Polio Cripples Nuclear Import

Nuclear transport pathways use soluble receptors to mediate the translocation of protein and cargo through the nuclear pore complex (NPC). In October 2005, investigators from around the globe braved the threat of hurricane Wilma and presented their latest findings at the Nucleocytoplasmic Transport Meeting at Jekyll Island, GA, USA. The meeting was organized by Drs Maureen Powers, Chuck Cole, and Douglass Forbes. Major themes within the meeting included architecture of the NPC, structural analysis of receptors, regulation of nuclear transport, models for translocation, and emerging roles for the NPC and lamina in gene expression. The transport of proteins and RNAs between the cytoplasm and nucleus is mediated by nuclear pore complexes (NPCs), large proteinaceous structures embedded in the double membrane of the nuclear envelope (NE) (1–4). The NPC structure is conserved in all eukaryotes and is composed of approximately 30 separate proteins termed nucleoporins (Nups), most of which are present in multiple copies per NPC. Nups provide docking sites for soluble transport receptors that mediate the translocation of protein and RNA cargoes through the NPC. The karyopherin family is composed of receptors for import (importins) and export (exportins). These receptors mediate most protein import and export pathways in the cell, as well as the export of some RNAs. Transport receptors recognize specific sequences termed nuclear localization signals (NLSs) or nuclear export signals (NESs) contained in the cargo protein. mRNAs are exported by a class of transport receptors that are distinct from the karyopherin family. An important regulator of nuclear transport is the small GTPase Ran, and its GTPase activating protein (RanGAP) and guanine nucleotide exchange factor (RanGEF or RCC1). Previous models of the NPC have been based primarily on low-resolution cryo-electron microscopy (EM) studies and the immuno-EM localization of a handful of NPC proteins (Figure 1A) (4). More recently, proteomic approaches have established the identities of virtually all the protein constituents of the NPC, setting the stage for a more complete picture of the NPC (5,6). Michael Rout (The Rockefeller University, New York, NY, USA) and colleagues used a computational approach to reconstruct the arrangement of 30 different Nups in the NPC. The model combined EM immunolocalization data with protein–protein interaction data obtained by immunoprecipitation of tagged Nups. This exciting model gives us the first glimpse of an integrated view of the locations of individual Nups and various subcomplexes of the NPC. Of course, solving the atomic structure of the NPC is one of the ‘holy grails’ of the field, and Thomas Schwartz (Massachusetts Institute of Technology, Boston, MA, USA) updated the meeting on his progress toward solving the structure of the p62 complex, a subcomplex located in the central channel of the NPC. Given its enormous size (>100 MDa), it might be necessary to piece together subcomplexes to solve the complete NPC structure. Antibody studies have allowed the localization of a small number of Nups that are asymmetrically positioned on either the nuclear or cytoplasmic sides of the NPC, and Nups with biased locations are good candidates for specialized transport functions. Two nucleoplasmic Nups in vertebrates are the large nucleoporins Nup153 and TPR. Volker Cordes (ZMBH, Heidelberg, Germany) used domain-specific antibodies to analyze the structural arrangement of TPR at the NPC by scanning and transmission EM. He found that different TPR domains correspond to distinct segments of the nuclear basket, with TPR homodimers forming a central loop to contact Nup153 at the NPC nuclear ring, while both N- and C-terminus of TPR project toward the nucleoplasm. Volker's antibody data provide strong evidence that TPR is the key component of the nuclear basket. Gallery of images illustrating some diverse aspects of nuclear structure and function.(A) NPCs on the surface of the NE membrane of Xenopus oocytes visualized by field emission scanning electron microscopy. Cytoplasmic filaments of the NPC appear as globular structures that line the perimeter of the pore. The image is courtesy of Dr Terry Allen. (B) Mitotic embryo fibroblast from a rae1+/–nup98+/– mouse imaged during anaphase B. While most of the chromosomes (green) have moved to the spindle poles, lagging chromosomes (red arrow) are visible and give rise to aneuploidy. Image is courtesy of Dr Jan van Duersen. (C) Model of transmembrane linkages between proteins in the cytoplasm and nucleus. Nesprins bind actin filaments in the cytoplasm and contact SUN domain proteins in the NE lumen. SUN domain proteins also contact lamins; these interactions are thought to provide a continuous linkage between the cytoskeleton and the nucleoskeleton. The image is courtesy of Dr Brian Burke and is reproduced from Crisp et al. (26) with permission from Rockefeller University