Karyopherin binding interactions and nuclear import mechanism of nuclear pore complex protein Tpr
© Ben-Efraim et al; licensee BioMed Central Ltd. 2009
Received: 1 June 2009
Accepted: 16 October 2009
Published: 16 October 2009
Tpr is a large protein with an extended coiled-coil domain that is localized within the nuclear basket of the nuclear pore complex. Previous studies  involving antibody microinjection into mammalian cells suggested a role for Tpr in nuclear export of proteins via the CRM1 export receptor. In addition, Tpr was found to co-immunoprecipitate with importins α and β from Xenopus laevis egg extracts , although the function of this is unresolved. Yeast Mlp1p and Mlp2p, which are homologous to vertebrate Tpr, have been implicated in mRNA surveillance to retain unspliced mRNAs in the nucleus[3, 4]. To augment an understanding of the role of Tpr in nucleocytoplasmic trafficking, we explored the interactions of recombinant Tpr with the karyopherins CRM1, importin β and importin α by solid phase binding assays. We also investigated the conditions required for nuclear import of Tpr using an in vitro assay.
We found that Tpr binds strongly and specifically to importin α, importin β, and a CRM1 containing trimeric export complex, and that the binding sites for importins α and β are distinct. We also determined that the nuclear import of Tpr is dependent on cytosolic factors and energy and is efficiently mediated by the importin α/β import pathway.
Based on the binding and nuclear import assays, we propose that Tpr is imported into the nucleus by the importin α/β heterodimer. In addition, we suggest that Tpr can serve as a nucleoporin binding site for importin β during import of importin β cargo complexes and/or importin β recycling. Our finding that Tpr bound preferentially to CRM1 in an export complex strengthens the notion that Tpr is involved in protein export.
Molecules are transported between the cytoplasm and the nucleus through nuclear pore complexes (NPCs), massive proteinaceous structures that span the double membrane of the nuclear envelope (NE). Molecules smaller than ~20-40 kDa in size can passively diffuse through the NPC. However most protein, and nucleic acid is transported by receptor and energy dependent mechanisms (reviewed in [5–8]).
Nucleocytoplasmic transport is mediated by shuttling transport receptors termed karyopherins or importins/exportins (reviewed in [5, 7]). In the extensively studied classical nuclear import pathway, cargoes carrying a basic amino acid-rich nuclear localization sequence (NLS) bind to the adaptor importin a, which in turn associates with the import receptor importin β that mediates transport into the nucleus. A second class of import cargo directly binds to importin β in the absence of an adaptor. In the classical nuclear export pathway, cargoes carrying a leucine-rich nuclear export signal (NES) bind to the exportin CRM1 together with RanGTP to be transported out of the nucleus.
The small GTPase Ran, which binds directly to both importins and exportins, plays a key role in determining the directionality of nuclear transport. The GTP-bound form of Ran is concentrated in the nucleus and the GDP-bound form predominates in the cytoplasm, due to the nuclear localization of the Ran guanine nucleotide exchange factor RCC1 (RanGEF) and the cytoplasmic localization of the Ran GTPase- activating protein (RanGAP). The binding of RanGTP to karyopherins modulates the affinity of the receptors for cargo. When an importin-cargo complex encounters RanGTP in the nucleus, RanGTP promotes the dissociation of cargo from the receptor as well as dissociation of the importin from nucleoporins, and the importin-RanGTP complex is recycled back to the cytoplasm. The converse is true for exportins: intranuclear RanGTP promotes the binding of cargo to exportins, and when the RanGTP-containing export complex encounters RanGAP in the cytoplasm, GTP hydrolysis results in release of the cargo and regeneration of the free exportin [9–11].
