Characterization of Ikappa Balpha Nuclear Import Pathway*

Pierre TurpinDagger §, Ronald T. Hayparallel , and Catherine DargemontDagger **

From the Dagger  Laboratoire de Transport nucléocytoplasmique, Unité Mixte de Recherche 144 Institut Curie-CNRS, 26, rue d'Ulm, 75248 Paris Cedex 05, France and the  Institute of Biomolecular Sciences, School of Biomedical Sciences, University of St. Andrews, The North Haugh, St. Andrews, KY169TS, Scotland

    ABSTRACT
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Abstract
Introduction
References

Ikappa Balpha controls the transcriptional activity of nuclear factor (NF)-kappa B by retaining it in the cytoplasm; but, when expressed in the nucleus, it can also inhibit the interaction of NF-kappa B with DNA and promote the export of NF-kappa B from the nucleus to the cytoplasm. Here, we report that Ikappa Balpha , when not bound to NF-kappa B, is constitutively transported to the nucleus, and we confirm that the interaction of Ikappa Balpha with NF-kappa B retains Ikappa Balpha in the cytoplasm. Nuclear import of Ikappa Balpha does not result from passive diffusion but from a specific energy-dependent transport process that requires the ankyrin repeats of Ikappa Balpha . Nuclear accumulation of Ikappa Balpha is dependent on importins alpha  and beta  as well as the small GTPase Ran, which are also responsible for the nuclear import mediated by basic nuclear localization sequences (NLS). However, these proteins are not sufficient to promote Ikappa Balpha nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of Ikappa Balpha which strongly reduce import of Ikappa Balpha but not of proteins containing basic NLS. These findings indicate that Ikappa Balpha is imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with ankyrin repeats of Ikappa Balpha .

    INTRODUCTION
Top
Abstract
Introduction
References

The Rel/NF-kappa B1 transcription factors are critical regulators of genes involved in diverse cellular processes such as immune and inflammatory responses as well as cell proliferation and apoptosis (1, 2). These factors share a Rel homology domain responsible for the dimerization, nuclear localization, and DNA binding functions (3, 4). In most unstimulated cells, NF-kappa B dimers are held in an inactive state in the cytoplasm by Ikappa B inhibitory proteins that mask their nuclear localization sequence (NLS) (5-8). In response to various stimuli, recently identified protein kinase cascades are activated, resulting in the phosphorylation of Ikappa B proteins on two serine residues at their amino-terminal domain (9-14). This modification triggers polyubiquitination of Ikappa B proteins, which then undergo rapid degradation via the 26 S proteasome (1). As a consequence, the NLS of NF-kappa B is exposed, and the transcription factor translocates to the nucleus where it activates responsive genes (15). In particular, NF-kappa B induces efficient resynthesis of Ikappa Balpha through the activation of Ikappa Balpha mRNA transcription (16-18). Newly synthesized Ikappa Balpha accumulates in the nucleus where it negatively regulates NF-kappa B-dependent transcription by inhibiting the NF-kappa B/DNA interaction and by transporting NF-kappa B back to the cytoplasm (19, 20). This latter function of Ikappa Balpha is conferred by a leucine-rich nuclear export sequence, located in its COOH-terminal region, which is homologous to the nuclear export signal found in HIV-1 Rev and PKI (the inhibitor of the catalytic subunit of protein kinase A) (20-23). Such nuclear export sequences are specifically recognized by the nuclear protein CRM1 (exportin-1) (24-27), which promotes the transport of nuclear export sequence-containing proteins and in particular NF-kappa B·Ikappa Balpha complexes from the nucleus to the cytoplasm.

Ikappa Balpha is composed of a surface-exposed NH2-terminal region, not essential for binding to RelA (p65), followed by a central, protease-resistant domain containing five ankyrin repeats and a compact, highly acidic COOH-terminal region connected to the core by a flexible linker (28). Both the central ankyrin domain and the linker region are essential for the interaction of Ikappa Balpha with Rel factors (28-31). Ikappa Balpha lacks an SV40 large T antigen- or nucleoplasmin-like NLS (basic NLS) or any other motif described to serve as NLS (8). Besides the ability of newly synthesized Ikappa Balpha to localize in the nucleus, it has been reported that Ikappa Balpha overexpressed from a transfected plasmid or microinjected into the cytoplasm also distributes to both the cytoplasmic and nuclear compartments (8, 20). Its molecular mass (37 kDa), which is below the theoretical cutoff of the nuclear pore complex, led to the hypothesis that Ikappa Balpha might enter the nucleus by diffusion (8, 32).

Different pathways have been described to account for the nuclear import of various types of karyophilic proteins (33, 34). Proteins carrying a basic amino acid stretch NLS interact with a heterodimeric NLS receptor composed of two subunits, importins alpha  and beta  or karyopherins alpha  and beta 1 (35-44); for alternative nomenclatures, see Ref. 34. hnRNP A1 import depends on another motif called M9, which is recognized by transportin or karyopherin beta 2 (45-47), whereas other members of the karyopherin beta  family, karyopherins beta 3 and beta 4, are responsible for the nuclear import of ribosomal proteins (48-51). Recognition of karyophilic substrates by these karyopherins beta  leads to the targeting of karyophilic proteins to the nuclear pore complex, a specialized and elaborated structure of the nuclear envelope through which the exchange of macromolecules between the nucleus and cytoplasm occurs (34). Karyopherin beta  family members share a common NH2-terminal binding motif for RanGTP (52), a small GTPase essential for most nucleocytoplasmic transport pathways (53-56). Ran is thought to be distributed asymmetrically between the nucleus and cytoplasm with the GTP-bound form in the nucleus and the GDP-bound form in the cytoplasm. Both GTPase Ran and the factor p10 or NTF2 (57-59) mediate the translocation of karyopherin-karyophilic protein complexes in the nucleus. Interaction of these complexes with RanGTP in the nucleus promotes the release of karyophilic proteins in this compartment and subsequent recycling of the NLS receptors (42, 60, 61).

The goal of the present study was to characterize precisely the requirements for nuclear localization of Ikappa Balpha as well as the molecular mechanisms underlying this transport process. We found that NF-kappa B-free Ikappa Balpha localizes constitutively in the cytoplasm and in the nucleus, and we confirm that the interaction of Ikappa Balpha with NF-kappa B retains Ikappa Balpha in the cytoplasm. Moreover we demonstrate that the nuclear import of Ikappa Balpha does not result from passive diffusion but rather from an energy-dependent process that is mediated by the ankyrin repeats of Ikappa Balpha . Nuclear import of Ikappa Balpha requires importins alpha  and beta  (karyopherins alpha  and beta 1) as well as the GTPase Ran. However, these proteins are not sufficient to promote Ikappa Balpha nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of Ikappa Balpha which strongly reduce import of Ikappa Balpha but not of proteins containing a basic amino acid stretch NLS. These findings indicate that Ikappa Balpha is imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with ankyrin repeats of Ikappa Balpha .

    EXPERIMENTAL PROCEDURES

Cells and Culture Conditions-- Adherent or S3 suspension HeLa cells were maintained in exponential growth in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

DNA Transfections-- For transient expression experiments, HeLa cells were transfected by electroporation as described previously (20) and cultured subsequently for 24 h before analysis.

