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PubMed2001

The nucleoporin Nup60p functions as a Gsp1p-GTP-sensitive tether for Nup2p at the nuclear pore complex.

The nucleoporins Nup60p, Nup2p, and Nup1p form part of the nuclear basket structure of the Saccharomyces cerevisiae nuclear pore complex (NPC). Here, we show that these necleoporins can be isolated from yeast extracts by affinity chromatography on karyopherin Kap95p-coated beads. To characterize Nup60p further, Nup60p-coated beads were used to capture its interacting proteins from extracts. We find that Nup60p binds to Nup2p and serves as a docking site for Kap95p-Kap60p heterodimers and Kap123p. Nup60p also binds Gsp1p-GTP and its guanine nucleotide exchange factor Prp20p, and functions as a Gsp1p guanine nucleotide dissociation inhibitor by reducing the activity of Prp20p. Yeast lacking Nup60p exhibit minor defects in nuclear export of Kap60p, nuclear import of Kap95p-Kap60p-dependent cargoes, and diffusion of small proteins across the NPC. Yeast lacking Nup60p also fail to anchor Nup2p at the NPC, resulting in the mislocalization of Nup2p to the nucleoplasm and cytoplasm. Purified Nup60p and Nup2p bind each other directly, but the stability of the complex is compromised when Kap60p binds Nup2p. Gsp1p-GTP enhances by 10-fold the affinity between Nup60p and Nup2p, and restores binding of Nup2p-Kap60p complexes to Nup60p. The results suggest a dynamic interaction, controlled by the nucleoplasmic concentration of Gsp1p-GTP, between Nup60p and Nup2p at the NPC.

Active Transport, Cell Nucleus↗
PubMed2002

The yeast nuclear pore complex functionally interacts with components of the spindle assembly checkpoint.

Aphysical and functional link between the nuclear pore complex (NPC) and the spindle checkpoint machinery has been established in the yeast Saccharomyces cerevisiae. We show that two proteins required for the execution of the spindle checkpoint, Mad1p and Mad2p, reside predominantly at the NPC throughout the cell cycle. There they are associated with a subcomplex of nucleoporins containing Nup53p, Nup170p, and Nup157p. The association of the Mad1p-Mad2p complex with the NPC requires Mad1p and is mediated in part by Nup53p. On activation of the spindle checkpoint, we detect changes in the interactions between these proteins, including the release of Mad2p (but not Mad1p) from the NPC and the accumulation of Mad2p at kinetochores. Accompanying these events is the Nup53p-dependent hyperphosphorylation of Mad1p. On the basis of these results and genetic analysis of double mutants, we propose a model in which Mad1p bound to a Nup53p-containing complex sequesters Mad2p at the NPC until its release by activation of the spindle checkpoint. Furthermore, we show that the association of Mad1p with the NPC is not passive and that it plays a role in nuclear transport.

Antimetabolites↗
PubMed2003

The nuclear pore complex: nucleocytoplasmic transport and beyond.

Over the past two years, it has become evident that there is an unexpected link between nuclear pore complex structure and dynamics, nucleocytoplasmic transport and chromosome segregation. In addition, a tomographic three-dimensional reconstruction of native nuclear pore complexes preserved in thick amorphous ice has unveiled a number of new structural features of this supramolecular machine. These data, together with some of the elementary physical principles that underlie nucleocytoplasmic transport, will be discussed in this review.

Active Transport, Cell Nucleus↗
PubMed2000

Nuclear pore complexes in the organization of silent telomeric chromatin.

The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These 'silent' domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80. This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

Antigens, Nuclear↗
PubMed1993

The nuclear pore complex.

Over the past years, significant progress has been made both in the analysis of the structural and molecular organization of the nuclear pore complex (NPC) and the mechanism of nuclear transport. In this minireview, I will focus on some of the recent developments in this field. Structural studies employing high resolution EM have revealed a detailed view of the three-dimensional organization of the NPC. In addition, an isolation procedure which yields highly enriched NPCs from yeast has given insight into the molecular complexity of the NPC organization. By biochemical, immunological and genetic approaches, a series of novel pore proteins were identified. Exploiting yeast as a genetic system, several mutants defective in nuclear import of proteins and export of RNA were selected. By in vitro nuclear transport assays, soluble cytoplasmic factors including NLS (nuclear localization sequence) binding proteins and heat shock proteins required for nuclear accumulation were found. The aim of the future research must be to put these various components of the NPC and nuclear transport machinery in a topological and functional context.