The framework of the NPC consists of eight central spokes flanked by nuclear and cytoplasmic rings, forming a ring-spoke assembly that surrounds a central transport channel. Extending outward from the ring-spoke assembly are ~50-100-nm-long nuclear fibrils, which are joined in a basket-like structure ("nuclear basket"), and ~35-50-nm-long cytoplasmic fibrils (reviewed in [12, 13]). The NPC of both mammals and yeast comprise ~30 different nucleoporins, which are present at integral multiples of 8 copies, consistent with the 8-fold rotational symmetry of the NPC framework. Within the NPC, nucleoporins are typically organized in distinct subcomplexes that are localized to specific regions of the NPC . Approximately 1/3 of the nucleoporins contain multiple copies of the FG (phenylalanine-glycine) di-amino acid repeat. These FG repeats are clustered in domains ("FG domains") that are intrinsically unstructured. The FG domains appear to form the major diffusion barrier of the NPC [14, 15], and also serve as the key interaction sites for karyopherins during their transit through the NPC [12, 16].
In addition to undergoing reversible disassembly during mitosis in higher eukaryotes, NPCs are assembled throughout interphase in concert with NE growth . Moreover many nucleoporins have intranuclear pools that appear to undergo dynamic exchange with NPC localized forms . It is plausible that many if not most nucleoporins are imported into the nucleus by receptor-mediated pathways, but this process has not been studied in detail.
A conserved component of the NPC is the protein Tpr (for translocated promoter region)  and its homologs. Mammalian Tpr is a 267 kDa structurally bipartite protein comprising 2,349 amino acids. Its N-terminal 1,600 residue domain associates in a dimer to form a parallel two-stranded coiled-coil interrupted periodically along its length. The C-terminal domain comprising ~800 amino acids is highly acidic and is predicted to be unstructured . Tpr homologs have been characterized in Xenopus laevis , Saccharomyces cerevisiae (myosin-like proteins 1 and 2; Mlp1p and Mlp2p) , Drosophila melanogaster  and Arabidopsis thaliana . In mammalian cells, Tpr is localized to the nucleoplasmic fibrils of the NPC [1, 25] and is suggested to act as the main architectural element of the nuclear basket . Mammalian Tpr is tethered to the NPCs through interaction with Nup153 , whereas in yeast, Mlp1p and Mlp2p have been suggested to be anchored to the NPC by interactions with Nic96, or with Nup60 . Numerous functions have been attributed to vertebrate Tpr and its yeast homologs Mlp1p and Mlp2p in addition to a role in NPC architecture. These involve mRNA export [27–30], nuclear protein export [1, 31], silent telomeric chromatin organization and telomere length control [32–34], spindle pole assembly in yeast , unspliced RNA retention [4, 36] and localization and stabilization of a desumoylating enzyme Ulp1 [37, 38]. In addition, Drosophila Tpr has been linked to mitotic spindle organization and spindle checkpoint control .
In mammalian cells, classical nuclear protein import is not detectably affected in Tpr depleted cells [1, 40], whereas classical nuclear export is found to be significantly inhibited . Yeast cells carrying a double deletion of Mlp1p and Mlp2p display markedly slower import of a model cargo , but protein export has not been examined. A biochemical study conducted with Xenopus Laevis egg extracts demonstrates that importin β and importin α co-immunoprecipitate with Tpr . Whether this interaction is direct or indirect was not investigated, and its biological significance remains unresolved. To gain further insight into the role of Tpr in nucleocytoplasmic trafficking of protein, we investigated the interaction of Tpr with the karyopherins involved in classical nuclear import and export using quantitative binding assays. Our findings indicate that Tpr binds specifically and with relatively high affinity to these nuclear transport receptors, and support the notion that Tpr provides a docking site for importin α/β and CRM1 in nuclear import and export. Furthermore, the results of our binding analysis together with in vitro nuclear import assays indicate that the nuclear import of Tpr is efficiently mediated by the classical importin α/β pathway.
Results and Discussion
Characterization of the binding of CRM1, importin β and importin α to Tpr
Dissociation constants for Tpr interaction with: CRM1, importin β or importin α.