Preparation of HeLa Cell Cytosol-- HeLa cell cytosol was prepared as described by Paschal and Gerace (58). 109 exponentially growing HeLa S3 cells were collected by centrifugation at 300 × g for 10 min. The cells were washed twice with phosphate-buffered saline and once with lysis buffer (5 mM Hepes, pH 7.4; 5 mM potassium acetate, pH 7.4; 2 mM magnesium acetate; 1 mM EGTA; 2 mM dithiothreitol; and protease inhibitors: 10 µg/ml each aprotinin, leupeptin, pepstatin; and 200 µg/ml 4-(2-aminoethyl)benzenesulfonyl fluoride (Uptima)). The cell pellet was resuspended in 1 volume of lysis buffer and disrupted in a tight fitting stainless steel homogenizer (as judged by phase-contrast microscopy). The homogenate was diluted with 0.1 volume of 10 × transport buffer (20 mM Hepes, pH 7.4, 110 mM potassium acetate, pH 7.4, 2 mM magnesium acetate, 0.5 mM EGTA, 1 mM dithiothreitol, and protease inhibitors) and centrifuged at 40,000 × g for 30 min at 4 °C. The supernatant was centrifuged further at 100,000 × g for 1 h. The resulting supernatant (~20 mg/ml as measured with the protein assay kit (Bio-Rad)) was aliquoted, frozen in liquid N2, and stored at -80 °C.

To prepare depleted cytosol, 100 µl of HeLa cell cytosol (2 mg of proteins) was incubated overnight at 4 °C with 10 µg of GST or 20 µg of GST-Ikappa Balpha (68-243) immobilized on 20 µl of glutathione-Sepharose beads (Amersham Pharmacia Biotech). Mixtures were subsequently centrifuged, and the postcentrifugation supernatant is defined as depleted cytosol.

Nuclear Import Assay-- Digitonin-permeabilized HeLa cells were prepared according to Adam et al. (62). Cells grown on coverslips were permeabilized with 55 µg/ml digitonin (Sigma) in transport buffer. A standard 50-µl nuclear import assay was performed in transport buffer containing an energy-regenerating system (1 mM ATP, 0.5 mM GTP, 10 mM creatine phosphate, and 0.4 unit/ml creatine phosphokinase), 15 µg/ml BSA-NLS-FITC, 30 µl of HeLa cell cytosol (~20 mg/ml), and 40 µg/ml or 10 µg/ml SV5-tagged versions of wt Ikappa Balpha or GST-Ikappa Balpha (68-243), respectively. The reaction was allowed to proceed for 45 min at 30 °C. Similar results were obtained with an untagged version of Ikappa Balpha (data not shown).

NLS, SLN Peptides, and Preparation of Transport Substrate (BSA-NLS-FITC)-- Peptides containing the SV40 large T antigen wt NLS (cgggPKKKRKVED) or reverse NLS (SLN; cgggDEVKRKVED) were synthesized with an NH2-terminal Cys for chemical coupling reactions followed by a (Gly)3 linker. BSA was conjugated to peptides and FITC according to Görlich et al. (38). It should be noted that the resulting fusion proteins contain more than 10 NLS or SLN peptides.

Immunofluorescence Microscopy-- For indirect immunofluorescence analysis, transfected HeLa cells were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 10 min. Digitonin-permeabilized HeLa cells were fixed with 2% paraformaldehyde and 0.025% glutaraldehyde and permeabilized with 0.1% Triton X-100 for 5 min. Monoclonal antibodies to SV5-Pk-tag (63) or to Ikappa Balpha (10B (28)) and polyclonal antibodies to NF-kappa B p65 (C-20, Santa Cruz) or to GST (64) were applied for 30 min followed by a 30-min incubation with Texas red or FITC-conjugated donkey anti-mouse or anti-rabbit IgG (Jackson). Coverslips were mounted in phosphate-buffered saline containing 50% glycerol. Confocal laser scanning microscopy and immunofluorescence analysis were performed with a TCS4D confocal microscope based on a DM microscope interfaced with a mixed gas argon-krypton laser (Leica Laser Technik). Fluorescence acquisitions were performed with the 488 nm and 568 nm laser lines to excite FITC and Texas red dyes, respectively, with a × 100 oil immersion PL APO objective. Data presented on a same figure were registered at the same laser and multiplier settings. To quantify fluorescence intensity, optical slices of 20 different cells/condition were recorded as 512 × 512-pixel images with the same preset parameters. A region of interest corresponding to the nuclear area was then created in each cell, and the mean density was calculated within this area using NIH image software. For measurement of Ikappa Balpha or GST-Ikappa Balpha (68-243) nuclear content, values obtained with nuclei incubated in the absence of Ikappa Balpha or GST-Ikappa Balpha (68-243) and stained with both primary and secondary antibodies were considered as negative controls and subtracted from the values obtained for nuclei incubated with the import substrate. For measurement of BSA-NLS-FITC nuclear content, areas registered outside the cells were considered as background. All data were saved in different series, and statistical analysis (mean intensities and standard deviation) was performed.

Plasmids-- DNA encoding the SV5-tagged version of Ikappa Balpha sequences for amino acids 1-317 or 68-317 were amplified by polymerase chain reaction using the pcDNA3-Ikappa Balpha ctag vector (20) as template and cloned into the BamHI/XbaI restriction sites of the eukaryotic expression vector pEGFP-C1 (CLONTECH) generating pEGFP-wt Ikappa Balpha ctag or pEGFP-Ikappa Balpha (68-317) ctag, respectively. Sequences encoding amino acids 68-265 or 68-243 of Ikappa Balpha were amplified using pcDNA3-Ikappa Balpha ctag vector as a template and cloned into the BamHI/EcoRI restriction sites of pcDNA3 ctag (pcDNA3-Ikappa Balpha ctag without the BamHI/EcoRI-wt Ikappa Balpha fragment) generating pcDNA3-Ikappa Balpha (68-265) ctag or pcDNA3-Ikappa Balpha (68-243) ctag, respectively. The corresponding BamHI/XbaI restriction fragments were inserted into BamHI/XbaI-cleaved pEGFP-C1 vector generating pEGFP-Ikappa Balpha (68-265) ctag or pEGFP-Ikappa Balpha (68-243) ctag, respectively. The plasmid encoding 4Nbeta C has been described previously (65).

To generate the SV5-tagged GST-Ikappa Balpha (68-243) in bacteria, a BamHI/EcoRI restriction fragment encoding amino acids 68-243 of Ikappa Balpha was generated using pcDNA3-Ikappa Balpha (68-243) ctag and inserted into BamHI/EcoRI-cleaved pGEX ctag vector (63) generating pGEX-Ikappa Balpha (68-243) ctag.

Expression of Recombinant Proteins-- Recombinant SV5-tagged wt Ikappa Balpha was produced and purified as described previously (66). pGEX-2T (Amersham Pharmacia Biotech) or pGEX-Ikappa Balpha (68-243) ctag plasmids were transformed into Escherichia coli DH5alpha or BL21(DE3) respectively. Cells were grown to an A600 of 0.4 and induced with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 3 h at 37 °C. Recombinant GST or SV5-tagged GST-Ikappa Balpha (68-243) were purified on glutathione-Sepharose beads (Amersham Pharmacia Biotech), eluted with glutathione, and dialyzed at 4 °C against transport buffer. The protein concentration was determined with the protein assay kit (Bio-Rad), and the sample was aliquoted, quick frozen in liquid N2, and stored at -80 °C. NF-kappa B p65 (amino acids 12-317) was expressed in bacteria and purified as described (67).

