The p53-induced factor Ei24 inhibits nuclear import through an importin β–binding–like domain

The p53-induced protein Ei24 can bind specifically to importin-β1 and importin-α2 to impede their normal role in nuclear import.


Introduction
Nuclear protein import is dependent on NLSs, which are recognized by members of the importin (IMP) superfamily of nuclear transport receptors (Poon and Jans, 2005). The best characterized pathway involves the recognition of an NLS-containing cargo by IMP1 directly or the IMP/1 heterodimer, where IMP is an adaptor protein (Cingolani et al., 2002;Poon and Jans, 2005). In the absence of IMP1, IMP is "autoinhibited" through an intrinsic NLS within IMP's IMP-binding (IBB) domain, which binds to its NLS binding site (Kobe, 1999;Harreman et al., 2003a,b;Goldfarb et al., 2004). Binding of IMP1 to the IBB domain relieves IMP autoinhibition to permit accessibility to the NLS binding site (Cingolani et al., 1999;Kobe, 1999;Conti and Kuriyan, 2000;Goldfarb et al., 2004). IMP1 subsequently mediates passage of the IMP/ heterodimercargo complex through the nuclear envelope-embedded nuclear pore, before dissociation of the complex in the nucleus upon binding to IMP1 of the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form (Poon and Jans, 2005).
Mechanisms of regulation of nuclear protein import, central to signal transduction/transcriptional outcomes in the nucleus, include those mediated by a specialized class of diverse cytoplasmic proteins, negative regulators of nuclear import (NRNIs), which sequester molecules in the cytoplasm to prevent their nuclear import. Cytoplasmic retention of the NLS-containing transcription factors NF-B and Gli1, for example, is effected by specific NRNIs, such as inhibitor of B (IB) and suppressor of fused (Su(fu)), respectively, which prevent IMP recognition by NLS masking (Jacobs and Harrison, 1998;Ding et al., 1999;Bergqvist et al., 2006). Analogously, the BRCA1binding protein BRAP2 (Li et al., 1998) can negatively regulate the nuclear import of different cellular and viral proteins, dependent on phosphorylation flanking the NLS (Fulcher et al., 2010). Finally, a truncated form of IMP2 ("CanRch1") from T he etoposide-induced protein Ei24 was initially identified as a p53-responsive, proapoptotic factor, but no clear function has been described. Here, we use a nonbiased proteomics approach to identify members of the importin (IMP) family of nuclear transporters as interactors of Ei24 and characterize an IMPbinding-like (IBBL) domain within Ei24. We show that Ei24 can bind specifically to IMP1 and IMP2, but not other IMPs, and use a mutated IMP1 derivative to show that Ei24 binds to the same site on IMP1 as the IMP IBB.
Ectopic expression of Ei24 reduced the extent of IMP1or IMP/1-dependent nuclear protein import specifically, whereas specific alanine substitutions within the IBBL abrogated this activity. Induction of endogenous Ei24 expression through etoposide treatment similarly inhibited nuclear import in a mouse embryonic fibroblast model. Thus, Ei24 can bind specifically to IMP1 and IMP2 to impede their normal role in nuclear import, shedding new light on the cellular functions of Ei24 and its tumor suppressor role.  Table S1 for details). (B) Endogenous Ei24 or IgG immunoprecipitates (IP)/input lysates from HEK293T cells were resolved by SDS-PAGE a human breast cancer line has been reported to inhibit nuclear accumulation of the tumor suppressor p53 (Kim et al., 2000).
Here, we describe the ability of the etoposide-induced protein Ei24 (etoposide-induced mRNA 2.4 kb) to act as an NRNI for the first time. Ei24 is an ER-localized protein (Zhao et al., 2005(Zhao et al., , 2012 originally identified as a p53-induced proapoptotic gene in etoposide-treated NIH3T3 cells (Lehar et al., 1996). It has been shown to be able to bind to the antiapoptotic protein Bcl-2 (Zhao et al., 2005), play a role in autophagy (Tian et al., 2010;Zhao et al., 2012), and induce growth arrest/apoptosis (Gu et al., 2000), but very little is known about how Ei24 may mediate these diverse functions. To address this, we used a nonbiased proteomics approach, identifying members of the IMP superfamily as binding partners of Ei24. We show that Ei24 contains an "IBB-like" (IBBL) domain conferring strong interaction with IMP2 and IMP1 in a similar fashion to the IBB of IMP2. We also show that Ei24 is able to reduce the nuclear accumulation of IMP/1-and IMP1-dependent cargoes, dependent on key basic residues within the IBBL domain; induction of endogenous Ei24 expression through etoposide treatment has the same effect. Collectively, the findings indicate that Ei24 is a novel IBBL-containing NRNI, shedding new light on Ei24's various cellular functions.