Kd apparent (nM)
Too low to measure
176 ± 8 (3)
83 ± 5 (3)
63 ± 5 (3)
Importin β I178D
Too low to measure
21.5 ± 1.5 (3)
In studies conducted in our laboratory and others, import of classical NLS cargoes was found to be unaffected in Tpr depleted cells [1, 40]. However, a biochemical study conducted with Xenopus egg extracts demonstrated that importins α and β co-immunoprecipitate with Tpr . This study did not distinguish whether binding of importin β and importin α to Tpr was direct or via an unidentified bridging factor. Importin β binds directly to several FG repeat nucleoporins with apparent Kd values between 9 nM and 225 nM depending on the nucleoporin , reflecting binding interactions of the importin β import complex with the NPC during translocation. We examined whether Tpr was able to directly bind to importin β and/or importin α in a similar affinity range. The binding between importin β and Tpr (Fig. 2B) showed a saturable binding isotherm with a relatively high apparent affinity (Kd = 50 nM, Table 1), demonstrating a direct interaction between the two proteins. Similar binding was found for C-Tpr (Fig. 2B), indicating that importin β interacts with this region of Tpr.
Importin β is a superhelical solenoid of tandem HEAT repeats, which binds to nucleoporin FG repeats on its outer surface, and to RanGTP and importin α and other cargoes on its inner surface (reviewed in ). Since Tpr is a "cargo" that presumably needs to be imported into nuclei for interphase NPC assembly, the Tpr-importin β interaction that we have detected could reflect a hypothetical cargo-like binding of Tpr to importin β that is importin a-independent. Alternatively, it could reflect a possible FG repeat-like binding of importin β to Tpr involved in importin β trafficking. Indeed, Tpr contains 3 FG repeats in its C-terminal region that could provide such binding sites. To distinguish between these possibilities, we measured the affinity of the mutant importin β I178D for Tpr. This mutant has a decreased affinity for FG repeats of nucleoporins due to disruption of a conserved hydrophobic binding pocket on the outer surface of importin β . However, the mutation has no effect on the binding to either Ran or to various cargoes, including importin α. We could not measure any specific binding of importin β I178D for Tpr in the concentration range tested (Fig. 2B), indicating that the binding of Tpr involves the major FG-repeat binding pocket on the outer surface of importin β. This finding suggests that Tpr functions as a nucleoporin that provides a binding site for importin β during import of importin β-cargo complexes or during recycling of cargo-free importin β, rather than an importin α-independent import cargo.
Nuclear Import of C-Tpr is mediated by the importin α/β pathway
The potential involvement of importin α and importin β in the nuclear import of C-Tpr was studied in digitonin-permeabilized cells reconstituted with recombinant transport factors (Fig. 5). We used the import of BSA-NLS of SV40 T-antigen, which is imported by the importin α/β pathway, as a positive control. Nuclear import of C-Tpr was observed when the permeabilized cells were supplemented with exogenous recombinant importin α/β (Fig. 5) and was considerably diminished when either importin α or importin β were omitted (Fig. 5), similar to the control cargo BSA-NLS, which requires both importins α and β for nuclear import (Fig. 5). Nuclear import of fluorescently labeled C-Tpr was strongly diminished with a 10-fold molar excess of unlabeled C-Tpr (Fig. 5). Together these data support our main conclusion that a combination of importin α and importin β are sufficient for efficient nuclear import of C-Tpr. This import pathway could be involved in Tpr assembly in the NPC during interphase, as well as at the end of mitosis. Since assembly of Tpr in the NPC at the end of mitosis follows that of almost all other nucleoporins , it is likely that Tpr enters the nucleus through largely if not completely intact NPC and thus it plausibly utilizes a receptor-mediated pathway to enter the nucleus.
Our quantitative binding studies showing that Tpr selectively binds to CRM1 present in a trimeric export complex supports the notion that Tpr plays a direct role in protein export . Our binding studies further suggest that Tpr can provide a binding site for the import and/or recycling of importin β complexes. Finally, the results presented here establish that nuclear import of Tpr can efficiently occur by the importin α/β import pathway.