Expression vectors for His-tagged hSRP1-alpha , His-tagged IBB (pKW312) and wild-type untagged importin beta /p97 (pKW291) were provided by K. Weis (University of California, San Francisco), and proteins were expressed and purified as described (44, 68). Plasmid for GST-Ran expression was a gift from M. Dasso, and Ran wt was purified as described (NIH, Bethesda; 69). Expression and purification of RanQ69L were performed essentially as described (70) using the expression vector provided by C. Dingwall (Stony Brook, NY). His-tagged p10 expression and purification were done as described (41) using the p10 expression vector provided by G. Blobel (The Rockefeller Institute, New York).

    RESULTS

Both Endogenous and Overexpressed Ikappa Balpha Can Localize to the Nucleus in the Absence of Cell Stimulation-- To investigate the mechanisms responsible for the nuclear accumulation of Ikappa Balpha , we first addressed the question of whether the ability of Ikappa Balpha to be imported into the nucleus depends on cell activation. In unstimulated HeLa cells, Ikappa Balpha was distributed throughout both the nucleus and the cytoplasm, whereas NF-kappa B p65 was localized predominantly in the cytoplasmic compartment (Fig. 1A). This result suggests that even in the absence of cell activation, a fraction of Ikappa Balpha which does not interact with p65 is expressed in the nucleus. To confirm this result, HeLa cells were transfected with a plasmid encoding a tagged version of Ikappa Balpha which could be detected with an anti-tag antibody (Fig. 1B, panel a). Overexpressed Ikappa Balpha displayed a cytoplasmic as well as a nuclear localization in HeLa cells. This distribution has also been observed recently in fibroblasts lacking Rel proteins (66), indicating that the ability of Ikappa Balpha to be expressed in the nucleus is not dependent on the presence of endogenous NF-kappa B family members. To establish whether the nuclear localization of Ikappa Balpha was caused by serum activation of the cells, transfected HeLa cells were treated with 100 µg/ml cycloheximide for 1 h, leading to the complete disappearance of the protein in both nuclear and cytoplasmic compartments (Fig. 1B, panel b). Cycloheximide was then removed, and cells were incubated for 2 additional h without serum. Under this unstimulated condition, newly synthesized Ikappa Balpha accumulated in both the nucleus and the cytoplasm (Fig. 1B, panel c), indicating that nuclear localization of overexpressed Ikappa Balpha could occur in the absence of stimulation of HeLa cells. Cells were then incubated with tumor necrosis factor-alpha for 30 min leading to degradation of Ikappa Balpha (Fig. 1B, panel d). Tumor necrosis factor-alpha was then removed, and newly synthesized Ikappa Balpha again accumulated in both cytoplasmic and nuclear compartments (Fig. 1B, panel e).


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Fig. 1.   Constitutive localization of Ikappa Balpha in both nuclear and cytoplasmic compartments. Panel A, endogenous Ikappa Balpha or NF-kappa B p65 was localized in HeLa cells by indirect immunofluorescence using Ikappa Balpha mouse monoclonal antibodies and NF-kappa B p65 rabbit polyclonal antibodies. Panel B, transfected epitope-tagged wt Ikappa Balpha was immunolocalized using an anti-tag monoclonal antibody followed by a FITC-conjugated anti-mouse antibody. Transfected HeLa cells cultured in the presence of serum (a) were washed and treated for 1 h with 100 µg/ml cycloheximide (CHX; b). This drug was then removed, and the cells were incubated in the absence of serum for 2 h (c) followed by a 30-min stimulation with tumor necrosis factor (TNF)-alpha (d) and a 30-min chase (e). Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection. Data were recorded at the same laser and multiplier settings.

These results show that in HeLa cells, endogenous as well as overexpressed Ikappa Balpha can localize to the nucleus in the absence of cell stimulation or after treatment with tumor necrosis factor-alpha .

Nuclear Import of Ikappa Balpha Is an Active Process Inhibited by NF-kappa B p65-- To establish whether nuclear localization of Ikappa Balpha was caused by passive diffusion or by a specific nuclear import mechanism, properties of Ikappa Balpha nuclear transport were analyzed using semipermeabilized cells (62). Digitonin-permeabilized HeLa cells were incubated for 45 min at 30 °C with recombinant wt Ikappa Balpha in the presence of HeLa cytosol, an energy source (ATP, GTP, and an ATP-regenerating system), and BSA-NLS-FITC as a control for import of a karyophilic protein (Fig. 2). Under this experimental condition both BSA-NLS-FITC and Ikappa Balpha accumulated in the nucleus. Accumulation of either BSA-NLS-FITC or wt Ikappa Balpha did not occur when the cytosol was replaced by BSA or when apyrase was added to cytosol (Fig. 2 and Table I), indicating that the nuclear import of wt Ikappa Balpha as BSA-NLS-FITC was not the result of diffusion into the nucleus but was rather a specific cytosol- and energy-dependent nuclear import process. Replacement of HeLa cytosol by BSA (or other inhibitory conditions, see Fig. 4) led to the interaction of Ikappa Balpha with cytoplasmic structures including the nuclear envelope. This effect was probably caused by the intrinsic ability of the NH2-terminal region of Ikappa Balpha to promote retention on similar structures under certain experimental conditions but was not relevant for nuclear import activity (data not shown).


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Fig. 2.   Nuclear import of Ikappa Balpha in semipermeabilized HeLa cells requires both cytosol and energy and is inhibited by p65. In the left panel, digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and recombinant wt Ikappa Balpha in the presence of HeLa cytosol (12 mg/ml) and an energy-regenerating system (ATP), BSA (12 mg/ml), and ATP, HeLa cytosol, and apyrase (Sigma, 24 units/ml) or HeLa cytosol, ATP, and recombinant p65(12-317) (20 µg/ml) as indicated. In the right panel, digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC only, in the presence of HeLa cytosol (12 mg/ml) and an energy-regenerating system (ATP). After incubation, cells were processed for indirect immunofluorescence with a tag-specific monoclonal antibody followed by a Texas red-conjugated donkey anti-mouse antibody. Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection.

                              
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Table I
Nuclear import of BSA-NLS-FITC, Ikappa Balpha , and GST-Ikappa Balpha (68-243) in semipermeabilized HeLa cells

In unstimulated cells, Ikappa Balpha has been shown to prevent nuclear import of NF-kappa B by masking its NLS (5, 6, 8). Moreover, nuclear localization of Ikappa Balpha is inhibited in vivo by overexpressed NF-kappa B members p65 or p50 (8). To investigate whether NF-kappa B can control the nuclear accumulation of Ikappa Balpha , nuclear import of Ikappa Balpha was analyzed in semipermeabilized cells in the presence of extracts, energy-regenerating system, and recombinant Rel domain of p65 (amino acids 12-317; p65(12-317)). The Rel domain is necessary and sufficient for association with Ikappa Balpha , dimerization, DNA binding, and nuclear localization (28). As shown in Fig. 2, p65(12-317) inhibited the nuclear import of Ikappa Balpha without affecting that of BSA-NLS-FITC. This result indicates that the domains involved in the nuclear import of Ikappa Balpha and p65 are mutually masked when these proteins interact. Therefore, nuclear accumulation of Ikappa Balpha is an active process that is inhibited by NF-kappa B p65.

