Characterization of IκBα Nuclear Import Pathway*

IκBα controls the transcriptional activity of nuclear factor (NF)-κB by retaining it in the cytoplasm; but, when expressed in the nucleus, it can also inhibit the interaction of NF-κB with DNA and promote the export of NF-κB from the nucleus to the cytoplasm. Here, we report that IκBα, when not bound to NF-κB, is constitutively transported to the nucleus, and we confirm that the interaction of IκBα with NF-κB retains IκBα in the cytoplasm. Nuclear import of IκBα does not result from passive diffusion but from a specific energy-dependent transport process that requires the ankyrin repeats of IκBα. Nuclear accumulation of IκBα is dependent on importins α and β 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 IκBα nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of IκBα which strongly reduce import of IκBα but not of proteins containing basic NLS. These findings indicate that IκBα 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 IκBα.

The Rel/NF-B 1 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-B dimers are held in an inactive state in the cytoplasm by IB inhibitory proteins that mask their nuclear localization sequence (NLS) (5)(6)(7)(8). In response to various stimuli, recently identified protein kinase cascades are activated, resulting in the phosphorylation of IB proteins on two serine residues at their amino-terminal domain (9 -14). This modification triggers polyubiquitination of IB proteins, which then undergo rapid degradation via the 26 S proteasome (1). As a consequence, the NLS of NF-B is exposed, and the transcription factor translocates to the nucleus where it activates responsive genes (15). In particular, NF-B induces efficient resynthesis of IB␣ through the activation of IB␣ mRNA transcription (16 -18). Newly synthesized IB␣ accumulates in the nucleus where it negatively regulates NF-B-dependent transcription by inhibiting the NF-B/DNA interaction and by transporting NF-B back to the cytoplasm (19,20). This latter function of IB␣ 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-B⅐IB␣ complexes from the nucleus to the cytoplasm.
IB␣ is composed of a surface-exposed NH 2 -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 IB␣ with Rel factors (28 -31). IB␣ 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 IB␣ to localize in the nucleus, it has been reported that IB␣ 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 IB␣ 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 ␣ and ␤ or karyopherins ␣ and ␤1 (35)(36)(37)(38)(39)(40)(41)(42)(43)(44); for alternative nomenclatures, see Ref. 34. hnRNP A1 import depends on another motif called M9, which is recognized by transportin or karyopherin ␤2 (45)(46)(47), whereas other members of the karyopherin ␤ family, karyopherins ␤3 and ␤4, are responsible for the nuclear import of ribosomal proteins (48 -51). Recognition of karyophilic substrates by these karyopherins ␤ 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 ␤ family members share a common NH 2 -terminal binding motif for RanGTP (52), a small GTPase essential for most nucleocytoplasmic transport pathways (53)(54)(55)(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 IB␣ as well as the molecular mechanisms underlying this transport process. We found that NF-B-free IB␣ localizes constitutively in the cytoplasm and in the nucleus, and we confirm that the interaction of IB␣ with NF-B retains IB␣ in the cytoplasm. Moreover we demonstrate that the nuclear import of IB␣ does not result from passive diffusion but rather from an energy-dependent process that is mediated by the ankyrin repeats of IB␣. Nuclear import of IB␣ requires importins ␣ and ␤ (karyopherins ␣ and ␤1) as well as the GTPase Ran. However, these proteins are not sufficient to promote IB␣ nuclear translocation. Factor(s) can be removed selectively from cell extracts with ankyrin repeats of IB␣ which strongly reduce import of IB␣ but not of proteins containing a basic amino acid stretch NLS. These findings indicate that IB␣ 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 IB␣.

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). 10 9 exponentially growing HeLa S3 cells were collected by centrifugation at 300 ϫ 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 g/ml each aprotinin, leupeptin, pepstatin; and 200 g/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 ϫ 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 ϫ g for 30 min at 4°C. The supernatant was centrifuged further at 100,000 ϫ g for 1 h. The resulting supernatant (ϳ20 mg/ml as measured with the protein assay kit (Bio-Rad)) was aliquoted, frozen in liquid N 2 , and stored at Ϫ80°C.