Recombinant baculovirus particles containing the mammalian Tpr cDNA were prepared using the Bac-to-Bac Baculovirus Expression System (Gibco BRL). The cDNA of Tpr was cloned into the Sal I site of the donor plasmid pFastBac HTc containing an N-terminal 6×His tag. The recombinant donor plasmid was transformed into DH10Bac Escherichia coli containing the viral backbone, where recombination generated a recombinant bacmid containing His-Tpr inserted into the viral backbone. Mammalian C-Tpr encompassing amino acids 1626-2348 was constructed by inserting HindIII fragment into HindIII site of pET30c vector, and the construct was analyzed for correct orientation.
Expression and Purification of Recombinant Proteins
N-terminal His-tagged Tpr was expressed in SF9 cells at 27°C for 45 hours. Cells were lysed in RIPA buffer (1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM sodium phosphate, pH 7.2, 2 mM beta-mercaptoethanol and 2 mM EDTA) with mammalian protease inhibitor cocktail (1:100, Sigma). Cell lysates were cleared at 14,000 × g and supernatants were purified on Talon affinity resin (Clontech). Only micorgram quantities of full-length Tpr could be obtained by this method. From Western blot analysis, we found that a significant amount of an immunoreactive band comigrating with purified full-length Tpr did not bind to the Talon resin, suggesting either that the His-tag was cleaved from this fraction, or that the His-tag was inaccessible, possibly because Tpr in the unbound fraction was present in small aggregates. Recombinant C-Tpr (comprising residues 1626-2348) was expressed in Escherichia coli BL21+ cells and grown at 37°C in 2 × LB medium to an OD600 of 0.5-0.6. Expression was induced by the addition of 0.2 mM IPTG for 3 h at 37°C. Cells were resuspended in 50 mM Tris buffer, pH 7.5 supplemented with 300 mM NaCl, 2 mM DTT, 1 mg/ml lysozyme, 10 μg/ml DNase, and the protease inhibitor cocktail. The suspension was sonicated (3 × 20 s), and centrifuged at 100,000 × g for 30 min. The supernatant was loaded on a Sepharose Q column, washed extensively with the lysis buffer containing 300 mM NaCl, and then eluted with a gradient of 300-530 mM NaCl for 1 hour. Fractions containing C-Tpr were pooled; ammonium sulfate precipitated and run on a Superdex 75 column equilibrated in transport buffer. Expression and purification of His-importin α, ΔIBB-importin a, importin β, His-S-tag-importin β, Ran, nuclear transport factor 2 (NTF2) and GST-M9 (a cargo of the nuclear import receptor, transportin) was performed as previously described . These proteins were dialyzed into transport buffer  before use in the various experiments.
Microtiter Plate Binding Assay
Solid phase binding assays were carried out on microtiter plates (Maxisorp; Nunc) coated with 25 ng of Tpr or C-Tpr. Assays were conducted as previously described . Binding of His-importin α to Tpr or C-Tpr immobilized on microtiter plates was detected by an affinity-purified rabbit pAb raised against human His-importin α. Binding of His-S-tag-importin β to Tpr or to C-Tpr on microtiter plates was detected by an affinity-purified pAb raised against S-tag. Binding of CRM1 to Tpr in the presence or absence of a synthetic peptide comprising the NES derived from PKI (ELALKLAGLDIN)  and RanGTP was detected by affinity purified rabbit pAb raised against a C-terminal peptide derived from CRM1. Horseradish peroxidase-conjugated secondary antibodies were used for colorimetric detection (Pierce Chemical Co) using 3,3', 5,5'-tetramethylbenzidine as substrate (Calbiochem). The three components: CRM1, NES and RanGTP were used at equimolar concentrations in the binding experiments.
Competition assays between ΔIBB-importin α and importin β for binding to Tpr were carried out on microtiter plates (Maxisorp; Nunc) coated with 25 ng Tpr. Different ratios of ΔIBB-importin a and importin β were mixed together and incubated with immobilized Tpr. The experiments were carried out in duplicates. One repeat was probed with antibodies against S-tag to detect importin β and the other repeat was probed with antibodies against ΔIBB-importin α. Results are presented as percentage of highest level of binding.