Nuclear Import of Ikappa Balpha Is Mediated by Its Ankyrin Repeats-- None of the previously described nuclear localization signals can be recognized in the sequence of Ikappa Balpha . To define the motif responsible for its nuclear import, nucleocytoplasmic distribution of fusion proteins between the green fluorescent protein (GFP) and different domains of Ikappa Balpha was analyzed. These fusion proteins had predicted molecular masses greater than 50 kDa and therefore were not able to diffuse into the nucleus. Plasmids encoding GFP fused to tagged version of wild-type Ikappa Balpha (GFP-wt Ikappa Balpha ), Ikappa Balpha lacking the NH2-terminal domain (GFP-Ikappa Balpha (68-317)) or both NH2- and COOH-terminal domains (GFP-Ikappa Balpha (68-265)), or Ikappa Balpha ankyrin repeats (GFP-Ikappa Balpha (68-243)) (Fig. 3A) were generated and transiently transfected in HeLa cells. Overexpressed proteins were detected both with GFP fluorescence and with a specific anti-tag antibody.


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Fig. 3.   Nuclear import of Ikappa Balpha is mediated by its ankyrin repeats. Panel A, schematic drawing of Ikappa Balpha and SV5-tagged GFP or beta -galactosidase-Ikappa Balpha fusion proteins. The positions of the amino acids that delimit the different domains of Ikappa Balpha (NH2-terminal domain, residues 1-68; ankyrin repeats domain, residues 68-243; linker domain, residues 243-265; and COOH-terminal domain, residues 265-317) are indicated in bold, and the names of the overexpressed proteins are indicated on the left. Panel B, epitope-tagged GFP or beta -galactosidase-Ikappa Balpha fusion proteins were transiently expressed in HeLa cells, stained using a tag-specific antibody (upper) or visualized directly with GFP fluorescence (lower). For the GFP-wt Ikappa Balpha fusion protein, cells expressing high levels of the exogenous proteins are also shown (see inset). Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection. Panel C, upper, digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either 40 µg/ml recombinant GST-Ikappa Balpha (68-243) or 20 µg/ml GST in the presence of HeLa cytosol (12 mg/ml) and an ATP-regenerating system. After incubation, cells were processed for indirect immunofluorescence with an anti-GST polyclonal antibody or a monoclonal anti-tag antibody followed by a Texas red-conjugated donkey anti-rabbit antibody or a Texas red-conjugated donkey anti-mouse antibody respectively. Lower, digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and recombinant GST-Ikappa Balpha (68-243) in the presence of HeLa cytosol (12 mg/ml) and ATP, BSA (12 mg/ml), and ATP or HeLa cytosol and apyrase (Sigma, 24 units/ml) as indicated. After incubation, cells were processed for indirect immunofluorescence with an anti-GST polyclonal antibody followed by a Texas red-conjugated donkey anti-rabbit antibody. Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection.

The subcellular localization of GFP-wt Ikappa Balpha depended on its level of expression (Fig. 3B). In cells expressing low amounts of GFP-wt Ikappa Balpha , this protein was essentially cytoplasmic, but in cells expressing higher amounts, this protein partitioned equally between nucleus and cytoplasm (Fig. 3B, compare the inset with the rest of the panel). However when the NH2-terminal domain of Ikappa Balpha (GFP-Ikappa Balpha (68-317)) or both NH2- and COOH-terminal domains (GFP-Ikappa Balpha (68-265) and GFP-Ikappa Balpha (68-243)) were deleted, the resulting fusion proteins were localized in both the nucleus and the cytoplasm whatever their expression level. It should be noted that the immunofluorescence signal obtained with these proteins was more intense in the nucleus than in the cytoplasm. Because both GFP-wt Ikappa Balpha and GFP-Ikappa Balpha (68-317) are able to interact with NF-kappa B (data not shown), the different distribution of these two protein suggests that the NH2-terminal domain of Ikappa Balpha prevents the nuclear accumulation of GFP-wt Ikappa Balpha . Both NH2- and COOH-terminal domains of Ikappa Balpha were also fused to beta -galactosidase (Fig. 3A; 43) but none of these regions was able to direct beta -galactosidase to the nucleus (Fig. 3B). These results indicate that the ankyrin repeats of Ikappa Balpha are necessary and sufficient to target this protein in the nucleus of intact cells and confirm a recent study showing that the nuclear localization of Ikappa Balpha requires the integrity of hydrophobic residues within the second ankyrin repeat (66).

To confirm that the ankyrin repeats were responsible for the nuclear import of Ikappa Balpha , a recombinant fusion protein between GST and the ankyrin repeats of Ikappa Balpha (GST-Ikappa Balpha (68-243)) was produced in bacteria and tested for its ability to be imported to the nucleus in semipermeabilized HeLa cells. GST-Ikappa Balpha (68-243) visualized using either anti-GST or anti-SV5 tag antibodies was imported efficiently into nuclei in a cytosol- and energy-dependent manner (Fig. 3C, upper and lower panels). In contrast, GST is not accumulated in the nucleus in the presence of cytosol and energy (Table I and Fig. 3C, upper panel). This result confirms, by the permeabilized cells assay, that the nuclear import of Ikappa Balpha is mediated by its ankyrin repeats.

Nuclear Import of Ikappa Balpha Is a GTPase Ran-dependent Process-- To gain insight into the molecular mechanism allowing Ikappa Balpha to enter the nucleus, the involvement of the small GTPase Ran was investigated. Indeed, Ran is involved not only in the nuclear import of basic NLS-containing proteins but also in the nuclear import of snRNPs, hnRNPs as well as in nuclear export of both RNAs and proteins (53, 54, 71, 72).

Nuclear import of either wt Ikappa Balpha or GST-Ikappa Balpha (68-243) and BSA-NLS-FITC in semipermeabilized cells was analyzed in the presence of HeLa extracts, an energy-regenerating system, and GTPgamma S, a nonhydrolyzable analog of GTP (Table I and Fig. 4, A and B). GTPgamma S at a 5 mM concentration strongly inhibited nuclear accumulation of these proteins, indicating that GTP hydrolysis is required for wt Ikappa Balpha nuclear import. To test the involvement of Ran in this process, a nuclear import assay was performed in the presence of either a recombinant GTPase-deficient Ran mutant (RanQ69L) or recombinant wild-type Ran (Ran wt). Nuclear import of BSA-NLS-FITC, wt Ikappa Balpha and GST-Ikappa Balpha (68-243) was blocked by RanQ69L, whereas transport of these proteins was only slightly impaired in the presence of wt Ran (Table I and Fig. 4, A and B). In contrast, neither RanQ69L nor Ran wt affected diffusion into the nucleus (data not shown). These results indicate that the GTPase Ran is necessary for the ankyrin repeat-mediated nuclear import of Ikappa Balpha .