Press. A subset of Nups contain repeats of the amino acids phenylalanine-glycine, and many studies have established that these FG repeats are binding sites for the karyopherins moving through the central channel of the NPC (1,2,7). It is estimated that the central channel of the NPC contains several hundred FG repeats. There is much debate within the field as to what this channel looks like and how it mediates a selective gating mechanism. Models include a central tube lined with FG repeats that acts as an affinity gate (also called virtual gating), or a sieve-like channel where disordered FG repeats mediate hydrophobic interactions between Nups creating a gel-like phase that generates a permeability barrier within the NPC (also called the selective phase model) (1,2). Michael Rexach (University of California, Santa Cruz, CA, USA) used a new bead-based, real-time equilibrium-binding assay to test for low-affinity interactions between FG regions of Nups. Binding required phenylalanine residues in FG motifs and was disrupted by hexanediol, an alcohol known to weaken the NPC permeability barrier in transport assays. He concluded that FG regions of Nups interact with each other, supporting the ‘selective phase’ model for NPC gating. Dirk Gorlich (ZMBH, Heidelberg, Germany) reported on efforts by his group to address the properties of the central channel envisioned to be important for the selective phase model. He surprised the meeting by showing that a single FG nucleoporin, Nsp1p, can assemble into a three-dimensional ‘hydrogel’ with molecular sieve-like properties. Dirk also showed that a mutant version of Nsp1p where the >50 FG repeats had been mutated fails to form the ‘hydrogel’. These results were consistent with the idea that interactions between hydrophobic repeats in Nups could play structural and functional roles in the permeability barrier. Future experiments will determine whether this ‘hydrogel’ displays other properties of the central channel and help address longstanding questions regarding the central channel, including how gating of the NPC is achieved. Earlier studies by several groups have indicated that different karyopherins display distinct binding specificities for FG Nups in vitro. An extensive collection of FG-domain deletion strains created by the Wente laboratory is being used to probe the interactions between different karyopherins and the central channel in Saccharomyces cerevisiae (8). It has been shown that Kap104p-mediated translocation is more sensitive than Kap95p/Kap60p-mediated translocation to certain FG-domain deletions (8). David Goldfarb (University of Rochester, Rochester, NY, USA) presented recent experiments that are most consistent with the notion that Kap95p/Kap60p does better than Kap104p in these FG-domain deletion strains, because it competes better for remaining FG-binding sites. This advantage may normally occur under stress, because elevated levels of Hsp70 favor the translocation of Kap95p/Kap60p over Kap104p. The karyopherin family comprises the major family of transport receptors. In yeast, there are 14 members of this family, and in human cells, there are more than 20 members (1,2). Although the amino acid sequence identities are low, each Kap has similar large size (approximately 100 kDa) and are all predicted to consist of multiple (approximately 20) HEAT motifs. The HEAT motif is a helix-loop-helix structure that owes its name to the proteins in which it was originally identified (9). Kaps contain an N-terminal domain that interacts with RanGTP and nucleoporins. Kaps also bind directly (or via adapters) to the NLSs or NESs of their cargoes. The crystal structures of several karyopherins, empty, with cargo, and with RanGTP, have all been solved. At the meeting, several groups focused on further analysis of karyopherins at the atomic level. The overall theme in these talks was that karyopherins have great conformational flexibility and that members of this family can have different conformations despite the fact that they all are composed of tandem HEAT repeats. Murray Stewart (Medical Research Council, Cambridge, UK) discussed the crystal structure of Kap95p. He proposed that this importin is flexible when binding to cargoes, but the interaction with RanGTP, which precludes cargo binding, introduces inflexibility into the protein. Murray continued this theme in discussing his structure of the Cse1p, the receptor that mediates nuclear export of importin-α (10). Elena Conti's laboratory (EMBL, Heidelberg, Germany) has solved the structure of unliganded Cse1p and found it to form a relatively rigid ring-like structure. In this closed conformation, the binding sites for either importin-α or RanGTP are distorted. Indeed, Cse1p binds either importin-α or Ran with micromolar affinity, while the simultaneous interaction of Cse1p with importin-α and RanGTP occurs with nanomolar affinity and is correlated with the open conformation of Cse1p observed in Murray's