Ei24 interacts with specific IMPs and shares homology with IMP2
Previous studies have implicated Ei24 in growth arrest, apoptosis, and autophagy (Polyak et al., 1997;Gu et al., 2000;Zhao et al., 2005Zhao et al., , 2012Tian et al., 2010). We applied a nonbiased proteomics approach to identify potential interacting proteins of Ei24 from human embryonic kidney HEK293T cells transfected to express FLAG-tagged Ei24 (FLAG-Ei24), subjected to immunoprecipitation (IP) using anti-FLAG or -Ei24 antibodies ( Fig. 1 A) with preimmune serum as a control (Fig. S1 A). Mass spectrometric analysis identified Ei24, as well as several other proteins enriched in the anti-FLAG and -Ei24 immunoprecipitates, including the IMP superfamily members IMP1 and Ran-binding protein 7 (IMP7; Fig. 1 A and Table S1). CoIP of endogenous Ei24 under high stringency conditions followed by Western analysis using specific antibodies confirmed that IMP1, IMP2, and IMP7, but not IMP2 or IMP4, were complexed to Ei24 under physiological conditions ( Fig. 1 B and not depicted).
Perusal of the human and mouse Ei24 sequence revealed a conserved, basic 51-amino acid region ( Fig. 1 C) with 33% similarity to IMP2's IBB, a highly basic domain that is recognized specifically by IMP1 to facilitate formation of the IMP/1 heterodimer (Cingolani et al., 1999). We named this the IBBL domain of Ei24, and first tested whether it can confer binding to IMP1 and/or IMP2 in a similar fashion to the IBB of IMP. Consistent with this idea, endogenous Ei24 was found to colocalize with IMP1, IMP2, and IMP7, particularly in the perinuclear region of HeLa cells treated with etoposide to up-regulate Ei24 expression (Fig. 1 D; see Fig. S1, C and D, indicating a significant approximately twofold increase in the extent of colocalization, concentrated to a marked extent at the ER). Importantly, proteinase K digestion of preparations of ER from subcellular fractionation experiments of cells expressing N-terminally FLAG-tagged Ei24 indicated that the IBBL was exposed to the cytosol (Fig. S1 B), which is consistent with the idea that Ei24-IMP interaction in the cytoplasm/at the ER may occur under normal physiological conditions.

RanGTP can effect IMP1-dependent dissociation from Ei24
After IMP-mediated transport into the nucleus, binding of RanGTP to IMP1 dissociates the transport complex by displacing IMP2's IBB from IMP1 (Görlich et al., 1996b;Lee et al., 2005). To test if RanGTP can release endogenous IMP1 from Ei24, GFP-tagged Ei24 (GFP-Ei24) was immunoprecipitated in the absence or presence of GTPS, a nonhydrolyzable form of GTP that maintains Ran in the GTP-bound state, followed by Western analysis and densitometry (Fig. 2, A and B). As expected, IMP2 was coprecipitated with the control protein GFP-IMP1 in the absence of GTPS, but to a significantly (P < 0.05) lower extent in its presence. Importantly, GTPS decreased GFP-Ei24 binding to IMP1 (P < 0.01); similar results were observed upon the addition of recombinant Ran loaded with GTPS but not GDP to lysates from cells expressing GFP-Ei24 (Fig. 2 C). In contrast to the results for IMP1, pull-down of IMP2 by GFP-Ei24 was unaffected by GTPS (Fig. 2, A and B), whereas GFP-Ei24 did not coprecipitate other IMP homologues such as IMP2 or IMP13; GFP alone did not coprecipitate any IMP, which implies that the observed interactions are specific. These results support the idea that RanGTP can inhibit Ei24-IMP1 binding, which is consistent with Ei24's IBBL conferring interaction with IMP1 in an analogous fashion to the IBB of IMP.