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Fig. 4.   Nuclear import of Ikappa Balpha involves the GTPase Ran. Digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either recombinant wt Ikappa Balpha (panel A) or recombinant GST-Ikappa Balpha (68-243) (panel B) in the presence of HeLa cytosol and an ATP-regenerating system in the absence (control) or in the presence of 5 mM GTPgamma S, 3 µM RanQ69L, or 3 µM Ran wt as indicated. After incubation, cells were processed for indirect immunofluorescence with either a tag-specific monoclonal antibody followed by a Texas red-conjugated donkey anti-mouse antibody for detection of wt Ikappa Balpha or an anti-GST rabbit polyclonal antibody followed by a Texas red-conjugated donkey anti-rabbit antibody for detection of GST-Ikappa Balpha (68-243). Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection.

Nuclear Import of Ikappa Balpha Requires Both Importins alpha  and beta -- Different hypotheses could account for the Ran-dependent nuclear transport of Ikappa Balpha . Ikappa Balpha might enter the nucleus by interacting with a NLS-containing protein (piggy-back), through a direct interaction with one of the component of the NLS import machinery or by using another Ran-dependent import pathway. To distinguish between these mechanisms, the ability of a peptide corresponding to the SV40 large T antigen NLS to compete with the nuclear import of wt Ikappa Balpha or GST-Ikappa Balpha (68-243) and BSA-NLS-FITC was analyzed. As shown in Fig. 5, A and B, NLS but not SLN peptide (reversed NLS sequence), completely blocked transport of the analyzed karyophilic proteins in the nucleus of permeabilized cells, indicating that nuclear import of Ikappa Balpha required the basic NLS receptor, importin alpha  (See also Table I). Because importin beta  binds importin alpha  to mediate basic NLS-dependent import, the involvement of the importin beta  in the nuclear uptake of Ikappa Balpha was then analyzed. For this purpose, a recombinant protein corresponding to the importin beta -binding site of importin alpha  (IBB) reported previously to act as a competitive inhibitor of the basic NLS nuclear import machinery (68, 73), was added in the nuclear import assay. This recombinant protein blocked nuclear import of BSA-NLS-FITC as well as nuclear accumulation of wt Ikappa Balpha or GST-Ikappa Balpha (68-243) (Table I and Fig. 5, A and B) but did not affect diffusion into the nucleus (data not shown). Taken together, these results indicate that importins alpha  and beta  are required for the ankyrin repeat-mediated nuclear import of Ikappa Balpha . Therefore the involvement of another Ran-dependent pathway would be minimal in such a system.


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Fig. 5.   Nuclear import of Ikappa Balpha requires the basic NLS receptor (importins alpha /beta ). Digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either recombinant wt Ikappa Balpha (panel A) or recombinant GST-Ikappa Balpha (68-243) (panel B) in the presence of HeLa cytosol and an ATP-regenerating system in the absence (control) or in the presence of 1 mM NLS peptide, 1 mM SLN peptide, or IBB (5 µM in panel A and 20 µM in panel B), as indicated. After incubation, cells were processed for indirect immunofluorescence with either a tag-specific monoclonal antibody followed by a Texas red-conjugated donkey anti-mouse antibody for detection of wt Ikappa Balpha or an anti-GST polyclonal antibody followed by a Texas red-conjugated donkey anti-rabbit antibody for detection of GST-Ikappa Balpha (68-243). Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection.

Ikappa Balpha Is Imported into the Nucleus by a Piggy-back Mechanism-- To distinguish a basic NLS-dependent piggy-back mechanism and a direct interaction of Ikappa Balpha with the basic NLS nuclear import machinery, we analyzed the nuclear import properties of BSA-NLS-FITC and either wt Ikappa Balpha or GST-Ikappa Balpha (68-243) in the presence of an energy-regenerating system and the recombinant purified nuclear import machinery consisting of the importins alpha  and beta , Ran, and p10. Although the addition of these recombinant proteins was sufficient to promote nuclear accumulation of BSA-NLS-FITC, neither wt Ikappa Balpha nor GST-Ikappa Balpha (68-243) was imported in this experimental condition (Fig. 6A).


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Fig. 6.   Ikappa Balpha is imported in the nucleus by a piggy-back mechanism. Panel A, digitonin-permeabilized HeLa cells were incubated at 30 °C for 45 min with BSA-NLS-FITC and either recombinant wt Ikappa Balpha or recombinant GST-Ikappa Balpha (68-243) in the presence of an ATP-regenerating system and the recombinant basic NLS nuclear import machinery consisting of 1 µM importin alpha , 400 nM importin beta , 1.4 µM Ran wt, and 1.5 µM p10. Panel B, digitonin-permeabilized HeLa cells were incubated at 30 °C for 30 min with BSA-NLS-FITC and recombinant wt Ikappa Balpha in the presence of an ATP-regenerating system and HeLa cytosol previously incubated with either GST or GST-Ikappa Balpha (68-243) beads (depleted cytosol). After incubation, cells were processed for indirect immunofluorescence with a tag-specific monoclonal antibody followed by a Texas red-conjugated donkey anti-mouse antibody. Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection.

Cytosol was prepared for transport assays as usual except that, prior to the assay, it was incubated with either GST or GST-Ikappa Balpha (68-243) immobilized on glutathione-Sepharose beads. Depletion of cytosol with GST had no effect on the ability of the cytosol to support nuclear import of either BSA-NLS-FITC or wt Ikappa Balpha . However, cytosol depleted with GST-Ikappa Balpha (68-243), although still able to support nuclear import of BSA-NLS-FITC, was strongly affected in its Ikappa Balpha nuclear import activity (Fig. 6B). This result shows that cytosol contains cellular factor(s) able to bind ankyrin repeats of Ikappa Balpha and essential for the nuclear import of Ikappa Balpha but not of proteins containing a basic amino acid stretch NLS. Ikappa Balpha is thus likely imported in the nucleus by a piggy-back mechanism that involves additional protein(s) containing a basic NLS and able to interact with ankyrin repeats of Ikappa Balpha .

    DISCUSSION

In response to distinct external stimuli, specific transcription factors are activated which function to regulate the activity of unique sets of target genes. The regulated nuclear import of transcription factors provides an efficient mechanism to restrict their access to the transcriptional machinery. However, in any case of signal-mediated transcriptional regulation, termination of a regulated transcriptional event is also required to turn off the cellular response and return to a "resting" state. In principle, different mechanisms could operate to turn off a transcriptional response, each of which could have distinct consequences. Recently, it has been recognized that nuclear export allows a transcriptional response not only to be terminated but also to be subsequently reinitiated with minimal delay. The NF-kappa B transcription factor constitutes a well documented example of this phenomenon. The transcriptional activity of NF-kappa B is regulated mainly by its subcellular localization, which is determined by the level of expression as well as by the nucleocytoplasmic distribution of the Ikappa B proteins. In particular, Ikappa Balpha retains NF-kappa B in an inactive form in the cytoplasm (5, 6, 8), but it can also enter the nucleus where it inhibits NF-kappa B/DNA interaction and transports NF-kappa B back to the cytoplasm (19, 20). To define more clearly the physiological conditions that lead to the nuclear expression of Ikappa Balpha and subsequent termination of the NF-kappa B-dependent transcription, it was necessary to characterize the requirements and mechanisms accounting for Ikappa Balpha nuclear import.