structure. This conformational regulation provides a rationale for why Cse1p fails to interact with importin-α in the cytosol, in the absence of RanGTP. Reporting on a low-resolution study of conformations of different transport factors, Elena dismissed the idea of a general importin-like or exportin-like structure. For example, the exportin Xpo-t is more similar in overall structure to importin-β than it is to the exportin Cse1 (11). It appears that the most common feature of karyopherins is their flexibility in acquiring distinct conformations depending on whether they are empty or interacting with RanGTP or cargo. Conformational differences were to be expected due to acidic loops and insertions found between HEAT repeats in assorted family members. Lucy Pemberton (University of Virginia, Charlottesville, VA, USA) reported that a single karyopherin, Kap114p, can bind multiple cargoes simultaneously in vitro, and hopefully future structures will address how these 7interactions take place at the atomic level. Collectively, these studies suggest that we still have a lot to learn about the complexity of karyopherin–cargo interactions. There was a very lively session describing efforts to analyze the mechanism of nuclear transport at the single-molecule level, as well as efforts to model nuclear transport. Although much is known about individual components of the transport machinery, and many important binding interactions have been characterized, tracking individual cargoes as they transit through the NPC should provide clues about the transport mechanism. Ulrich Kubitschek (University of Bonn, Bonn, Germany) used single-molecule far-field fluorescence microscopy to visualize nuclear transport through NPCs of both permeabilized and living cells. Intracellular single-molecule detection in the nanometer range is feasible using this technique. Binding of cargo reduced the dwell times of the transport receptors in the NPC, suggesting that cargo has an important influence on karyopherin interactions with Nups. Seigfried Musser (Texas A&M, College Station, TX, USA) presented his measurements of import efficiency, which is the number of molecules that are transported divided by the number of molecules that interact with an NPC. In the permeabilized cell system, he found that both import efficiency and translocation time were dependent on the concentration of the importin-β, as import efficiency was higher and the translocation time was faster with increasing importin-β concentrations. Seigfried speculated that the cargo-free importin-β concentration within the NPC may regulate nucleocytoplasmic transport rates. Ruti Kapon (Weizmann Institute of Science, Rehovot, Israel) examined receptor-mediated import through the NPC using a non-equilibrium statistical mechanics model where all reactions are modeled using kinetic rates. Her model suggested that the selectivity of the channel against receptor-free cargo, in itself, leads to accumulation of the cargo in the destination compartment even if the translocation process itself is unbiased. A major question in the field is how the NPC coordinates import and export through the same channel. Ruti suggested that synchronization of import and export is based on stochastic fluctuations in the NPC. She posited the NPC is unidirectional for a characteristic time, but it can spontaneously change its directionality. Thus, for given periods, an individual NPC can be dedicated to either import or export. Experimental analysis is required to establish the validity of these models. Ian Macara (University of Virginia, Charlottesville, VA, USA) presented his ongoing studies that combine computer simulation and microinjection assays to monitor fluorescent cargoes and recombinant receptors in real time. Ian discussed his previous observation that in living cells, high levels of importin-β or RanGEF inhibit import of an NLS-cargo (12). Excess RanGEF probably sequesters Ran in the nucleus, whereas excess importin-β may deplete Ran from the nucleus by co-opting it for futile nucleoplasmic shuttling cycles. In his new studies, Ian used a shuttling NES-NLS cargo that can reach steady-state localization in the cell. Excess importin-β and Crm1 decreased the N:C ratio of the reporter, whereas importin-α increased the N:C ratio. Components of the Ran system had little effect most likely because the reporter reflected both import and export function. There was an interesting discussion regarding how importin-α-dependent transport is less efficient and energetically more costly given the need to recycle importin-α to the cytoplasm, but importin-α proteins endow the cell with greater flexibility in terms of binding diverse cargoes (13). Nuclear export of mRNA is mediated principally by Mex67p/TAP/NXF1, a highly conserved receptor family that is unrelated to the karyopherin family of import and export receptors (14). Functional coupling between mRNA export and upstream reactions (transcription and mRNA