Ei24 binds to IMPs with high affinity
We next tested whether Ei24's IBBL can bind directly to IMPs in vitro, comparing results to those of CanRch1, a form of IMP2 truncated at residue 89 that includes the IBB but not the NLS-binding domain (Kim et al., 2000). An AlphaScreen binding assay (Wagstaff and Jans, 2006) was performed using purified bacterially expressed His 6 -IMPs, biotinylated GST-CanRch1 and -Ei24N (2-225 aa) proteins ( Fig. 3 and Table 1).
before Western analysis using the specific antibodies indicated. (C) Multiple sequence alignment of the human and mouse Ei24 IBBL domains (predicted to form an -helical structure using Protein Homology/Analogy Recognition Engine version 2.0) together with IMP2/CanRch1, performed as described in Materials and methods. Numbers indicate the portion of the amino acid residues (single letter code) within the respective proteins. Gray and black shading indicates similar and identical residues, respectively. Sites of targeted mutation in this study are indicated by asterisks. (D) HeLa cells treated with 50 µM etoposide or DMSO vehicle control for 16 h were fixed and immunostained using specific antibodies for endogenous IMP1, IMP2, IMP7, or Ei24, and counterstained with DAPI. Merged images are shown at higher magnification (high mag., bottom panels). Yellow coloration in merged images indicates colocalization; quantitative analysis is presented in Fig. S1, C and D. Bars, 20 µm. reduced (>80%) binding to both Ei24N and CanRch1 (Fig. 3), which indicates that the Ei24 IBBL requires the same residues on IMP1 for high-affinity binding as those interacting with the prototypical IBB. Ei24N and CanRch1 also bound to IMP2IBB (truncated form of IMP2 lacking the IBB) in near identical fashion to IMP1 in terms of both maximal binding and K d (4 nM); binding to full-length (FL) IMP2 wild Ei24 2-225 corresponds to a clinically derived, breast cancer truncated form of Ei24 that lacks the C-terminal 133 residues but retains the IBBL (Gentile et al., 2001).
Ei24N resembled CanRch1 in binding to IMP1 with high affinity (5 nM); in contrast, a mutant of IMP1 unable to bind the IBB ("IBBm," mutated at residues W430/W472/ W864; Koerner et al., 2003), showed significantly (P < 0.0001) Figure 2. IMP1 recognition by Ei24 is inhibited in the presence of GTPS or recombinant RanGTP. (A) Lysates from HeLa-Bcl XL cells expressing GFP, GFP-Ei24, or GFP-IMP1 fusion proteins prepared 20 h after transfection were subjected to IP with GFP-Trap resin in the absence or presence of 1.7 mM GTPS. Western analysis was performed on input and immunoprecipitates (IP: GFP) using the specific antibodies indicated. (B) Densitometric analysis was performed on images such as those shown in A for binding of endogenous IMP1 (left) and IMP2 (right) to GFP-Ei24 or GFP-IMP1, as indicated. Pooled results (n ≥ 2) representing the mean ± SD (error bars) for IMP bound relative to no GTPS treatment (No add.) are shown; p-values (Student's t test) denote significant differences. NS, not significant. (C) Lysates from HeLa-Bcl XL cells expressing GFP-Ei24 or GFP alone were incubated for 20 min with 3 µM recombinant of Ran loaded with GTPS or GDP, before IP and Western blot analysis as in A. Ei24 negatively regulates nuclear import • Lieu et al.
IMP (Catimel et al., 2001), which is consistent with FL IMP's documented autoinhibited state (Kobe, 1999;Harreman et al., 2003a,b;Goldfarb et al., 2004). The comparable K d of 7 nM for the Ei24 IBBL and CanRch1 binding to FL and IBB truncated IMP2 indicates that the binding site for the type (WT) was more than twofold lower ( Fig. 3 and Table 1). These results concur with previous studies reporting a very similar K d for binding of the IMP IBB to either IMP1 or IMP2IBB (Harreman et al., 2003a,b), as well as strongly reduced binding of the IBB to FL compared with IBB-deleted Figure 3. Like CanRch1, Ei24 can bind directly to IMP1 and IMPIBB with high affinity. (A) 30 nM of biotinylated (B)-GST-Ei24(N) or B-GST-CanRch1 was added to increasing concentrations of IMP1 (top), IMP2 (bottom), or their mutant derivatives, as indicated, and an AlphaScreen assay was performed (see Materials and methods). Results are for a single experiment (representative of three separate assays), with values calculated as the percentage of binding to IMP1 WT or IMP2IBB, with apparent dissociation constants (K d ) indicated. (B) Pooled data represent the mean percentage of maximal binding (±SEM, error bars; n ≥ 3) to Ei24N/CanRch1 for each IMP relative to that of IMP1 WT; p-values are shown for significant differences. IBBm, IBB-binding mutant. p53 nuclear import is consistent with previous observations for CanRch1 (Kim et al., 2000), and implies that the C terminus/ ER localization of Ei24 is not essential for inhibition of p53 nuclear import.