To prepare depleted cytosol, 100 l of HeLa cell cytosol (2 mg of proteins) was incubated overnight at 4°C with 10 g of GST or 20 g of GST-IB␣(68 -243) immobilized on 20 l 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 g/ml digitonin (Sigma) in transport buffer. A standard 50-l 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 g/ml BSA-NLS-FITC, 30 l of HeLa cell cytosol (ϳ20 mg/ml), and 40 g/ml or 10 g/ml SV5-tagged versions of wt IB␣ or GST-IB␣(68 -243), respectively. The reaction was allowed to proceed for 45 min at 30°C. Similar results were obtained with an untagged version of IB␣ (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 NH 2 -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 IB␣ (10B (28)) and polyclonal antibodies to NF-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 ϫ 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 ϫ 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 IB␣ or GST-IB␣(68 -243) nuclear content, values obtained with nuclei incubated in the absence of IB␣ or GST-IB␣(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.
Expression vectors for His-tagged hSRP1-␣, His-tagged IBB (pKW312) and wild-type untagged importin ␤/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).

Both Endogenous and Overexpressed IB␣ Can Localize to the Nucleus in the Absence of Cell
Stimulation-To investigate the mechanisms responsible for the nuclear accumulation of IB␣, we first addressed the question of whether the ability of IB␣ to be imported into the nucleus depends on cell activation. In unstimulated HeLa cells, IB␣ was distributed throughout both the nucleus and the cytoplasm, whereas NF-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 IB␣ 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 IB␣ which could be detected with an anti-tag antibody (Fig.  1B, panel a). Overexpressed IB␣ displayed a cytoplasmic as well as a nuclear localization in HeLa cells. This distribution has also been observed recently in fibroblasts lacking Rel pro-teins (66), indicating that the ability of IB␣ to be expressed in the nucleus is not dependent on the presence of endogenous NF-B family members. To establish whether the nuclear localization of IB␣ was caused by serum activation of the cells, transfected HeLa cells were treated with 100 g/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 IB␣ accumulated in both the nucleus and the cytoplasm (Fig. 1B, panel c), indicating that nuclear localization of overexpressed IB␣ could occur in the absence of stimulation of HeLa cells. Cells were then incubated with tumor necrosis factor-␣ for 30 min leading to degradation of IB␣ (Fig. 1B, panel d). Tumor necrosis factor-␣ was then removed, and newly synthesized IB␣ again accumulated in both cytoplasmic and nuclear compartments (Fig. 1B,  panel e).
These results show that in HeLa cells, endogenous as well as overexpressed IB␣ can localize to the nucleus in the absence of cell stimulation or after treatment with tumor necrosis factor-␣.
Nuclear Import of IB␣ Is an Active Process Inhibited by NF-B p65-To establish whether nuclear localization of IB␣ was caused by passive diffusion or by a specific nuclear import mechanism, properties of IB␣ nuclear transport were analyzed using semipermeabilized cells (62). Digitonin-permeabilized HeLa cells were incubated for 45 min at 30°C with recombinant wt IB␣ 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 IB␣ accumulated in the nucleus. Accumulation of either BSA-NLS-FITC or wt IB␣ 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 IB␣ as BSA-NLS-FITC was not the result of diffusion into the nucleus but was rather a specific cytosol-and energydependent nuclear import process. Replacement of HeLa cytosol by BSA (or other inhibitory conditions, see Fig. 4) led to the interaction of IB␣ with cytoplasmic structures including the nuclear envelope. This effect was probably caused by the intrinsic ability of the NH 2 -terminal region of IB␣ to promote retention on similar structures under certain experimental conditions but was not relevant for nuclear import activity (data not shown).