processing) is mediated by several multiprotein complexes. The TREX complex consists of proteins involved in transcription elongation and mRNA export and includes the THO protein complex as well as the mRNA export factors Yra1p/Aly and Sub2p/Uap56 (14). There were several talks that addressed the links between mRNA transcription, processing and nuclear export. Andres Aguilera (University of Seville, Spain) showed that the THO protein complex functions in co-transcriptional mRNP formation, and he described a new component of the THO complex, Tho1p. Tho1p is a conserved RNA-binding protein that interacts with transcribed chromatin in a THO- and RNA-dependent manner. These results suggested that Tho1p, similar to Sub2p, assembles onto nascent mRNA during transcription and participates in mRNP biogenesis and nuclear export. Nuclear export of mRNA is also coordinated with mRNA splicing and 5′ capping. Robin Reed (Harvard Medical School, Boston, MA, USA) showed that the human TREX complex is recruited to the 5′-end of mRNA in a splicing- and cap-dependent manner. The TREX complex binds to the 5′-methyl cap-binding protein, CBP80, an interaction that provides a plausible mechanism for TREX recruitment to the 5′-end of mRNA. The findings presented by Robin suggest a model of mRNA export that differs from models from Melissa Moore's laboratory, which were based on coupling the export machinery to the exon junction complex (15). Francoise Stutz (University of Geneva, Geneva, Switzerland) continued the theme of coupled reactions by describing links between the RNA export factor Yra1p/Aly, and Mlp1p and Mlp2p, yeast homologs of TPR that face the nucleoplasm. Previously, Francoise identified genetic interactions between the MLP1/2 and YRA1 that suggested that the Mlp1/2p proteins provide a scaffold at the NPC for Yra1p-dependent docking of mRNPs (16). Yra1p appears to directly regulate transcription, as recruitment of TBP to an induced promoter is decreased in a yra1 mutant. Francoise found that this effect could be reversed in a yra1/mlp2 double mutant, suggesting that Mlp1/2p negatively regulates the transcriptional function of Yra1p. Collectively, these talks emphasized the close links between nuclear export, transcription and RNA processing and suggested that the NPC may regulate transcription by several mechanisms. John Aitchison (Institute for Systems Biology, Seattle, WA, USA) focused on the role of nucleoporin Nup2p, which displays dynamic associations with the NPC. John investigated the previous observation that Nup2p has boundary activity when tethered to chromatin (17). John and colleagues used a variety of assays, including chromatin immunoprecipitation and gene arrays, to show that Nup2p regulates gene expression and gene silencing. Another component of the nuclear transport machinery, the RanGEF Prp20p, was found to have similar chromatin-related functions. The human homolog of Prp20p is known to interact with core histones, and as Nup2p interacts with both the NPC and Prp20p, these data argue for linkage between the NPC and chromatin. As the association of Nup2p with the NPC is transient, the data suggest that the NPC might play an anchoring role and help direct the partitioning of chromatin into inactive and active regions. The mRNA export events downstream of TREX and TAP/Nxf1/Mex67p are poorly defined. Some exported hnRNPs are enormous, suggesting that this must be an energy-requiring process. RanGTP does not appear to regulate this process, suggesting that other important regulatory mechanisms might exist. Three talks discussed Dbp5p, an RNA helicase that plays a role in mRNA export and transiently associates with the cytoplasmic side of the NPC. Karsten Weis (University of California, Berkeley, CA, USA) showed that the DExD/H-box protein Dbp5p has intrinsically low ATPase activity and is unable to associate stably with RNA in the absence of ATP, suggesting that Dbp5p alone is an inactive enzyme. He also showed that the NPC-associated protein Gle1p the ATPase activity of Dbp5p and presented a model in which these proteins as a complex to mRNA export. Wente University, USA) reported a similar that Gle1p and Dbp5p but in was to show that the the activity of Dbp5p by in the of the This is interesting in of analysis of the role of in nuclear transport, where showed that cytoplasmic is required for mRNA export Collectively, these findings to how small molecules may regulate mRNA export. Chuck Medical School, USA) focused on the role of Dbp5p in mRNA processing at distinct from export through the NPC. mRNA is increased in a mutant, and a of Dbp5p with He described new between Dbp5p and which are cytoplasmic structures that are to be necessary for mRNA and these data the model that Dbp5p may play important roles in mRNA export and processing both at the NPC and in the factors that play important roles in mRNA export have been and (University of California, CA, USA) described analysis of the yeast mRNA export factor