Ei24 is a general inhibitor of IMP1and IMP2/1-mediated nuclear import
To test the ability of Ei24 to inhibit nuclear protein import generally, DsRed2-Ei24FL and DsRed2-Ei24N were coexpressed with constructs where GFP is fused to either the nuclear targeting signal of chicken anemia virus viral protein 3 (VP3; Wagstaff and Jans, 2006) or telomeric-repeat binding factor TRF1 (Forwood and Jans, 2002), both of which are dependent on IMP1 for nuclear import (Fig. 5, A and B; and Fig. S2). Results were compared with those for DsRed2-CanRch1. Although GFP-VP3 was strongly nuclear when expressed alone, increased cytoplasmic fluorescence was evident in cells coexpressing DsRed2-tagged Ei24FL, Ei24N, or CanRch1 ( Fig. 5 A), with quantitative analysis confirming a significant (P < 0.05) reduction (up to 30%) in the Fn/c (Fig. 5 B). A similar trend was observed for GFP-TRF1, which indicates a significant (P < 0.001) decrease in the Fn/c in the presence of DsRed2-tagged Ei24FL, Ei24N, or Can-Rch1, compared with in their absence (Fig. S2).
We also assessed the ability of Ei24 and CanRch1 to inhibit IMP/1-mediated nuclear import, observing a significant (P < 0.0001) up to 65% reduction in the Fn/c for GFP-p53 upon ectopic expression of DsRed2-tagged Ei24FL, Ei24N, or CanRch1 compared with that of GFP-p53 expressed alone (Fig. 5, A and B), with similar results for the prototypical IMP/1recognized cargo GFP-T-ag NLS (Fig. S2). Overexpression of DsRed2-Ei24 or -CanRch1 did not affect nuclear accumulation of GFP-aF10 (Fig. 5, A and B). Collectively, the results indicate that the N terminus of Ei24 is sufficient to inhibit nuclear import specifically mediated by either IMP1 alone or IMP/1. A polyarginine sequence within the IBBL domain is required for IMP1 binding and nuclear import inhibition Harreman et al. (2003b) previously reported that mutations to the polybasic "RRRR" motif in the IMP IBB domain greatly reduce the affinity of binding to IMP1; this motif is present within the IBBL domain (RRRR 62 ) from mouse and human Ei24 (Fig. 1 C). To test the contribution of these residues to Ei24 binding to IMP1, a FLAG-Ei24-encoding construct in which RRRR 62 was mutated to alanine (RRRRm) was tested for its ability to bind IMP1 in HEK293T cells compared with that of the comparable WT construct. Whereas FLAG-Ei24 WT clearly interacted with endogenous IMP1, the mutant derivative RRRRm (Fig. 5 C) or derivative lacking the entire IBBL domain (not depicted) significantly (P < 0.0001) reduced the levels of immunoprecipitated IMP1, which indicates that the RRRR motif within the IBBL is required for Ei24 binding to IMP1. To assess the effect of the RRRRm mutation on Ei24's ability to inhibit nuclear import, HeLa-Bcl XL cells were transfected to express GFP-p53, -VP3, or -aF10 together with FLAG-Ei24 WT or the mutant derivatives RRRRm or RKQ 79 (RKQm) as a control (Fig. 5, D and E). Nuclear accumulation of Ei24 IBBL is the NLS-binding site of IMP. IMP binding to GST alone was negligible (unpublished data), underlining the specificity of the interactions. All results were consistent with the idea that the Ei24 IBBL can bind directly to IMP1, in comparable fashion to the IMP IBB itself.