Role of NF-kappa B in the Cytoplasmic Retention of Ikappa Balpha -- In the present report, we show that when Ikappa Balpha is not bound to NF-kappa B, it is constitutively imported into the nucleus both in vivo and in vitro. Cytoplasmic NF-kappa B-free Ikappa Balpha exists when the Ikappa Balpha expression level exceeds the amount of NF-kappa B or when these proteins are distributed differentially between the nucleus and cytoplasm. Overexpression of Ikappa Balpha by transient transfection with plasmid encoding Ikappa Balpha (8, 20) and physiological situations such as NF-kappa B-induced de novo synthesis of Ikappa Balpha (19) or dissociation of the NF-kappa B·Ikappa Balpha complex by phosphorylation of Ikappa Balpha tyrosine 42 (74) have been reported to produce NF-kappa B-free Ikappa Balpha . Subcellular distribution of Ikappa Balpha has been investigated in some of these conditions and found to be both cytoplasmic and nuclear (75). In contrast, when Ikappa Balpha is overexpressed as an NF-kappa B-bound form, it localizes exclusively in the cytoplasm (8). These data suggest that interaction of NF-kappa B with Ikappa Balpha in the cytoplasm could either prevent diffusion of Ikappa Balpha into the nucleus by forming a complex that is unable to translocate freely through the nuclear pore or mask a region of Ikappa Balpha involved in its nuclear import. Using semipermeabilized cells, we show here that Ikappa Balpha does not pass to the nucleus by diffusion but is transported there by an extract- and energy-dependent pathway. Both in vitro and in vivo, the ankyrin repeats appear sufficient to promote the nuclear import of Ikappa Balpha . This result confirms a recent study showing that the nuclear localization of Ikappa Balpha requires the integrity of hydrophobic residues within the second ankyrin repeat (66). It has been well documented that the five ankyrin repeats of Ikappa Balpha bind the NF-kappa B Rel homology domain, and the sixth degenerated ankyrin repeat, also called the "linker" region, also participates in the interaction with NF-kappa B (28, 30, 76). The Rel homology domain of p65 prevents not only the transport of Ikappa Balpha but also the nuclear accumulation of GST-Ikappa Balpha (68-243) in the nucleus of semipermeabilized cells (a higher concentration of p65 is necessary to inhibit nuclear import of GST-Ikappa Balpha (68-243); data not shown). Taken together, these results demonstrate that NF-kappa B retains Ikappa Balpha in the cytoplasm by masking its ankyrin repeats, which are essential for nuclear import. In unstimulated cells, cytoplasmic retention of NF-kappa B·Ikappa Balpha complexes is therefore caused by a mutual masking of the sequences responsible for the nuclear import of both proteins.

Ikappa Balpha Nuclear Import Pathway-- Ankyrin repeats could allow NF-kappa B-free Ikappa Balpha to enter the nucleus via a novel nuclear import pathway or, alternatively, interact directly or indirectly (piggy-back) with known nuclear import receptors. The present report shows that nuclear import of Ikappa Balpha requires GTP hydrolysis by Ran as well as the basic NLS receptors, importins alpha  and beta . The whole purified recombinant nuclear import machinery (importins alpha /beta , Ran, and p10) was however not sufficient to target Ikappa Balpha or GST-Ikappa Balpha (68-243) into the nucleus. When HeLa cytosol was submitted to a GST affinity column, the flow-through was able to promote nuclear import of both BSA-NLS-FITC and Ikappa Balpha . In contrast, the flow-through resulting from a GST-Ikappa Balpha (68-243) affinity column was not affected in its ability to induce BSA-NLS-FITC nuclear import but was unable to promote nuclear import of Ikappa Balpha . We thus propose that ankyrin repeats of Ikappa Balpha probably interact with additional component(s) containing a basic NLS that is recognized by the basic NLS receptor (piggy-back mechanism). Similar piggy-back mechanisms accounting for protein nuclear import have been reported already. For example, the 46-kDa subunit of the mouse DNA primase does not have an NLS but enters nuclei upon interaction with the 54-kDa subunit, which carries a basic NLS (77). Although NF-kappa B itself contains an NLS and binds Ikappa Balpha ankyrin repeats, the Rel homology domain of p65 was unable to target Ikappa Balpha or GST-Ikappa Balpha (68-243) into the nucleus of semipermeabilized cells in the presence of the recombinant basic NLS nuclear import machinery (data not shown). Moreover, overexpressed wt Ikappa Balpha displays identical subcellular distribution in fibroblasts expressing or lacking p50, p52, p65, or c-Rel (66), indicating that NF-kappa B is therefore unlikely to be responsible for the nuclear import of Ikappa Balpha .

The partner of Ikappa Balpha required for import may specifically recognize ankyrin repeats of Ikappa Balpha or, alternatively, structures shared by other ankyrin repeat-containing proteins. The presence of ankyrin repeats is a common characteristic of Ikappa B proteins, and, interestingly, some of them have been shown to localize in the nucleus. Ikappa B members display an expression pattern depending on cell types and different affinities for the NF-kappa B members (for review, see Refs. 3 and 4). Ikappa Bbeta has no NLS and interacts with the same subset of NF-kappa B proteins as Ikappa Balpha . Upon certain stimuli, Ikappa Bbeta is degraded and subsequently resynthesized, but it accumulates as a hypophosphorylated protein. Interaction of this newly synthesized Ikappa Bbeta with NF-kappa B fails to mask both the NLS and DNA binding domain of NF-kappa B and therefore leads to a complex able to enter the nucleus by piggy-back and activate transcription (78, 79). Bcl-3, an Ikappa B protein containing two basic NLS in its NH2-terminal domain, is expressed predominantly in the nucleus of lymphoid cells and binds NF-kappa B p50 and p52 homodimers. This interaction does not mask NF-kappa B NLS and results in the nuclear import of (p50)2·Bcl-3 complexes. It has been reported that this transport can be ensured either by p50 NLS or by Bcl-3 NLS (80). From these data, it appears clearly that the ankyrin repeats of Bcl-3 or Ikappa Bbeta are not involved in the physiological nuclear import of these proteins, although the intrinsic ability of these repeats to localize in the nucleus has been reported recently (66). In particular, when Ikappa Bbeta is overexpressed from a transfected vector in HeLa cells, it localizes both in the cytoplasm and in the nucleus (data not shown). On the other hand, some ankyrin repeat-containing proteins other than from the Ikappa B family have also been shown to be expressed in the nucleus. For example, oncogenic intracellular forms of NOTCH can still be detected in the nucleus when their two putative NLS have been deleted (81, 82). In addition, a 37-kDa fragment composed of the ankyrin repeats of the recently identified 104-kDa diacylglycerol kinase DGK-IV/DGK-zeta and lacking a recognizable NLS accumulates in the nucleus (83). It has been reported recently that ankyrin repeats of 53BP2 and GABPbeta but not Notch1 are able, when fused to a reporter, to target the resulting fusion protein to the nucleus (66). Whether a common protein or protein family is responsible for the nuclear import of ankyrin repeat-containing proteins remains to be elucidated.

    ACKNOWLEDGEMENTS

We are grateful to Drs. R. Goldsteyn, C. Maison, and J. Salamero for a critical reading of the manuscript. We thank Drs. K. Weis, M. Dasso, C. Dingwall, G. Blobel, M. Rodriguez, and T. Galli for the generous gift of expression vectors coding for importins alpha  and beta  as well as IBB, GST-Ran, RanQ69L, p10, 4Nbeta C, and anti-GST antibodies, respectively. We thank Ellis Jaffray for technical support in protein production and purification.