This protein is an membrane protein that to the Although is not thought to be a of the NPC, it interacts with several which is consistent with a role in transport. She showed that interacts with mRNPs in and is required for the NE localization of the mRNA export factor She proposed a model where this membrane protein provides binding sites to the NPC to help mRNPs and in the for export. these studies suggest that mRNA export is a process a of factors that appear to regulate this process from transcription to talks addressed nucleocytoplasmic transport of components required for protein Medical USA) discussed the import and export of which are one of the most of In previous showed that in the nucleus in the exportin mutant can be into the nucleus in a mechanism that is dependent on Ran and the importin that of might be of an mRNA or it could be of a mechanism for given that in the nucleus in to (University of TX, USA) focused on nuclear export of the which on as an for export. of the from the cytoplasmic GTPase and the protein In in the nucleus, and accumulation in the These suggest a mechanism of the export complex can occur in the focused on the nuclear export and cytoplasmic processing of RNA component of the He showed that processing of the of the in occurs in the cytoplasm nuclear export. This is a of models that show that processing is in the nucleus. This is necessary for the activity of the in and might be important for interaction with in the nucleus. consist of core proteins and an RNA and are for biogenesis and mRNA It is not known how these but University, New York, NY, USA) the role of the protein in the process. between nucleus and cytoplasm and one of the core to the nascent is by core protein, to in and transcription factors of import and export as a of cytoplasmic signals with nuclear import export can have a influence on the nuclear concentration of transcription factors The receptor and (University of Virginia, Charlottesville, VA, USA) presented showing that export is by of a to the export (University of Germany) described his molecular analysis of nuclear export of nuclear factor of of nucleoporins or export of RNA export or protein and are on the cytoplasmic side of the NPC these Nups may be the sites where export complexes are and into the nucleoporins a that has on a number of transcription factors and The of the that this has the stage for how regulates protein USA) reported that reduced levels of in the in the nuclear transport of a transcription factor that is a downstream of transcription factors to on nuclear import mediated by karyopherins, but the of the protein can an that direct interaction with John USA) presented findings from and yeast that a model nuclear localization of high group transcription factors as than the observation that nuclear transport can be correlated with the in cells, the of whether there are in the nuclear transport machinery during in is University, used in and to show that there are in the expression of importin-α and importin-β receptors during This that nuclear transport might be at the of receptor during links between cell the import receptor and the RNA-binding protein were reported by University, of the import was shown to accumulation in the cytoplasm of in As can bind and factors and the effect of cytoplasmic was to and protein cell The study of and their regulatory mechanisms has many aspects of cell and the field of nuclear transport has been of the first export signals to be is by the human protein and to be a cargo for cell nuclear export. an RNA export function that is for the it is a David (University of Virginia, Charlottesville, VA, USA) presented his new results from a to of the small molecules in the display micromolar of function in cells. David an that to one of the small of that function can be identified in this of of regulation in has to a based on signals that nuclear export of RNA to the The transport in which this is by the export receptor The is to be to higher but the for RNA export signals in mRNAs has been by the in these signals at the sequence level. have for the of RNA export (University of Virginia, Charlottesville, VA, USA) used a system to for and identified in the human The have sequence to the with one the for a which that mediates nuclear export of its mRNA. In previous USA) had identified a nucleotide RNA transport and showed that its activity is to the from used a to in in the mouse RNA export, like RNA export, on the appears to contact with the structural between and should provide into the important of of mRNAs by direct mechanisms. Two talks presented at the meeting addressed how to the nucleus. In the of proteins are and the is through the NPC into the cell nucleus. (The Research CA, USA) has to the import mechanism and has identified a key role for protein which contains NLSs and the interaction between and cell import

Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231–239, 1998.© 1998 Wiley-Liss, Inc.