Endogenous Ei24 can inhibit p53 nuclear import
To test the effect of high-affinity interaction of Ei24 with IMPs on IMP2/1-dependent nuclear import, a p53 WT and knockout (KO) murine embryonic fibroblast (MEF) system was used together with etoposide treatment (Liang and Clarke, 1999;Kim et al., 2000), which markedly up-regulates Ei24 expression in WT MEFs but not in MEFs lacking p53 (p53 KO; Fig. 4, A and B). GFP-p53 was expressed in DMSO or etoposidetreated p53 WT and KO MEFs (Fig. 4 C), with GFP-aF10(696-794 aa), the nuclear import of which is IMP independent (Cai et al., 2002), and GFP alone as controls. Quantitative analysis to determine the nuclear-to-cytoplasmic fluorescence ratio (Fn/c) revealed that etoposide-treated WT MEFs significantly (P < 0.005) reduced (60%) the level of GFP-p53 nuclear accumulation compared with in its absence (Fig. 4 D); p53 KO MEFs showed no such effect. Notably, etoposide treatment of WT MEFs increased the number of GFP-p53 cytoplasmic aggregates compared with in its absence (Fig. 4 C), in contrast to KO MEFs, which showed few aggregates, implying that this localization may relate to Ei24 action. No aggregates were observed for the GFP-aF10 or GFP controls, which were unaffected by etoposide treatment (Fig. 4 D), underlining the specificity of the effect. p53-dependent up-regulation of Ei24 in response to etoposide can thus lead to inhibition of IMP/1mediated nuclear accumulation of GFP-p53, which implies that Ei24 may function in a negative feedback loop to contribute to dampening p53 activity in DNA damage (Lohrum et al., 2001;Nie et al., 2007). Reducing p53 nuclear access would in turn stem Ei24 up-regulation and initiate a return to steady state.

Cell culture and transfection
The HEK293T, HeLa, and HeLa-Bcl XL cell lines were all maintained in DMEM supplemented with 10% FCS, penicillin, and streptomycin in a humidified incubator with 5% CO 2 atmosphere at 37°C. HeLa-Bcl XL cells are resistant to apoptosis induced by Ei24 overexpression (Gu et al., 2000). Cells were transfected at 70-80% confluency using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

Confocal laser scanning microscopy (CLSM)
Cells were imaged 16-20 h after transfection on an imaging system (Yokogawa CSU10 based CLSM system; Ultraview; PerkinElmer) with an EM charge-coupled device camera (Andor Technology) and a 100×/1.4 NA oil immersion objective lens (Olympus) or an inverted CLSM system (C1 Inverted; Nikon) using a 100×/1.4 NA oil immersion objective lens (Nikon; Monash Micro Imaging). Cells were imaged in phenol red-free DMEM (Life Technologies), and live-cell imaging was routinely performed on a stage heated to 37°C. The Andor iQ and NIS-Elements version 4.10 software was used for image acquisition on the Ultraview (PerkinElmer) and CLSM (C1 Inverted; Nikon) systems, respectively. Digitized images were subsequently analyzed using the ImageJ 1.33u software (National Institutes of Health) to calculate the nuclear (F n ) to cytoplasmic (F c ) fluorescence ratio (F n/c ), corrected for by subtracting the background fluorescence (F b ). ImageJ software was also used to alter the brightness and contrast levels uniformly across all samples in the same assay to enhance visibility and for pseudocoloring where appropriate.