    FOOTNOTES

* This work was supported in part by grants from the Association de Recherche contre le Cancer and the European Communities Concerted Action Project Rocio II.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a fellowship from the Ministere de l'Education Nationale.

parallel Supported by the Medical Research Council.

** To whom correspondence should be addressed. Tel.: 33-1-4234-6366; Fax: 33-1-4234-6367; E-mail: dargemon{at}curie.fr.

    ABBREVIATIONS

The abbreviations used are: NF-kappa B, nuclear factor kappa B; NLS, nuclear localization sequence(s); HIV-1, human immunodeficiency virus type 1; GST, glutathione S-transferase; BSA, bovine serum albumin; FITC, fluorescein isothiocyanate; wt, wild-type; GFP, green fluorescent protein; hnRNP, heterogenous nuclear ribonucleoprotein; IBB, importin beta  binding site; GTPgamma S, guanosine 5'-3- O-(thio)triphosphate.

    REFERENCES
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Abstract
Introduction
References
  1. Baeuerle, P. A., and Baltimore, D. (1996) Cell 87, 13-20[Medline] [Order article via Infotrieve]
  2. Hay, R. T. (1993) Biochem. Soc. Trans. 21, 926-930[Medline] [Order article via Infotrieve]
  3. Liou, H. C., and Baltimore, D. (1993) Curr. Opin. Cell Biol. 5, 477-487[Medline] [Order article via Infotrieve]
  4. Verma, I. M., Stevenson, J. F., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735[CrossRef][Medline] [Order article via Infotrieve]
  5. Ganchi, P. A., Sun, S. C., Greene, W. C., and Ballard, D. W. (1992) Mol. Biol. Cell 3, 1339-1352[Abstract]
  6. Beg, A. A., Ruben, S. M., Scheinman, R. I., Haskill, S., Rosen, C. A., and Baldwin, A. J. (1992) Genes Dev. 6, 1899-1913[Abstract]
  7. Henkel, T., Zabel, U., van Zee, K., Muller, J. M., Fanning, E., and Baeuerle, P. A. (1992) Cell 68, 1121-1133[Medline] [Order article via Infotrieve]
  8. Zabel, U., Henkel, T., Silva, M. S., and Baeuerle, P. A. (1993) EMBO J. 12, 201-211[Abstract]
  9. Verma, I. M., and Stevenson, J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11758-11760[Free Full Text]
  10. Stancovski, I., and Baltimore, D. (1997) Cell 91, 299-302[Medline] [Order article via Infotrieve]
  11. Di Donato, J. A., Hayakawa, M., Rothwarf, D. M., Zandi, E., and Karin, M. (1997) Nature 388, 548-554[CrossRef][Medline] [Order article via Infotrieve]
  12. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
  13. Woronicz, J. D., Gao, X., Cao, Z., Rothe, M., and Goeddel, D. V. (1997) Science 278, 866-869[Abstract/Free Full Text]
  14. Zandi, E., Rothwarf, D. M., Delhase, M., Hayakawa, M., and Karin, M. (1997) Cell 91, 243-252[Medline] [Order article via Infotrieve]
  15. Henkel, T., Machleidt, T., Alkalay, I., Kronke, M., Ben-Neriah, Y., and Baeuerle, P. A. (1993) Nature 365, 182-185[CrossRef][Medline] [Order article via Infotrieve]
  16. Chiao, P. J., Miyamoto, S., and Verma, I. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 28-32[Abstract]
  17. Le Bail, O., Schmidt Ullrich, R., and Israel, A. (1993) EMBO J. 12, 5043-5049[Abstract]
  18. Sun, S. C., Ganchi, P. A., Ballard, D. W., and Greene, W. C. (1993) Science 259, 1912-1915[Medline] [Order article via Infotrieve]
  19. Arenzana-Seisdedos, F., Thompson, J., Rodriguez, M. S., Bachelerie, F., Thomas, D., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2689-2696[Abstract]
  20. Arenzana-Seisdedos, F., Turpin, P., Rodriguez, M., Thomas, D., Hay, R. T., Virelizier, J. L., and Dargemont, C. (1997) J. Cell Sci. 110, 369-378[Abstract/Free Full Text]
  21. Wen, W., Meinkoth, J. L., Tsien, R. Y., and Taylor, S. S. (1995) Cell 82, 463-473[Medline] [Order article via Infotrieve]
  22. Fischer, U., Huber, J., Boelens, W. C., Mattaj, I. W., and Luhrmann, R. (1995) Cell 82, 475-483[Medline] [Order article via Infotrieve]
  23. Fritz, C. C., and Green, M. R. (1996) Curr. Biol. 6, 848-854[Medline] [Order article via Infotrieve]
  24. Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. (1997) Nature 390, 308-311[CrossRef][Medline] [Order article via Infotrieve]
  25. Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997) Cell 90, 1051-1060[Medline] [Order article via Infotrieve]
  26. Ossareh-Nazari, B., Bachelerie, F., and Dargemont, C. (1997) Science 278, 141-144[Abstract/Free Full Text]
  27. Stade, K., Ford, C. S., Guthrie, C., and Weis, K. (1997) Cell 90, 1041-1050[Medline] [Order article via Infotrieve]
  28. Jaffray, E., Wood, K. M., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2166-2172[Abstract]
  29. Blank, V., Kourilsky, P., and Israel, A. (1991) EMBO J. 10, 4159-4167[Abstract]
  30. Hatada, E. N., Naumann, M., and Scheidereit, C. (1993) EMBO J. 12, 2781-2788[Abstract]
  31. Inoue, J., Kerr, L. D., Kakizuka, A., and Verma, I. M. (1992) Cell 68, 1109-1120[Medline] [Order article via Infotrieve]
  32. Cressman, D. E., and Taub, R. (1993) Oncogene 8, 2567-2573[Medline] [Order article via Infotrieve]
  33. Ohno, M., Fornerod, M., and Mattaj, I. W. (1998) Cell 92, 327-336[Medline] [Order article via Infotrieve]
  34. Nigg, E. A. (1997) Nature 386, 779-787[CrossRef][Medline] [Order article via Infotrieve]
  35. Iovine, M. K., Watkins, J. L., and Wente, S. R. (1995) J. Cell Biol. 131, 1699-1713[Abstract]
  36. Görlich, D., Vogel, F., Mills, A. D., Hartmann, E., and Laskey, R. A. (1995) Nature 377, 246-248[CrossRef][Medline] [Order article via Infotrieve]
  37. Görlich, D., Kostka, S., Kraft, R., Dingwall, C., Laskey, R. A., Hartmann, E., and Prehn, S. (1995) Curr. Biol. 5, 383-392[Medline] [Order article via Infotrieve]
  38. Görlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Cell 79, 767-778[Medline] [Order article via Infotrieve]
  39. Imamoto, N., Shimamoto, T., Takao, T., Tachibana, T., Kose, S., Matsubae, M., Sekimoto, T., Shimonishi, Y., and Yoneda, Y. (1995) EMBO J. 14, 3617-3626[Abstract]