383 Preclinical antitumor activity of second generation analogs of SDX-101

The nuclear pore complex (NPC) mediates communication between the cytoplasm and nucleus in eukaryotic cells. Active transport of large polypeptides as well as passive diffusion of smaller (≈10 kD) macromolecules through the NPC can be inhibited by depletion of intracellular Ca2+ stores. However, the physiological relevance of this process for the regulation of nucleocytoplasmic trafficking is not yet clear. We expressed green fluorescent protein (GFP)–tagged glucocorticoid receptor (GR) and mitogen-activated protein (MAP) kinase–activated protein kinase 2 (MK2) to study the effect of Ca2+ store depletion on active transport in HM1 cells, a human embryonic kidney cell line stably transfected with the muscarinic M1 receptor. Dexamethasone-induced nuclear import of GR-GFP and anisomycin-induced nuclear export of GFP-MK2 was monitored by confocal microscopy. We found that store depletion by carbachol, thapsigargin or ionomycin had no effect on GR-GFP import, whereas pretreatment with 1,2-bis-(o-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid–acetoxymethyl ester (BAPTA-AM) attenuated import significantly. Export of GFP-MK2 was not influenced by any pretreatment. Moreover, carbachol stimulated GFP-MK2 translocation to the cytoplasm in the absence of anisomycin. These results demonstrate that Ca2+ store depletion in intact HM1 cells is not directly linked to the inhibition of active protein transport through the NPC. The inhibition of GR-GFP import but not GFP-MK2 export by BAPTA-AM presumably involves a depletion-independent mechanism that interferes with components of the nuclear import pathway.

Nup1p is a yeast nuclear pore complex protein (nucleoporin) required for nuclear protein import, mRNA export and maintenance of normal nuclear architecture. We have used a genetic approach to identify other proteins that interact functionally with Nup1p. Here we describe the isolation of seventeen mutants that confer a requirement for Nup1p in a background in which this protein is normally not essential. Some of the mutants require wild-type Nup1p, while others are viable in combination with specific nup1 alleles. Several of the mutants show nonallelic noncomplementation, suggesting that the products may be part of a hetero-oligomeric complex. One is allelic to srp1 which, although it was identified in an unrelated screen, was shown to encode a protein that is localized to the nuclear envelope (Yano, R., M. Oakes, M. Yamaghishi, J. A. Dodd, and M. Nomura. 1992. Mol. Cell. Biol. 12:5640- 5651). We have used immunoprecipitation and fusion protein precipitation to show that Srp1p forms distinct complexes with both Nup1p and the related nucleoporin Nup2p, indicating that Srp1p is a component of the nuclear pore complex. The distant sequence similarity between Srp1p and the beta-catenin/desmoplakin family, coupled with the altered structure of the nuclear envelope in nup1 mutants, suggests that Srp1p may function in attachment of the nuclear pore complex to an underlying nuclear skeleton.

We investigated the nuclear import mechanism of Cdc7, which is essential for the initiation of DNA replication. Here we report that importin-beta binds directly to Cdc7 via the Kinase Insert II domain, promoting its nuclear import. Although both importin-alpha and -beta bind to Cdc7 via the Kinase Insert II domain in a mutually independent manner, the binding affinity of Cdc7 for importin-beta is approximately 10 times higher than for importin-alpha at low protein concentrations of an equimolar ratio. Immunodepletion of importin-beta, but not importin-alpha, abrogates Cdc7 nuclear import, and the addition of importin-beta to the importin-depleted cytosol restores Cdc7 nuclear import. Furthermore, transduction of anti-importin-beta, but not anti-importin-alpha antibodies, into live cells inhibits Cdc7 nuclear import. Unexpectedly, we found that Cdc7 nuclear import is inhibited by competitive binding of importin-alpha to Cdc7. Further studies by site-directed mutagenesis suggest that Lys306 and Lys309 within the Kinase Insert II domain are critical for Cdc7 nuclear localization.

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.