Indirect immunofluorescence
Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) 16-20 h after transfection and permeabilized using 0.2% Triton X-100 (Sigma-Aldrich) in blocking buffer (1% BSA in PBS). Non-specific binding sites were blocked with blocking buffer and immunostained with primary antibodies anti-Ei24 (Ab35) at 1:100, anti-IMP1 (Abcam) at 1:500, anti-IMP2 (BD) at 1:200, the IMP1 nuclear import cargo GFP-VP3 was significantly (P < 0.05) reduced by Ei24 WT and the RKQm mutant, in stark contrast to coexpression with RRRRm. Comparably, there was a significant (up to 35%) decrease in the Fn/c of GFP-p53 in the presence of Ei24 WT (P < 0.001) and RKQm (P < 0.05), respectively, but not RRRRm, relative to GFP-p53 alone (Fig. 5,  D and E). A similar trend was observed for the IMP/1recognized cargo, GFP-T-ag NLS (Fig. S2). No effect of Ei24 WT, RRRRm, or RKQm was detectable for nuclear accumulation of GFP-aF10, which is consistent with the idea that Ei24 inhibition of nuclear import is specific for IMP/1-and IMP1dependent cargoes. Thus, Ei24's conserved RRRR 62 motif, but not RKQ 79 , is central to Ei24-dependent inhibition of nuclear import through specific binding to IMP1.
In summary, this is the first study to shed light on the function of the ER-localized p53-induced factor Ei24, showing that it possesses a novel IBBL domain that retains key properties of the prototypical IBB of IMP in terms of ability to bind to IMP1, as well as to IMP, through the same binding site as that used to bind the IBB. Instead of facilitating nuclear import, however, Ei24 inhibits IMP/1-and 1-dependent nuclear protein import by sequestering IMP1 and possibly IMP in the cytoplasm/at the ER. We postulate that there is a fine balance between IMPdependent nuclear import and inhibition thereof by Ei24, enabling fine tuning of the nuclear import efficiency of important proteins such as p53 during normal cell function, as well as during stress such as DNA damage. Perturbation of the balance between these import pathways can clearly be effected by changes in expression of Ei24, as shown here (Fig. 1 D and Fig. 4). Relatively small changes in the cellular pool of IMP1 and possibly IMP can significantly impact the nuclear transport efficiency of key nuclear import cargoes as shown here, and in turn, presumably, autophagy/stress responses and/or apoptosis (Gu et al., 2000).
Increased nuclear IMP levels have been reported to correlate with poor prognoses for patients with advanced breast cancer (Gluz et al., 2008), with nuclear import specifically enhanced in cancerous/transformed cells (Kuusisto et al., 2012). Clearly, deregulated IMP/1-mediated nuclear import is closely linked to tumorigenesis. The role of Ei24 in inhibiting IMPdependent nuclear protein import in this context may be absolutely critical, as well as the basis of Ei24's tumor suppressor properties, and is the focus of future work in this laboratory.

Materials and methods
Plasmid constructs FLAG-Ei24 (and mutant derivatives thereof), GFP-Ei24, DsRed2-Ei24, GFP-p53, GFP-VP3, GFP-T-ag NLS, GFP-TRF1, and GFP-aF10 were expressed under the control of a cytomegalovirus (CMV) promoter encoded by single assay representative of three separate experiments; p-values denote significant differences. (C) Lysates from HEK293T cells transfected to express FLAG-Ei24 WT, RRRRm mutant derivative, or vector alone were subjected to immunoprecipitation using anti-FLAG M2 antibody. Western analysis was performed on input and immunoprecipitates (IP: FLAG) using specific antibodies against IMP1 or Ei24, with -actin as a control. Densitometric analysis was performed on images such as those shown in C; results are for the mean ± SEM (error bars n = 3) for binding (%) relative to that for WT. (D) CLSM images of HeLa-Bcl XL cells transfected to coexpress the indicated GFP fusion proteins in the absence or presence of FLAG-Ei24 derivatives were fixed 20 h after transfection before being immunostained using an anti-FLAG M2 antibody. Bars, 10 µm. (E) Quantitative analysis of the extent of nuclear accumulation for the various GFP fusion proteins. Results shown are for the mean ± SEM (error bars; n ≥ 40) for a single assay representative of three independent experiments. Statistical analysis was performed as in B. 10 times. Precleared lysate was added to 10 µg anti-Ei24 (Sigma-Aldrich) or rabbit IgG (Santa Cruz Biotechnology, Inc.) antibody and 50 µl Protein A/G plus agarose slurry (Santa Cruz Biotechnology, Inc.), and incubated overnight at 4°C. Immunoprecipitates were washed with RIPA buffer and eluted in 3× Laemmli sample buffer. HeLa-Bcl XL cells grown in 6-cm 2 dishes expressing GFP-Ei24 or GFP-IMP1 fusion proteins were lysed with dilution buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.5 mM EDTA), 0.5% NP-40 (Sigma-Aldrich), and Complete EDTA-free protease inhibitors (Roche). In some experiments, lysates were preincubated with a final concentration of 1.7 mM GTPS (Sigma-Aldrich) or 3 µM recombinant Ran reconstituted with GTPS or GDP for 20 min on ice. Immunocomplexes were bound to and eluted from the GFP-Trap resin (ChromoTek) according to the manufacturer's instructions. Protein from whole-cell extracts or immunoprecipitates were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Pall). Specific proteins were detected on membranes probed with anti-GFP (Roche) at 1:1,000, anti-IMP1 (provided by D. Görlich,, Max Planck Institute, Göttingen, Germany; or from Abcam) at 1:1,000, anti-IMP4 (KPNA3; Abcam) at 1:500, anti-IMP2 (BD) at 1:1,000, anti-IMP2 (BD) at 1:500, anti-IMP13 (rabbit polyclonal antibody generated against the synthetic peptides LPEEFQTSRLPQYRKGLVR and EQKDTF-SQQILRERVNKRRVK; Tao et al., 2006) at 1:1,000, anti-FLAG antibody (Sigma-Aldrich), or anti-BiP (Cell Signaling Technology), together with species-specific HRP-conjugated secondary antibody (EMD Millipore). Protein bands were visualized using Western Lightning ECL reagent (PerkinElmer) and chemiluminescence detection on x-ray film (Fujifilm). Densitometric analysis of protein bands was performed on digitized images of immunoblots using the ImageJ 1.33u software (Kaur et al., 2013).

