experiments between purified polypeptides revealed that gp62 exhibits in vitro at least two types of binding sites for a cytosolic N-acetylglucosaminyltransferase resulting in a sequential glycosylation of the protein. High affinity binding sites for this glycosyltransferase were located in the amino-terminal domain between amino acids 248-341 of mouse gp62, whereas low affinity binding sites were scattered in the entire amino-terminal half. The segment containing amino acids 248-341 was able to compete with in vitro O-glycosylation of gp62 subdomains containing low affinity binding sites. This gp62 segment was also able to compete with O-glycosylation of other unrelated cellular proteins in vitro.

Amino Acid Sequence↗
PubMed1994

An NLS is sufficient to engage facilitated translocation by the nuclear pore complex and subsequent intranuclear binding.

We investigated the nuclear transport of a fusion protein consisting of a nuclear localization signal linked to beta-galactosidase, normally a cytoplasmic protein. We microinjected the radiolabeled fusion protein into the cytoplasm of living Xenopus oocytes or supplied it directly to the surface of the oil-isolated oocyte nucleus and measured its transport into the nucleus. Our data confirm that a nuclear localization signal is sufficient to entrain a protein's facilitated transport through the nuclear pore complex and its subsequent nuclear accumulation. Moreover, nuclear envelope micropuncture experiments determine that the fusion protein's accumulation results from its intranuclear binding, demonstrating that no specific region of a transported protein--other than the nuclear localization signal itself--is required for facilitated transport and intranuclear binding. Finally, we present evidence that the intranuclear binding of a transported protein requires not only its nuclear localization signal, but also its prior facilitated transport through the nuclear pore complex.

Amino Acid Sequence↗
PubMed2004

Yeast nuclear pore complexes have a cytoplasmic ring and internal filaments.

The nuclear pore complex (NPC) controls transport of macromolecules across the nuclear envelope. It is large and complex but appears to consist of only approximately 30 different proteins despite its mass of > 60MDa. Vertebrate NPC structure has been analyzed by several methods giving a comprehensive architectural model. Despite our knowledge of yeast nucleoporins, structural data is more limited and suggests the basic organization is similar to vertebrates, but may lack some peripheral and other components. Using field emission scanning electron microscopy to probe NPC structure we found that the yeast, like higher eukaryotic, NPCs contain similar peripheral components. We can detect cytoplasmic rings and evidence of nucleoplasmic rings in yeasts. A filamentous basket is present on the nucleoplasmic face and evidence for cytoplasmic filaments is shown. We observed a central structure, possibly the transporter, that which may be linked to the cytoplasmic ring by internal filaments. Immuno-gold labeling suggested that Nup159p may be attached to the cytoplasmic ring, whereas Nup116p may be associated, partly, with the cytoplasmic filaments. Analysis of a Nup57p mutant suggested a role in maintaining the stability of cytoplasmic components of the NPC. We conclude that peripheral NPC components appear similar in yeasts compared to higher organisms and present a revised model for yeast NPC structural composition.

Animals↗
PubMed2001

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.

Active Transport, Cell Nucleus↗
PubMed2003

Characterisation of nuclear pore complex oxalate binding protein from human kidney.

Both rat and human kidney nuclei exhibited time and pH dependent oxalate or histone-oxalate uptake which was inhibited by anion transport inhibitor, 4,4'-dithiocyanostilbene-2,2'-disulphonic acid. Sodium chloride had no effect. Nuclear membrane had oxalate binding at pH 7.4. Extraction of nuclear membrane by Triton-high salt mixture showed maximal oxalate binding activity with nuclear pore complex while nuclear lamin had no oxalate binding. The rat and human kidney nuclear pore complex showed oxalate binding of 144 and 220 pmoles/mg protein respectively. Subsequent purification of the protein on diethyl amino ethyl-Sephadex A 50 column and Sephadex G-200 column yielded 4-fold purification. The protein revealed a molecular weight of 205 kDa on SDS-PAGE. The protein was found to be saturable at 2 microM oxalate and had a Kd of 2.98 pM and a Bmax of 197 pmoles. Antibody for 205 kD was separated from primary biliary cirrhosis serum containing auto antibody against 205 kDa using affinity column chromatography. The oxalate binding activity as well as the nuclear uptake of oxalate or histone-oxalate were inhibited by its antibody.

Animals↗
PubMed2004

Dynamic properties of nuclear pore complex proteins in gp210 deficient cells.

Gp210, an integral membrane protein of the nuclear pore complex (NPC), is believed to be involved in NPC biogenesis. To test this hypothesis, we have investigated dynamic properties of the NPC and distribution of NPC proteins in NIH/3T3 cells lacking gp210. POM121 (the other integral NPC protein) and NUP107 (of the NUP107/160 complex) were correctly distributed at the nuclear pores in the absence of gp210. Furthermore, fluorescence recovery after photobleaching experiments showed that POM121 and NUP107 remained stably associated at the NPCs. We conclude that gp210 cannot be required for incorporation of POM121 or NUP107 or be required for maintaining NPC stability.

3T3 Cells↗
PubMed1999

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

Amino Acid Sequence↗
PubMed1990

The NUP1 gene encodes an essential component of the yeast nuclear pore complex.

Monoclonal antibodies generated against a family of related nuclear pore complex proteins (nucleoporins) from rat liver nuclei cross-react with several proteins in the yeast S. cerevisiae and show punctate nuclear envelope staining similar to the pattern seen in mammalian cells. We have cloned a gene encoding one of these proteins (NUP1) and have confirmed the localization of the NUP1 protein to the pore complex by immunofluorescence, using an epitope-tagged construct to differentiate it from other members of this family. The NUP1 protein is essential for cell viability, and overexpression from the yeast GAL10 promoter prevents further cell growth. The central domain of NUP1 consists of a series of degenerate repeats similar to those found in the nucleoskeletal protein NSP1, a protein that cross-reacts with monoclonal antibodies against NUP1. We propose that the repetitive domain is a feature common to the nucleoporins.

Amino Acid Sequence↗
PubMed1996

Targeting and function in mRNA export of nuclear pore complex protein Nup153.

Nup153 is a large (153 kD) O-linked glyco-protein which is a component of the basket structure located on the nucleoplasmic face of nuclear pore complexes. This protein exhibits a tripartite structure consisting of a zinc finger domain flanked by large (60-70 kD) NH2- and COOH-terminal domains. When full-length human Nup153 is expressed in BHK cells, it accumulates appropriately at the nucleoplasmic face of the nuclear envelope. Targeting information for Nup153 resides in the NH2-terminal domain since this region of the molecule can direct an ordinarily cytoplasmic protein, pyruvate kinase, to the nuclear face of the nuclear pore complex. Overexpression of Nup153 results in the dramatic accumulation of nuclear poly (A)+ RNA, suggesting an inhibition of RNA export from the nucleus. This is not due to a general decline in nucleocytoplasmic transport or to occlusion or loss of nuclear pore complexes since nuclear protein import is unaffected. While overexpression of certain Nup153 constructs was found to result in the formation of unusual intranuclear membrane arrays, this structural phenotype could not be correlated with the effects on poly (A)+ RNA distribution. The RNA trafficking defect was, however, dependent upon the Nup153 COOH-terminal domain which contains most of the XFXFG repeats. It is proposed that this region of Nup153, lying within the distal ring of the nuclear basket, represents a docking site for mRNA molecules exiting the nucleus.

Animals↗