In unstimulated cells, IB␣ has been shown to prevent nuclear import of NF-B by masking its NLS (5,6,8). Moreover, nuclear localization of IB␣ is inhibited in vivo by overexpressed NF-B members p65 or p50 (8). To investigate whether NF-B can control the nuclear accumulation of IB␣, nuclear import of IB␣ 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 IB␣, dimerization, DNA binding, and nuclear localization (28). As shown in Fig. 2, p65(12-317) inhibited the nuclear import of IB␣ without affecting that of BSA-NLS-FITC. This result indicates that the domains involved in the nuclear import of IB␣ and p65 are mutually masked when these proteins interact. Therefore, nuclear accumulation of IB␣ is an active process that is inhibited by NF-B p65.
Nuclear Import of IB␣ Is Mediated by Its Ankyrin Repeats-None of the previously described nuclear localization signals can be recognized in the sequence of IB␣. To define the motif responsible for its nuclear import, nucleocytoplasmic distribu- was localized in HeLa cells by indirect immunofluorescence using IB␣ mouse monoclonal antibodies and NF-B p65 rabbit polyclonal antibodies. Panel B, transfected epitope-tagged wt IB␣ 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 g/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)-␣ (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.
tion of fusion proteins between the green fluorescent protein (GFP) and different domains of IB␣ 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 IB␣ (GFP-wt IB␣), IB␣ lacking the NH 2 -terminal domain (GFP-IB␣(68 -317)) or both NH 2 -and COOH-terminal domains (GFP-IB␣(68 -265)), or IB␣ ankyrin repeats (GFP-IB␣(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.
The subcellular localization of GFP-wt IB␣ depended on its level of expression (Fig. 3B). In cells expressing low amounts of GFP-wt IB␣, 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 NH 2terminal domain of IB␣ (GFP-IB␣(68 -317)) or both NH 2and COOH-terminal domains (GFP-IB␣(68 -265) and GFP-IB␣(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 IB␣ and GFP-IB␣(68 -317) are able to interact with NF-B (data not shown), the different distribution of these two protein suggests that the NH 2 -terminal domain of IB␣ prevents the nuclear accumulation of GFP-wt IB␣. Both NH 2 -and COOHterminal domains of IB␣ were also fused to ␤-galactosidase ( Fig. 3A; 43) but none of these regions was able to direct ␤-galactosidase to the nucleus (Fig. 3B). These results indicate that the ankyrin repeats of IB␣ 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 IB␣ 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 IB␣, a recombinant fusion protein between GST and the ankyrin repeats of IB␣ (GST-IB␣(68 -243)) was produced in bacteria and tested for its ability to be imported to the nucleus in semipermeabilized HeLa cells. GST-IB␣(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 IB␣ is mediated by its ankyrin repeats.
Nuclear Import of IB␣ Is a GTPase Ran-dependent Process-To gain insight into the molecular mechanism allowing IB␣ 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 IB␣ or GST-IB␣(68 -243) and BSA-NLS-FITC in semipermeabilized cells was analyzed in the presence of HeLa extracts, an energy-regenerating system, and

FIG. 2. Nuclear import of IB␣ 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 IB␣ 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 g/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. GTP␥S, a nonhydrolyzable analog of GTP (Table I and Fig. 4, A  and B). GTP␥S at a 5 mM concentration strongly inhibited nuclear accumulation of these proteins, indicating that GTP hydrolysis is required for wt IB␣ nuclear import. To test the involvement of Ran in this process, a nuclear import assay was performed in the presence of either a recombinant GTPasedeficient Ran mutant (RanQ69L) or recombinant wild-type Ran (Ran wt). Nuclear import of BSA-NLS-FITC, wt IB␣ and GST-IB␣(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 IB␣. Nuclear Import of IB␣ Requires Both Importins ␣ and ␤-Different hypotheses could account for the Ran-dependent nuclear transport of IB␣. IB␣ 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 IB␣ or GST-IB␣(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 IB␣ required the basic NLS receptor, importin ␣ (See also Table I). Because importin ␤ binds importin ␣ to mediate basic NLS-dependent import, the involvement of the importin ␤ in the nuclear uptake of IB␣ was then analyzed. For this purpose, a recombinant protein corresponding to the importin ␤-binding site of importin ␣ (IBB) reported previously to act as a competitive inhibitor of the basic NLS nuclear import machinery (68,73), was added in . 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, digitoninpermeabilized HeLa cells were incubated at 30°C for 45 min with BSA-NLS-FITC and either 40 g/ml recombinant GST-IB␣(68 -243) or 20 g/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-IB␣ (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 nuclear import assay. This recombinant protein blocked nuclear import of BSA-NLS-FITC as well as nuclear accumulation of wt IB␣ or GST-IB␣(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 ␣ and ␤ are required for the ankyrin repeat-mediated nuclear import of IB␣. Therefore the involvement of another Ran-dependent pathway would be minimal in such a system.