The Ran GTPase activating protein RanGAP1 plays an essential role in nuclear transport by stimulating RanGTP hydrolysis in the cytoplasmic compartment. In mammalian cells, unmodified RanGAP1 is predominantly cytoplasmic, whereas modification by small ubiquitin-related modifier protein (SUMO) targets RanGAP1 to the cytoplasmic filaments of nuclear pore complex (NPC). Although RanGAP1 contains nine putative nuclear export signals and a nuclear localization signal, little is known if RanGAP1 shuttles between the nuclear and cytoplasmic compartments and how its primary localization in the cytoplasm and at the NPC is regulated. Here we show that inhibition of CRM1-mediated nuclear export using RNAi-knockdown of CRM1 and inactivation of CRM1 by leptomycin B (LMB) results in nuclear accumulation of RanGAP1. LMB treatment induced a more robust redistribution of RanGAP1 from the cytoplasm to the nucleoplasm compared to CRM1 RNAi and also uniquely triggered a decrease or loss of RanGAP1 localization at the NPC, suggesting that LMB treatment is more effective in inhibiting CRM1-mediated nuclear export of RanGAP1. Our time-course analysis of LMB treatment reveals that the NPC-associated RanGAP1 is much more slowly redistributed to the nucleoplasm than the cytoplasmic RanGAP1. Furthermore, LMB-induced nuclear accumulation of RanGAP1 is positively correlated with an increase in levels of SUMO-modified RanGAP1, suggesting that SUMOylation of RanGAP1 may mainly take place in the nucleoplasm. Lastly, we demonstrate that the nuclear localization signal at the C-terminus of RanGAP1 is required for its nuclear accumulation in cells treated with LMB. Taken together, our results elucidate that RanGAP1 is actively transported between the nuclear and cytoplasmic compartments, and that the cytoplasmic and NPC localization of RanGAP1 is dependent on CRM1-mediated nuclear export.

Nuclear transport of proteins and RNA occurs through the nuclear pore complex and is mediated by a superfamily of transport receptors known collectively as karyopherins. Karyopherins bind to their cargoes by recognition of specific nuclear localization signals or nuclear export signals. Transport through the nuclear pore complex is facilitated by transient interactions between the karyopherins and the nuclear pore complex. The interactions of karyopherins with their cargoes are regulated by the Ras-related GTPase Ran. Ran is assisted in this process by proteins that regulate its GTPase cycle and subcellular localization. In this review, we describe several of the major transport pathways that are conserved in higher and lower eukaryotes, with particular emphasis on the role of Ran. We highlight the latest advances in the structure and function of transport receptors and discuss recent examples of steroid hormone receptor import and regulation by signal transduction pathways. Understanding the molecular basis of nuclear transport may provide insight into human diseases by revealing how nucleocytoplasmic trafficking regulates protein activity.

Nuclear pore complexes (NPCs) are multiprotein aqueous channels that penetrate the nuclear envelope connecting the nucleus and the cytoplasm. NPCs consist of multiple copies of roughly 30 different proteins known as nucleoporins (NUPs). Due to their essential role in controlling nucleocytoplasmic transport, NPCs have traditionally been considered as structures of ubiquitous composition. The overall structure of the NPC is indeed conserved in all cells, but new evidence suggests that the protein composition of NPCs varies among cell types and tissues. Moreover, mutations in various nucleoporins result in tissue-specific diseases. These findings point towards a heterogeneity in NPC composition and function. This unexpected heterogeneity suggests that cells use a combination of different nucleoporins to assemble NPCs with distinct properties and specialized functions.

Nuclear proteins like transcription factors and ribosomal proteins are synthesized in the cytoplasm and have to be transported into the nucleus to fulfill their functions. The transport of proteins >20–60 kD through the nuclear pore complex (NPC) into the nucleus is an active, energy-requiring process. Transport substrates are recognized by their transport proteins via certain signals. The best-characterized protein import pathway is the ‘classical’ nuclear localization signal-dependent pathway with importin α and β carrying the substrate to the NPC. The transport of the importin-substrate complex into the nucleus is regulated by the small GTPase Ran/TC4. During the last years more than ten proteins have been discovered which have already been proven or are very likely to be nuclear transport factors of distinct import pathways: members of the importin α protein family are very similar and transport in complex with importin β nuclear localization signal-bearing proteins into the nucleus. Members of the Ran-binding protein family show some weak similarity to importin β. Sharing a common domain at the amino terminus, they are able to bind RanGTP, a prerequisite for their function as nuclear import or export factors for distinct proteins or RNAs. However, Ran/TC4 seems to play a key regulatory role in all nuclear transport pathways described so far, although the molecular mechanism of the translocation step through the NPC is still unclear.