Amino Acid Sequence↗
PubMed1997

[Immunogold labeling electron microscopy showing vimentin filament anchored on nuclear pore complex].

The relationship between intermediate filament and nucleus is an important question to be solved. By combining sequential cell fractionation with immunoblotting, we showed that the intermediate filament protein in turkey erythrocyte is vimentin. Using pre-embedment immunogold labeling techniques together with sequential cell fractionation, we showed that cytoplasmic intermediate filaments in turkey erythrocyte are labeled specifically by rabbit anti-vimentin antibody-protein A-gold. Moreover, we showed that the cytoplasmic filaments anchored on nuclear pore complex were also labeled by rabbit anti-vimentin antibody-protein A-gold specifically. Our results demonstrated that cytoplasmic filaments anchored on nuclear pore complex was vimentin filament. The experiments indicated that vimentin filament may be anchored on nuclear pore complex by binding with Nup 180 and intermediate filament may be involved in nuclear transportation.

Animals↗
PubMed2000

An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: implications for annulate lamella formation.

Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. Söderqvist et al., 1997, Eur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP(3)) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP(3) had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP(3) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP(3) into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0. 1 to 2 microm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.

Animals↗
PubMed2002

Formation of Tap/NXT1 heterodimers activates Tap-dependent nuclear mRNA export by enhancing recruitment to nuclear pore complexes.

The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)(+) RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)(+) RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

Active Transport, Cell Nucleus↗
PubMed2001

Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization.

Tpr is a protein component of nuclear pore complex (NPC)-attached intranuclear filaments. Secondary structure predictions suggest a bipartite structure, with a large N-terminal domain dominated by heptad repeats (HRs) typical for coiled-coil--forming proteins. Proposed functions for Tpr have included roles as a homo- or heteropolymeric architectural element of the nuclear interior. To gain insight into Tpr's ultrastructural properties, we have studied recombinant Tpr segments by circular dichroism spectroscopy, chemical cross-linking, and rotary shadowing electron microscopy. We show that polypeptides of the N-terminal domain homodimerize in vitro and represent alpha-helical molecules of extended rod-like shape. With the use of a yeast two-hybrid approach, arrangement of the coiled-coil is found to be in parallel and in register. To clarify whether Tpr can self-assemble further into homopolymeric filaments, the full-length protein and deletion mutants were overexpressed in human cells and then analyzed by confocal immunofluorescence microscopy, cell fractionation, and immuno-electron microscopy. Surplus Tpr, which does not bind to the NPC, remains in a soluble state of approximately 7.5 S and occasionally forms aggregates of entangled molecules but neither self-assembles into extended linear filaments nor stably binds to other intranuclear structures. Binding to the NPC is shown to depend on the integrity of individual HRs; amino acid substitutions within these HRs abrogate NPC binding and render the protein soluble but do not abolish Tpr's general ability to homodimerize. Possible contributions of Tpr to the structural organization of the nuclear periphery in somatic cells are discussed.

Animals↗
PubMed2006

The nuclear pore complex: the gateway to successful nonviral gene delivery.

One of the limiting steps in the efficiency of nonviral gene delivery is transport of genetic material across the nuclear membrane. Trafficking of nuclear proteins from the cytoplasm into the nucleus occurs via the nuclear pore complex and is mediated by nuclear localization signals and their nuclear receptors. Several strategies employing this transport mechanism have been designed and explored to improve nonviral gene delivery. In this article, we review the mechanism of nuclear import through the nuclear pore complex and the strategies used to facilitate nuclear import of exogenous DNA and improve gene expression.

Active Transport, Cell Nucleus↗
PubMed2003

Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded.