TABLE I Nuclear import of BSA-NLS-FITC, IB␣, and GST-IB␣(68 -243) in semipermeabilized HeLa cells
IB␣ Is Imported into the Nucleus by a Piggy-back Mechanism-To distinguish a basic NLS-dependent piggy-back mechanism and a direct interaction of IB␣ with the basic NLS nuclear import machinery, we analyzed the nuclear import properties of BSA-NLS-FITC and either wt IB␣ or GST-IB␣ (68 -243) in the presence of an energy-regenerating system and the recombinant purified nuclear import machinery consisting of the importins ␣ and ␤, Ran, and p10. Although the addition of these recombinant proteins was sufficient to promote nuclear accumulation of BSA-NLS-FITC, neither wt IB␣ nor GST-IB␣(68 -243) was imported in this experimental condition (Fig. 6A).
Cytosol was prepared for transport assays as usual except that, prior to the assay, it was incubated with either GST or GST-IB␣(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 IB␣. However, cytosol depleted with GST-IB␣(68 -243), although still able to support nuclear import of BSA-NLS-FITC, was strongly affected in its IB␣ nuclear import activity (Fig. 6B). This result shows that cytosol contains cellular factor(s) able to bind ankyrin repeats of IB␣ and essential for the nuclear import of IB␣ but not of proteins containing a basic amino acid stretch NLS. IB␣ 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 IB␣.

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-B transcription factor constitutes a well documented example of this phenomenon. The transcriptional activity of NF-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 IB proteins. In particular, IB␣ retains NF-B in an inactive form in the cytoplasm (5,6,8), but it can also enter the nucleus where it inhibits NF-B/DNA interaction and transports NF-B back to the cytoplasm (19,20). To define more clearly the physiological conditions that lead to the nuclear expression of IB␣ and subsequent termination of the NF-B-dependent transcription, it was necessary to characterize the requirements and mechanisms accounting for IB␣ nuclear import.
Role 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 IB␣ or an anti-GST polyclonal antibody followed by a Texas red-conjugated donkey anti-rabbit antibody for detection of GST-IB␣ (68 -243). Cells were visualized with a confocal laser scanning microscope, and photographs correspond to the accumulation of four optical sections in one projection. present report, we show that when IB␣ is not bound to NF-B, it is constitutively imported into the nucleus both in vivo and in vitro. Cytoplasmic NF-B-free IB␣ exists when the IB␣ expression level exceeds the amount of NF-B or when these proteins are distributed differentially between the nucleus and cytoplasm. Overexpression of IB␣ by transient transfection with plasmid encoding IB␣ (8,20) and physiological situations such as NF-B-induced de novo synthesis of IB␣ (19) or dissociation of the NF-B⅐IB␣ complex by phosphorylation of IB␣ tyrosine 42 (74) have been reported to produce NF-Bfree IB␣. Subcellular distribution of IB␣ has been investigated in some of these conditions and found to be both cytoplasmic and nuclear (75). In contrast, when IB␣ is overexpressed as an NF-B-bound form, it localizes exclusively in the cytoplasm (8). These data suggest that interaction of NF-B with IB␣ in the cytoplasm could either prevent diffusion of IB␣ into the nucleus by forming a complex that is unable to translocate freely through the nuclear pore or mask a region of IB␣ involved in its nuclear import. Using semipermeabilized cells, we show here that IB␣ 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 IB␣. This result confirms a recent study showing that the nuclear localization of IB␣ requires the integrity of hydrophobic residues within the second ankyrin repeat (66). It has been well documented that the five ankyrin repeats of IB␣ bind the NF-B Rel homology domain, and the sixth degenerated ankyrin repeat, also called the "linker" region, also participates in the interaction with NF-B (28,30,76). The Rel homology domain of p65 prevents not only the transport of IB␣ but also the nuclear accumulation of GST-IB␣ (68 -243) in the nucleus of semipermeabilized cells (a higher concentration of p65 is necessary to inhibit nuclear import of GST-IB␣(68 -243); data not shown). Taken together, these results demonstrate that NF-B retains IB␣ in the cytoplasm by masking its ankyrin repeats, which are essential for nuclear import. In unstimulated cells, cytoplasmic retention of NF-B⅐IB␣ complexes is therefore caused by a mutual masking of the sequences responsible for the nuclear import of both proteins.