Mass spectrometry (MS)
Immunoprecipitated proteins transferred to nylon membranes were identified by Sypro Ruby Protein Gel Stain (Molecular Probes; Life Technologies), excised, reduced, and alkylated with iodoacetamide, and digested with trypsin. The unfractionated tryptic digest was subjected to MS using the 4700 Proteomics Analyzer (Applied Biosystems), which employs matrixassisted laser desorption/ionization (MALDI) in conjunction with tandem time-of-flight (TOF) mass analyzers. The digest was introduced into the instrument in a crystalline matrix of -cyano-4-hydroxycinnamic acid. Database searches were performed with GPS Explorer software (Applied Biosystems), using the Mascot search engine (Perkins et al., 1999). Protein assignments were made based on both the MS and MS/MS spectra. Top-ranking proteins that had a significant (P < 0.05) Mascot score from each band were identified (see Table S1 for detailed analyses). The protein scores were derived from ions scores as a nonprobabilistic basis for ranking protein hits. The Swiss-Prot database was used for protein identification.

Membrane topology analysis
Membrane topology for Ei24 was assessed using a modified method as described previously (Leighton and Schatz, 1995). In brief, cell pellets from N-terminally FLAG-tagged Ei24 transfected HEK293 cells were washed with cold 1× PBS and resuspended in buffer A (10 µM NaCl, 1.5 µM MgCl 2 , and 10 µM Tris, pH 7.5, containing complete proteinase inhibitor cocktail) for 1 h on ice. Cells were homogenized with a type B Dounce homogenizer until 80% of cells were lysed. An equal volume of buffer B (210 µM mannitol, 70 µM sucrose, 5 µM Tris, pH 7.5, and 1 µM EDTA, pH 7.5) was added immediately and the lysate was centrifuged at 700 g for 10 min at 4°C to remove nuclei/intact cells. The supernatant was transferred into a new tube and then centrifuged at 1,200 g for 15 min at 4°C to isolate ER pellets, which were then resuspended in buffer B and aliquoted in 200-µg protein amounts. ER fractions were incubated for 30 min on ice in the presence or absence of 1 mg/ml proteinase K and inactivated using cold trichloroacetic acid. After centrifugation, the fractions were analyzed by Western blotting (as described earlier in the Materials and methods).
Online supplemental material Fig. S1 (A and B) shows proteomic analysis of FLAG-Ei24 immunoprecipitates with a preimmune antibody control and Ei24's topology at the ER, with Table S1 showing MS analysis details. Fig. S1 (C and D) shows quantitative colocalization analysis of Ei24 and IMPs in etoposidetreated HeLa cells. Fig. S2 demonstrates that Ei24 can inhibit IMP1-and IMP2/1-mediated nuclear translocation of GFP-TRF1 and -T-ag NLS, respectively. Online supplemental material is available at http://www.jcb .org/cgi/content/full/jcb.201304055/DC1. anti-IMP7 (GeneTex) at 1:500, or anti-FLAG M2 (Sigma-Aldrich) at 1:200 for 90 min, followed by a 60-min incubation with Alexa Fluor 488-or Alexa Fluor 568-conjugated secondary antibodies (Molecular Probes) at 1:1,000. Coverslips were mounted onto glass slides with 4% (wt/vol) propyl-gallate in 90% glycerol (Sigma-Aldrich) or ProLong Gold antifade reagent with DAPI mounting media (Molecular Probes). Cells were imaged on the Ultraview or C1 Inverted CLSM systems using a 100×/1.4 NA oil immersion objective lens at room temperature. Digitized images were analyzed and processed using ImageJ software as described in the previous section.