Nuclear Import Assay
For analysis of nuclear import in digitonin-permeabilized adherent NRK cells, nuclear import in vitro assays, substrate visualization, and preparation of HeLa cytosol (by digitonin lysis) were carried out essentially as in . 5 mg of C-Tpr was coupled with Cy5™ (1 vial, Amersham Pharmacia Biotech. Inc.) for 30 minutes at room temperature and then separated from free dye by chromatography on a PD10 column (Amersham Pharmacia Biotech. Inc.) pre-equilibrated with transport buffer. Labeling of BSA with Cy3™ was performed similarly. Conjugation of labeled BSA to a peptide derived from the NLS of SV40 T- antigen (CGGGPKKKRKVEDI) was performed as in . Import reactions contained either 2.5 mg/ml HeLa cytosol or recombinant factors at the following concentrations: 100 nM His-importin α, 62.5 nM importin β, 450 n Ran, 500 nM NTF2. The import reaction mixture was supplemented with an energy-regenerating system and 1 mM GTP.
List of Abbreviations
bovine serum albumin
chromosome region maintenance 1
myosin-like protein 1
nuclear export signal
nuclear localization sequence
nuclear pore complex
nuclear transport factor 2
RanGTP binding protein
RanGTPase activating protein
Ran guanine nucleotide exchange factor
regulator of chromosome condensation 1
translocated promoter region
wheat germ agglutinin.
We thank Dr. Ralph H. Kehlenbach for preparing the polyclonal antibodies against the C-terminus of CRM1. This work was supported by grant GM41955 to L.G.
- Frosst P, Guan T, Subauste C, Hahn K, Gerace L: Tpr is localized within the nuclear basket of the pore complex and has a role in nuclear protein export. J Cell Biol. 2002, 156: 617-630. 10.1083/jcb.200106046.PubMed CentralView ArticlePubMedGoogle Scholar
- Shah S, Tugendreich S, Forbes D: Major binding sites for the nuclear import receptor are the internal nucleoporin Nup153 and the adjacent nuclear filament protein Tpr. J Cell Biol. 1998, 141: 31-49. 10.1083/jcb.141.1.31.PubMed CentralView ArticlePubMedGoogle Scholar
- Vinciguerra P, Iglesias N, Camblong J, Zenklusen D, Stutz F: Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export. EMBO J. 2005, 24: 813-823. 10.1038/sj.emboj.7600527.PubMed CentralView ArticlePubMedGoogle Scholar
- Galy V, Gadal O, Fromont-Racine M, Romano A, Jacquier A, Nehrbass U: Nuclear retention of unspliced mRNAs in yeast is mediated by perinuclear Mlp1. Cell. 2004, 116: 63-73. 10.1016/S0092-8674(03)01026-2.View ArticlePubMedGoogle Scholar
- Pemberton LF, Paschal BM: Mechanisms of receptor-mediated nuclear import and nuclear export. Traffic. 2005, 6: 187-198. 10.1111/j.1600-0854.2005.00270.x.View ArticlePubMedGoogle Scholar
- Tran EJ, Wente SR: Dynamic Nuclear Pore Complexes: Life on the Edge. Cell. 2006, 125: 1041-1053. 10.1016/j.cell.2006.05.027.View ArticlePubMedGoogle Scholar
- Stewart M: Molecular mechanism of the nuclear protein import cycle. Nature Reviews Molecular Cell Biology. 2007, 8: 195-208. 10.1038/nrm2114.View ArticlePubMedGoogle Scholar
- Mosammaparast N, Pemberton LF: Karyopherins: from nuclear-transport mediators to nuclear-function regulators. Trends Cell Biol. 2004, 14: 547-556. 10.1016/j.tcb.2004.09.004.View ArticlePubMedGoogle Scholar