IB␣ Nuclear Import Pathway-Ankyrin repeats could allow NF-B-free IB␣ 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 IB␣ requires GTP hydrolysis by Ran as well as the basic NLS receptors, importins ␣ and ␤. The whole purified recombinant nuclear import machinery (importins ␣/␤, Ran, and p10) was however not sufficient to target IB␣ or GST-IB␣(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 IB␣. In contrast, the flow-through resulting from a GST-IB␣(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 IB␣. We thus propose that ankyrin repeats of IB␣ probably interact with additional component(s) containing a basic NLS that is recognized by the basic NLS receptor (piggy-back mechanism). Similar piggyback 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-B itself contains an NLS and binds IB␣ ankyrin repeats, the Rel homology domain of p65 was unable to target IB␣ or GST-IB␣(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 IB␣ displays identical subcellular distribution in fibroblasts expressing or lacking p50, p52, p65, or c-Rel (66), indicating that NF-B is therefore unlikely to be responsible for the nuclear import of IB␣.
The partner of IB␣ required for import may specifically recognize ankyrin repeats of IB␣ or, alternatively, structures shared by other ankyrin repeat-containing proteins. The presence of ankyrin repeats is a common characteristic of IB proteins, and, interestingly, some of them have been shown to localize in the nucleus. IB members display an expression pattern depending on cell types and different affinities for the NF-B members (for review, see Refs. 3 and 4). IB␤ has no NLS and interacts with the same subset of NF-B proteins as FIG. 6. IB␣ 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 IB␣ or recombinant GST-IB␣ (68 -243) in the presence of an ATP-regenerating system and the recombinant basic NLS nuclear import machinery consisting of 1 M importin ␣, 400 nM importin ␤, 1.4 M Ran wt, and 1.5 M p10. Panel B, digitonin-permeabilized HeLa cells were incubated at 30°C for 30 min with BSA-NLS-FITC and recombinant wt IB␣ in the presence of an ATP-regenerating system and HeLa cytosol previously incubated with either GST or GST-IB␣(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. IB␣. Upon certain stimuli, IB␤ is degraded and subsequently resynthesized, but it accumulates as a hypophosphorylated protein. Interaction of this newly synthesized IB␤ with NF-B fails to mask both the NLS and DNA binding domain of NF-B and therefore leads to a complex able to enter the nucleus by piggy-back and activate transcription (78,79). Bcl-3, an IB protein containing two basic NLS in its NH 2 -terminal domain, is expressed predominantly in the nucleus of lymphoid cells and binds NF-B p50 and p52 homodimers. This interaction does not mask NF-B NLS and results in the nuclear import of (p50) 2 ⅐Bcl-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 IB␤ 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 IB␤ 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 IB 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-and lacking a recognizable NLS accumulates in the nucleus (83). It has been reported recently that ankyrin repeats of 53BP2 and GABP␤ 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.