Quantitative colocalization
Colocalization analysis was performed on digitized CLSM images using the ImageJ 1.33u software, where a defined Ei24 threshold signal above background levels (secondary antibody alone control) was overlaid onto that of the IMP signal to produce a merged image for colocalized Ei24 and IMP pixels. The pixel intensity of the merged image was expressed as a percentage of the total IMP signal to calculate the mean percentage of IMP colocalized with Ei24.
Protein expression, purification, and biotin labeling GST-Ei24N (2-225 aa), GST-CanRch1, and GST-Ran (WT/Q69L; Tachibana et al., 2000;Ogawa et al., 2012) fusion proteins were expressed in Escherichia coli strain BL21(DE3) containing a pRARE2 plasmid. Cultures were grown to an OD 600 of 0.6-1 and protein expression was induced with 0.1-1 mM IPTG for 6 h at 18°C. Pellets were lysed in 50 mM Tris, pH 8.0, 500 mM NaCl, 5 mg/ml lysozyme (Research Organics), and Complete EDTA-free protease inhibitors (Roche) before being sonicated three times for 30 s at 30-s intervals. Protein was purified from cleared lysate with equilibrated glutathione Sepharose 4B resin (GE Healthcare) and incubated at 4°C for 2 h, before elution with 10 mM of reduced glutathione in 50 mM Tris, pH 8, and 500 mM NaCl. Glutathione was removed by dialysis and protein concentrated using a Centricon centrifugal filter unit with an appropriate molecular weight cutoff (Amicon; EMD Millipore). For AlphaScreen assays, recombinant GST fusion proteins were labeled using the EZ-Link Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher Scientific), and free biotin was separated from labeled protein using a PD-10 Desalting Column (GE Healthcare), according to the manufacturer's instructions. For the GST-Ran WT and GST-Ran(Q69L) proteins, GST was removed using PreScission Protease (GE Healthcare) according to manufacturer's instructions, and the proteins then loaded with GDP or GTPS (Sigma-Aldrich), respectively, as described previously (Tachibana et al., 2000).
Hexa-histidine (His 6 )-tagged full-length IMP2 and IMPIBB (residues 67-503; Yang et al., 2010) were provided by S. Yang (Monash University). In brief, protein was expressed in E. coli strain BL21(DE3) grown to an OD 600 of 1.0 and induced with 1 mM IPTG for 4 h at 28°C. Proteins were purified by affinity chromatography using nickel-nitrilotriacetic acid agarose (QIAGEN) and eluted in 500 mM imidazole. Dialysis was performed to remove imidazole. His 6 -IMP1 WT and IBBm (containing mutations W430A/W472A/W864A) proteins were expressed in the E. coli ER1003 strain and purification was performed on chitin beads followed by gel filtration on a Superose-12 column as described previously (Koerner et al., 2003).

AlphaScreen binding assay
The binding affinity of biotinylated GST-Ei24N, GST-CanRch1 or GST alone to His 6 -tagged IMPs was determined using the bead-based Alpha-Screen assay (PerkinElmer), as described previously (Wagstaff and Jans, 2006). In brief, 30 nM of biotinylated GST-Ei24N, GST-CanRch1, or GST alone was incubated with increasing concentrations of His 6 -tagged IMPs. After incubation with nickel-chelating acceptor and streptavidin-coated donor beads, results were read on a Fusion plate reader (PerkinElmer). Triplicate values were averaged and sigmoidal titration curves (threeparameter sigmoidal fit) were plotted using the SigmaPlot graphing program (Systat Software Inc.) to determine the dissociation constant (K d ) and maximal binding (B max ) value. It should be noted that the sensitivity of the AlphaScreen binding assay results in lower estimated K d values than various other assays (e.g., Catimel et al., 2001;Forwood and Jans, 2002;Harreman et al., 2003a,b;Fulcher et al., 2010), but relative/comparative values are comparable (e.g., see the legend for Table 1).