- Weis K: Nucleocytoplasmic transport throughout the cell cycle. Cell. 2003, 112: 441-451. 10.1016/S0092-8674(03)00082-5.View ArticlePubMedGoogle Scholar
- Dasso M: The Ran GTPase: Theme and Variations. Curr Biol. 2002, 12: R502-R508. 10.1016/S0960-9822(02)00970-3.View ArticlePubMedGoogle Scholar
- Joseph J: Ran at a glance. J Cell Sci. 2006, 19: 3481-3484. 10.1242/jcs.03071.View ArticleGoogle Scholar
- Fahrenkrog B, Koser J, Aebi U: The nuclear pore complex: a jack of all trades?. TrendsBiochem Sci. 2004, 29: 175-182. 10.1016/j.tibs.2004.02.006.Google Scholar
- Schwartz TU: Modularity within the architecture of the nuclear pore complex. Curr Opin Struc Biol. 2005, 15: 221-226. 10.1016/j.sbi.2005.03.003.View ArticleGoogle Scholar
- Denning DP, Patel SS, Uversky V, Fink AL, Rexach M: Disorder in the nuclear pore complex: the FG repeat regions of nucleoporins are natively unfolded. Proc Natl Acad Sci USA. 2003, 100: 2450-2455. 10.1073/pnas.0437902100.PubMed CentralView ArticlePubMedGoogle Scholar
- Denning DP, Rexach MF: Rapid evolution exposes the boundaries of domain structure and function in natively unfolded FG nucleoporins. Mol Cell Proteomics. 2007, 6: 272-282.View ArticlePubMedGoogle Scholar
- Bayliss R, Littlewood T, Stewart M: Structural basis for the interaction between FxFG nucleoporin repeats and importin β in nuclear trafficking. Cell. 2000, 102: 99-108. 10.1016/S0092-8674(00)00014-3.View ArticlePubMedGoogle Scholar
- Antonin W, Ellenberg J, Dultz E: Nuclear pore complex assembly through the cell cycle: regulation and membrane organization. FEBS Lett. 2008, 582: 2004-16. 10.1016/j.febslet.2008.02.067.View ArticlePubMedGoogle Scholar
- Dultz E, Zanin E, Wurzenberger C, Braun M, Rabut G, Sironi L, Ellenberg J: Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells. J Cell Biol. 2008, 180: 857-65. 10.1083/jcb.200707026.PubMed CentralView ArticlePubMedGoogle Scholar
- Mitchell PJ, Cooper CS: Nucleotide sequence analysis of human Tpr cDNA clones. Oncogene. 1992, 7: 383-388.PubMedGoogle Scholar
- Hase ME, Kuznetsov NV, Cordes VC: Amino acid substitutions of coiled-coil protein Tpr abrogate anchorage to the nuclear pore complex but not parallel, in-register homodimerization. Mol Biol Cell. 2001, 12: 2433-2452.PubMed CentralView ArticlePubMedGoogle Scholar
- Cordes VC, Reidenbach S, Rackwitz HR, Franke WW: Identification of protein p270/Tpr as a constitutive component of the nuclear pore complex-attached intranuclear filaments. J Cell Biol. 1997, 136: 515-529. 10.1083/jcb.136.3.515.PubMed CentralView ArticlePubMedGoogle Scholar
- Kolling R, Nguyen T, Chen EY, Botstein DA: New yeast gene with a myosin-like heptad repeat structure. Mol Gen Genet. 1993, 237: 359-369.PubMedGoogle Scholar
- Zimowska G, Aris JP, Paddy MR: A Drosophila Tpr protein homolog is localized to both in the extrachromosomal channel network and to nuclear pore complexes. J Cell Sci. 1997, 110: 927-944.PubMedGoogle Scholar
- Rose A, Patel S, Meier I: The plant nuclear envelope. Planta. 2004, 218: 327-336. 10.1007/s00425-003-1132-2.View ArticlePubMedGoogle Scholar
- Krull S, Thyberg J, Bjorkroth B, Rackwitz HR, Cordes VC: Nucleoporins as components of the nuclear pore complex core structure and Tpr as the architectural element of the nuclear basket. Mol Biol Cell. 2004, 15: 4261-4277. 10.1091/mbc.E04-03-0165.PubMed CentralView ArticlePubMedGoogle Scholar