Nuclear lamins and nuclear pore complexes (NPCs) are major components of the nuclear envelope in metazoan cells. The objectives of this thesis were first to study interactions between nuclear lamins and the nuclear pore protein Nup153 (Chapters 2 and 3) and second to determine lamin assembly conditions of the Xenopus oocytes LIII in vitro (Chapter 4). Nuclear lamins are major constituents of the nuclear lamina underlying the nuclear periphery along with inner nuclear membrane proteins. The nuclear lamina provides stability and determines the nuclear architecture and spacing of the NPCs. NPCs form supramolecular assemblies that regulate nucleocytoplasmic transport. An overview of the functional aspects associated with the nuclear lamina and NPCs in health and disease is provided in Chapter 1. In depth analysis of the interaction of nuclear lamins with the nucleoporin Nup153 is revealed in Chapters 2 and 3. Using in vitro solution binding assays as well as immunoprecipitation assays, in chapter 2 we show direct associations between Nup153 and nuclear lamins. This work is explored even further in chapter 3 using binding assays and immunofluorescence microscopy as well as immunoprecipitation assays; we examined the interaction in the presence of lamin related mutations. Finally, in chapter 4 we established buffer conditions for LIII assembly in vitro, analysed by electron microscopy (EM) using glycerol spraying/low-angle rotary metal shadowing and negative staining. Our results presented in this thesis contribute to expand our current knowledge of the interactions of the NPCs with the nuclear lamins, as well as to increase our understanding of the impact of mutations in lamins that can cause laminopathies. In addition, the studies on the assembly conditions of LIII provide a vehicle for further characterisation of the influence of binding partners and the importance of lamin sub-fragments on the formation of higher order assemblies.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.

The nuclear pore complex (NPC) is a huge macromolecular assembly that selectively regulates the exchange of cargo between the nuclear and the cytoplasmic compartment in a cell. The transport of cargo is mediated by nuclear transport receptors (NTR’s), such as Karyopherin, via interacting with NPC proteins (Nucleoporins or Nups) containing highly disordered phenylalanine-glycine (FG) domains. This thesis is an investigation into the structure – function relationship of FG domains, which are located within the nuclear pore complex (NPC) and thus are fundamental to the NPC gating mechanism. Surface plasmon resonance (SPR) is used to measure Karyopherin – FG domain binding affinity and kinetics in vitro near physiological NTR concentrations. To contribute structural information in situ a novel SPR based method is applied that uses non-interacting molecules as innate structural probes. A major part of the thesis is thus dedicated to the proof of concept of using non-interacting molecules to determine the height of surface tethered molecular brushes. The conformation of close-packed surface tethered FG domains reveals molecular brush like extension and is directly impacted by Karyopherin binding. The findings predict that unlike FG centric models NTR’s form a key component of the NPC and NTR-occupancy controls selectivity and speed of nucleo-cytoplasmic transport. This has the important implication that NTR – FG domain binding avidity due to multivalent interactions does not hinder fast transport per se.

The movement of macromolecules from the cytoplasm into the nucleus occurs through nuclear pore complexes (NPCs) in the nuclear envelope. NPCs are large organelles that bridge the double membrane of the nuclear envelope forming aqueous channels that connect the cytoplasm and nucleoplasm. The aqueous channels of NPCs are sufficiently large to permit the diffusion of small macromolecules from one compartment to the other, but most nuclear proteins contain a nuclear localization sequence (NLS) and are actively imported into the nucleus. NLSs are recognized by soluble nuclear carriers in the cytoplasm which, after binding their cargo, dock at the NPC. Import cargo:carrier complexes then move through the NPC in an energy-independent fashion by sequential binding to and release from multiple carrier binding sites located throughout the length of the NPC. In this way, the import cargo is moved from the cytoplasmic to the nucleoplasmic face of the NPC. There, the small GTPase Ran disrupts the cargo:carrier complex resulting in the release of cargo into the nuclear interior. This review will focus on the mechanism by which Ran catalyzes cargo release during nuclear import.