Nuclear transport proceeds through nuclear pore complexes (NPCs) that are embedded in the nuclear envelope of eukaryotic cells. The Saccharomyces cerevisiae NPC is comprised of 30 nucleoporins (Nups), 13 of which contain phenylalanine-glycine repeats (FG Nups) that bind karyopherins and facilitate the transport of karyopherin-cargo complexes. Here, we characterize the structural properties of S. cerevisiae FG Nups by using biophysical methods and predictive amino acid sequence analyses. We find that FG Nups, particularly the large FG repeat regions, exhibit structural characteristics typical of "natively unfolded" proteins (highly flexible proteins that lack ordered secondary structure). Furthermore, we use protease sensitivity assays to demonstrate that most FG Nups are disordered in situ within the NPCs of purified yeast nuclei. The conclusion that FG Nups constitute a family of natively unfolded proteins supports the hypothesis that the FG repeat regions of Nups form a meshwork of random coils at the NPC through which nuclear transport proceeds.

Active Transport, Cell Nucleus↗
PubMed2000

Mapping interactions between nuclear transport factors in living cells reveals pathways through the nuclear pore complex.

The interactions between transport receptors and proteins of the nuclear pore complex (NPC) are fundamental to understanding nucleocytoplasmic transport. In order to delineate the path that a particular transport receptor takes through the NPC, we have employed fluorescence resonance energy transfer (FRET) between enhanced cyan and yellow fluorescent proteins (ECFP, EYFP) in living cells. A panel of yeast strains expressing functional receptor--ECFP and nucleoporin--EYFP fusions has been analyzed with a FRET assay. With this approach, we define points of contact in the NPC for the related importin Pse1/Kap121 and exportin Msn5. These data demonstrate the utility of FRET in mapping dynamic protein interactions in a genetic system. Furthermore, the data indicate that an importin and exportin have overlapping pathways through the NPC.

Bacterial Proteins↗
PubMed2003

Asymmetric distribution of nuclear pore complexes and the cytoplasmic localization of beta2-tubulin mRNA in Chlamydomonas reinhardtii.

Although it is generally accepted that nuclear architecture is an important determinant of nuclear activity, it is not clear whether cytoplasmic events, such as transcript localization and cell polarity, are affected by this architecture. Characterization of the nuclear architecture of the single-cell alga Chlamydomonas reinhardtii revealed a polarized nucleus, with nuclear pore complexes preferentially concentrated at the posterior side of the nucleus. Nuclear asymmetry was greatly exaggerated during the upregulation of genes encoding flagellar proteins, when nuclear pore complexes (NPCs) were observed to hyperpolarize to the posterior side of the nucleus while heterochromatin polarized to the anterior side. Interestingly, prior to deflagellation, the beta2-tubulin gene was preferentially located in the posterior region of the nucleus, and following deflagellation, beta2-tubulin transcripts accumulated posteriorly in polysome-rich cytoplasmic regions adjacent to the highest concentration of NPCs, suggesting a connection between nuclear architecture and cytoplasmic transcript localization.

Animals↗
PubMed1997

The location of the transport gate in the nuclear pore complex.

Signal-mediated nuclear transport is a gated process that occurs through a central transporter element located within the pore complex. The purpose of this investigation was to identify the region of the transporter that functions as the gate; i.e. the region that restricts passive diffusion of macromolecules through the pores. To accomplish this, small gold particles coated with polyethylene glycol (PEG; total particle diameter 40-70 A) or large PEG-particles (total diameter 110-270 A) were microinjected into the cytoplasm or nucleoplasm of Xenopus oocytes. Since PEG does not contain either nuclear import or export signals, it is assumed that the particles distribute by simple diffusion. The cells were fixed after 5 or 30 minutes and subsequently examined using TEM. The distribution of the particles located adjacent to and within the pore complexes was then mapped. The results obtained at both 5 and 30 minutes after cytoplasmic injections of small gold were basically the same. The particles readily entered the transporter but, on the average, were approximately 11 times more concentrated in the cytoplasmic half of this structure. The opposite distribution was observed following nuclear injections, i.e. the particles that were located in the transporter were approximately 7 times more numerous in the nuclear half. Our data indicate that there is a single transport gate located in the central domain of the transporter that restricts passive diffusion. The large particles that were injected into the cytoplasm migrated to the surface of the pore complex, but entered the transporter less frequently than small gold. Interestingly, the diffusion of large PEG-particles to the surface of the pores following nuclear injection was greatly restricted; however, this was not the case for similar size particles that were coated with protein containing nuclear export signals (NES). The latter results suggest that the NES is not only required for translocation, but also for migration within the nucleoplasm.