- Hase ME, Cordes VC: Direct interaction with nup153 mediates binding of Tpr to the pathway of the nuclear pore complex. Moll Cell Biol. 2003, 14: 1923-1940. 10.1091/mbc.E02-09-0620.View ArticleGoogle Scholar
- Kosova B, Pante N, Rollenhagen C, Podtelejnikov A, Mann M, Aebi U, Hurt E: Mlp2p, a component of nuclear pore attached intranuclear filaments, associates with nic96p. J Biol Chem. 2000, 275: 343-350. 10.1074/jbc.275.1.343.View ArticlePubMedGoogle Scholar
- Bangs P, Burke B, Powers C, Craig R, Purohit A, Doxsey S: Functional analysis of Tpr: identification of nuclear pore complex association and nuclear localization domains and a role in mRNA export. J Cell Biol. 1998, 143: 1801-1812. 10.1083/jcb.143.7.1801.PubMed CentralView ArticlePubMedGoogle Scholar
- Green DM, Johnson CP, Hagan H, Corbett AH: The C-terminal domain of myosin-like protein 1 (Mlp1p) is a docking site for heterogeneous nuclear ribonucleoproteins that are required for mRNA export. Proc Natl Acad Sci USA. 2003, 100: 1010-1015. 10.1073/pnas.0336594100.PubMed CentralView ArticlePubMedGoogle Scholar
- Xu XM, Rose A, Muthuswamy S, Jeong SY, Venkatakrishnan S, Zhao Q, Meier I: Nuclear pore anchor, the Arabidopsis Homolog of Tpr/Mlp1/Mlp2/Megator, Is Involved in mRNA Export and SUMO Homeostasis and Affects Diverse Aspects of Plant Development. Plant Cell. 2007, 19: 1537-1548. 10.1105/tpc.106.049239.PubMed CentralView ArticlePubMedGoogle Scholar
- Cornett J, Cao F, Wang CE, Ross CA, Bates GP, Li SH, Li JX: Polyglutamine expansion of huntingtin impairs its nuclear export. NatGenet. 2007, 37: 198-204.Google Scholar
- Galy V, Olivo-Marin JC, Scherthan H, Doye V, Rascalou N, Nehrbass U: Nuclear pore complexes in the organization of silent telomeric chromatin. Nature. 2000, 403: 108-112. 10.1038/47528.View ArticlePubMedGoogle Scholar
- Hediger F, Dubrana K, Gasser SM: Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control. J Struct Biol. 2002, 140: 79-91. 10.1016/S1047-8477(02)00533-6.View ArticlePubMedGoogle Scholar
- Feuerbach F, Galy V, Trelles-Sticken E, Fromont-Racine M, Jacquier A, Gilson E, Olivo-Marin JC, Scherthan H, Nehrbass U: Nuclear architecture and spatial positioning help establish transcriptional states of telomeres in yeast. Nat Cell Biol. 2000, 4: 214-221. 10.1038/ncb756.View ArticleGoogle Scholar
- Niepel M, Strambio-de-Castillia C, Fasolo J, Chait BT, Rout MP: The nuclear pore complex-associated protein, Mlp2p, binds to the yeast spindle pole body and promotes its efficient assembly. J Cell Biol. 2005, 170: 225-235. 10.1083/jcb.200504140.PubMed CentralView ArticlePubMedGoogle Scholar
- Casolari JM, Silver PA: Guardian at the gate: preventing unspliced pre-mRNA export. Trends Cell Biol. 2004, 14: 222-225. 10.1016/j.tcb.2004.03.007.View ArticlePubMedGoogle Scholar
- Zhao X, Wu CY, Blobel G: Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality. J Cell Biol. 2004, 167: 605-611. 10.1083/jcb.200405168.PubMed CentralView ArticlePubMedGoogle Scholar
- Palancade B, Liu X, Garcia-Rubio M, Aguilera A, Zhao X, Doye V: Nucleoporins Prevent DNA Damage Accumulation by Modulating Ulp1-dependent Sumoylation Processes. Mol Biol Cell. 2007, 18: 2912-2923. 10.1091/mbc.E07-02-0123.PubMed CentralView ArticlePubMedGoogle Scholar