  40. Moroianu, J., Blobel, G., and Radu, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 2008-2011[Abstract]
  41. Moroianu, J., Hijikata, M., Blobel, G., and Radu, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6532-6536[Abstract]
  42. Chi, N. C., Adam, E. J., Visser, G. D., and Adam, S. A. (1996) J. Cell Biol. 135, 559-569[Abstract]
  43. Radu, A., Blobel, G., and Moore, M. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1769-1773[Abstract]
  44. Weis, K., Mattaj, I. W., and Lamond, A. I. (1995) Science 268, 1049-1053[Medline] [Order article via Infotrieve]
  45. Siomi, H., and Dreyfuss, G. (1995) J. Cell Biol. 129, 551-560[Abstract]
  46. Pollard, V. W., Michael, W. M., Nakielny, S., Siomi, M. C., Wang, F., and Dreyfuss, G. (1996) Cell 86, 985-994[Medline] [Order article via Infotrieve]
  47. Aitchison, J. D., Blobel, G., and Rout, M. P. (1996) Science 274, 624-627[Abstract/Free Full Text]
  48. Deane, R., Schafer, W., Zimmermann, H. P., Mueller, L., Görlich, D., Prehn, S., Ponstingl, H., and Bischoff, F. R. (1997) Mol. Cell. Biol. 17, 5087-5096[Abstract]
  49. Schlenstedt, G., Smirnova, E., Deane, R., Solsbacher, J., Kutay, U., Görlich, D., Ponstingl, H., and Bischoff, F. R. (1997) EMBO J. 16, 6237-6249[Abstract/Free Full Text]
  50. Rout, M. P., Blobel, G., and Aitchison, J. D. (1997) Cell 89, 715-725[Medline] [Order article via Infotrieve]
  51. Yaseen, N. R., and Blobel, G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 4451-4456[Abstract/Free Full Text]
  52. Görlich, D., Dabrowski, M., Bischoff, F. R., Kutay, U., Bork, P., Hartmann, E., Prehn, S., and Izaurralde, E. (1997) J. Cell Biol. 138, 65-80[Abstract/Free Full Text]
  53. Melchior, F., Guan, T., Yokoyama, N., Nishimoto, T., and Gerace, L. (1995) J. Cell Biol. 131, 571-581[Abstract]
  54. Moore, M. S., and Blobel, G. (1993) Nature 365, 661-663[CrossRef][Medline] [Order article via Infotrieve]
  55. Izaurralde, E., Jarmolowski, A., Beisel, C., Mattaj, I. W., Dreyfuss, G., and Fischer, U. (1997) J. Cell Biol. 137, 27-35[Abstract/Free Full Text]
  56. Richards, S. A., Carey, K. L., and Macara, I. G. (1997) Science 276, 1842-1844[Abstract/Free Full Text]
  57. Nehrbass, U., and Blobel, G. (1996) Science 272, 120-122[Abstract]
  58. Paschal, B. M., and Gerace, L. (1995) J. Cell Biol. 129, 925-937[Abstract]
  59. Moore, M. S., and Blobel, G. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10212-10216[Abstract/Free Full Text]
  60. Rexach, M., and Blobel, G. (1995) Cell 83, 683-692[Medline] [Order article via Infotrieve]
  61. Görlich, D., Pante, N., Kutay, U., Aebi, U., and Bischoff, F. R. (1996) EMBO J. 15, 5584-5594[Abstract]
  62. Adam, S. A., Marr, R. S., and Gerace, L. (1990) J. Cell Biol. 111, 807-816[Abstract]
  63. Hanke, T., Szawlowski, P., and Randall, R. E. (1992) J. Gen. Virol. 73, 653-660[Abstract]
  64. Galli, T., Zahraoui, A., Vaidyanathan, V. V., Raposo, G., Tian, J. M., Karin, M., Niemann, H., and Louvard, D. (1998) Mol. Biol. Cell 9, 1437-1448[Abstract/Free Full Text]
  65. Kroll, M., Conconi, M. A., Desterro, M. J. P., Marin, A., Thomas, D., Friguet, B., Hay, R. T., Virelizier, J.-L., Arenzana-Seisdedos, F., and Rodriguez, M. S. (1997) Oncogene 15, 1841-1850[CrossRef][Medline] [Order article via Infotrieve]
  66. Rodriguez, M. S., Michalopoulos, I., Arenzana, S. F., and Hay, R. T. (1995) Mol. Cell. Biol. 15, 2413-2419[Abstract]
  67. Matthews, J. R., Nicholson, J., Jaffray, E., Kelly, S. M., Price, N. C., and Hay, R. T. (1995) Nucleic Acids Res. 23, 3393-3402[Abstract]
  68. Weis, K., Ryder, U., and Lamond, A. I. (1996) EMBO J. 15, 1818-1825[Abstract]
  69. Dasso, M., Seki, T., Azuma, Y., Ohba, T., and Nishimoto, T. (1994) EMBO J. 13, 5732-5744[Abstract]
  70. Klebe, C., Nishimoto, T., and Wittinghofer, F. (1993) Biochemistry 32, 11923-11928[Medline] [Order article via Infotrieve]
  71. Palacios, I., Weis, K., Klebe, C., Mattaj, I. W., and Dingwall, C. (1996) J. Cell Biol. 133, 485-494[Abstract]
  72. Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W., and Görlich, D. (1997) EMBO J. 16, 6535-6547[Abstract/Free Full Text]
  73. Görlich, D., Henklein, P., Laskey, R. A., and Hartmann, E. (1996) EMBO J. 15, 1810-1817[Abstract]
  74. Imbert, V., Rupec, R. A., Livolsi, A., Pahl, H. L., Traenckner, E. B., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auberger, P., Baeuerle, P. A., and Peyron, J. F. (1996) Cell 86, 787-798[Medline] [Order article via Infotrieve]
  75. Spiecker, M., Peng, H. B., and Liao, J. K. (1997) J. Biol. Chem. 272, 30969-30974[Abstract/Free Full Text]
  76. Haskill, S., Beg, A. A., Tompkins, S. M., Morris, J. S., Yurochko, A. D., Sampson-Johannes, A., Mondal, K., Ralph, P., and Baldwin, A. S., Jr. (1991) Cell 65, 1281-1289[Medline] [Order article via Infotrieve]
  77. Mizuno, T., Okamoto, T., Yokoi, M., Izumi, M., Kobayashi, A., Hachiya, T., Tamai, K., Inoue, T., and Hanaoka, F. (1996) J. Cell Sci. 109, 2627-2636[Abstract/Free Full Text]
  78. Tran, K., Merika, M., and Thanos, D. (1997) Mol. Cell. Biol. 17, 5386-5399[Abstract]
  79. Suyang, H., Phillips, R., Douglas, I., and Ghosh, S. (1996) Mol. Cell. Biol. 16, 5444-5449[Abstract]
  80. Zhang, Q., Di Donato, J. A., Karin, M., and McKeithan, T. W. (1994) Mol. Cell. Biol. 14, 3915-3926[Abstract]
  81. Aster, J. C., Robertson, E. S., Hasserjian, R. P., Turner, J. R., Kieff, E., and Sklar, J. (1997) J. Biol. Chem. 272, 11336-11343[Abstract/Free Full Text]
  82. Kopan, R., Schroeter, E. H., Weintraub, H., and Nye, J. S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1683-1688[Abstract/Free Full Text]
  83. Goto, K., and Kondo, H. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11196-11201[Abstract/Free Full Text]


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