Amino Acid Sequence↗
PubMed2006

Systematic deletion and mitotic localization of the nuclear pore complex proteins of Aspergillus nidulans.

To define the extent of the modification of the nuclear pore complex (NPC) during Aspergillus nidulans closed mitosis, a systematic analysis of nuclear transport genes has been completed. Thirty genes have been deleted defining 12 nonessential and 18 essential genes. Several of the nonessential deletions caused conditional phenotypes and self-sterility, whereas deletion of some essential genes caused defects in nuclear structure. Live cell imaging of endogenously tagged NPC proteins (Nups) revealed that during mitosis 14 predicted peripheral Nups, including all FG repeat Nups, disperse throughout the cell. A core mitotic NPC structure consisting of membrane Nups, all components of the An-Nup84 subcomplex, An-Nup170, and surprisingly, An-Gle1 remained throughout mitosis. We propose this minimal mitotic NPC core provides a conduit across the nuclear envelope and acts as a scaffold to which dispersed Nups return during mitotic exit. Further, unlike other dispersed Nups, An-Nup2 locates exclusively to mitotic chromatin, suggesting it may have a novel mitotic role in addition to its nuclear transport functions. Importantly, its deletion causes lethality and defects in DNA segregation. This work defines the dramatic changes in NPC composition during A. nidulans mitosis and provides insight into how NPC disassembly may be integrated with mitosis.

Active Transport, Cell Nucleus↗
PubMed1994

Nup107 is a novel nuclear pore complex protein that contains a leucine zipper.

We have previously described procedures for the isolation of potential nuclear pore complex proteins (nucleoporins or Nups) from rat liver nuclear envelopes and their subsequent subfractionation into those binding to wheat germ agglutinin (WGA) and those that do not. One of these non-WGA-reactive proteins, termed Nup155, was previously molecularly cloned and sequenced and by immunoelectron microscopy shown to be a bona fide nucleoporin. Here we have characterized a second protein of the non-WGA-reactive type and show that it is a Nup as well. Molecular cloning and sequencing revealed that the protein has a calculated molecular mass of 107.2 kDa. It is, therefore, termed Nup107 (for nucleoporin of 107 kDa). Like other Nups that have so far been analyzed, Nup107 contains an abundance of kinase consensus sites. However, the primary structure of Nup107 is unique and not similar to other proteins in the data banks. Its most striking feature is a leucine zipper in its carboxyl-terminal region.

Amino Acid Sequence↗
PubMed2001

Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells.

The nuclear pore complex (NPC) and its relationship to the nuclear envelope (NE) was characterized in living cells using POM121-green fluorescent protein (GFP) and GFP-Nup153, and GFP-lamin B1. No independent movement of single pore complexes was found within the plane of the NE in interphase. Only large arrays of NPCs moved slowly and synchronously during global changes in nuclear shape, strongly suggesting mechanical connections which form an NPC network. The nuclear lamina exhibited identical movements. NPC turnover measured by fluorescence recovery after photobleaching of POM121 was less than once per cell cycle. Nup153 association with NPCs was dynamic and turnover of this nucleoporin was three orders of magnitude faster. Overexpression of both nucleoporins induced the formation of annulate lamellae (AL) in the endoplasmic reticulum (ER). Turnover of AL pore complexes was much higher than in the NE (once every 2.5 min). During mitosis, POM121 and Nup153 were completely dispersed and mobile in the ER (POM121) or cytosol (Nup153) in metaphase, and rapidly redistributed to an immobilized pool around chromatin in late anaphase. Assembly and immobilization of both nucleoporins occurred before detectable recruitment of lamin B1, which is thus unlikely to mediate initiation of NPC assembly at the end of mitosis.

Animals↗
PubMed2002

Is the central channel of nuclear pore complex really the exclusive gateway for macromolecular transport?

Examination of many nuclear pore complexes revealed in some of them very thin filaments presumably RNA with accompanying proteins, directing from inner nucleus to the edge of the complex. On one representative micrograph it is shown that this strand continues in the same direction through central part of the complex most probably through the peripheral channel. Next route of the strand is through the tunnel of a hollow rod - subunit of the cytoplasmic ring.

Animals↗