- Lince-Faria M, Maffini S, Orr B, Ding Y, Florindo C, Sunkel CE, Tavares A, Johansen J, Johansen KM, Maiato H: Spatiotemporal control of mitosis by the conserved spindle matrix protein Megator. J Cell Biol. 2009, 184: 647-57. 10.1083/jcb.200811012.PubMed CentralView ArticlePubMedGoogle Scholar
- Shibata S, Matsuoka Y, Yoneda Y: Nucleocytoplasmic transport of proteins and poly(A)+ RNA in reconstituted Tpr-less nuclei in living mammalian cells. Genes Cells. 2002, 7: 421-434. 10.1046/j.1365-2443.2002.00525.x.View ArticlePubMedGoogle Scholar
- Strambio-de-Castillia C, Blobel G, Rout MP: Proteins connecting the nuclear pore complex with the nuclear interior. J Cell Biol. 1999, 144: 839-855. 10.1083/jcb.144.5.839.PubMed CentralView ArticlePubMedGoogle Scholar
- Fornerod M, Van Deursen J, Van Baa S, Reynolds A, Davis D, Mutri KG, Fransen JG: The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J. 1997, 16: 807-816. 10.1093/emboj/16.4.807.PubMed CentralView ArticlePubMedGoogle Scholar
- Askjaer P, Bachi A, Wilm M, Bisschoff FR, Weeks DL, Ogniewski V, Ohno M, Nierhrs C, Kjems J, Mattaj I, Fornerod M: RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase. Mol Cell Biol. 1999, 9: 6276-6285.Google Scholar
- Kehlenbach RH, Assheuer R, Kehlenbach A, Becker J, Gerace L: Stimulation of nuclear export and inhibition of nuclear import by a Ran mutant deficient in binding to Ran-binding protein 1. J Biol Chem. 2001, 276: 14524-14531.PubMedGoogle Scholar
- Ben-Efraim I, Gerace L: Gradient of Increasing Affinity of Importin β for Nucleoporins along the Pathway of Nuclear Import. J Cell Biol. 2001, 152: 411-418. 10.1083/jcb.152.2.411.PubMed CentralView ArticlePubMedGoogle Scholar
- Rexach M, Blobel G: Protein import into nuclei: association and dissociation reactions involving transport substrate, transport factors, and nucleoporins. Cell. 1995, 83: 683-692. 10.1016/0092-8674(95)90181-7.View ArticlePubMedGoogle Scholar
- Hu W, Jans DA: Efficiency of importin α/β mediated nuclear localization sequence recognition and nuclear import. J Biol Chem. 1999, 274: 15820-15827. 10.1074/jbc.274.22.15820.View ArticlePubMedGoogle Scholar
- Cordes VC, Hase ME, Muller L: Molecular segments of protein Tpr that confer targeting and association with the nuclear pore complex. ECR. 1998, 245: 43-56.View ArticleGoogle Scholar
- Bodoor K, Shaikh S, Salina D, Raharjo WH, Bastos R: Sequential recruitment of NPC proteins to the nuclear periphery at the end of mitosis. Cell Sci. 1999, 112: 2253-2264.Google Scholar
- Lyman SK, Guan T, Bednenko J, Wodrich H, Gerace L: Influence of cargo size on Ran and energy requirements for nuclear protein import. J Cell Biol. 2002, 159: 55-67. 10.1083/jcb.200204163.PubMed CentralView ArticlePubMedGoogle Scholar
- Melchior F, Paschal B, Evans J, Gerace L: Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor. J Cell Biol. 1993, 123: 1649-1659. 10.1083/jcb.123.6.1649.View ArticlePubMedGoogle Scholar
- Wen W, Meinkoth JL, Tsien RY, Taylor SS: Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995, 82: 463-473. 10.1016/0092-8674(95)90435-2.View ArticlePubMedGoogle Scholar
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