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Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes

2000; Springer Nature; Volume: 19; Issue: 16 Linguagem: Inglês

10.1093/emboj/19.16.4362

1460-2075

Gerd Lipowsky,

Trace Elements in Health

Article15 August 2000free access Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes Gerd Lipowsky Gerd Lipowsky ZMBH, D-37073 Göttingen, Germany Search for more papers by this author F.Ralf Bischoff F.Ralf Bischoff DKFZ, 69120 Heidelberg, Germany Search for more papers by this author Petra Schwarzmaier Petra Schwarzmaier ZMBH, D-37073 Göttingen, Germany Search for more papers by this author Regine Kraft Regine Kraft MDC, 13122 Berlin-Buch, Germany Search for more papers by this author Susanne Kostka Susanne Kostka MDC, 13122 Berlin-Buch, Germany Search for more papers by this author Enno Hartmann Enno Hartmann Universität Göttingen Abteilung Biochemie II, D-37073 Göttingen, Germany Search for more papers by this author Ulrike Kutay Ulrike Kutay ZMBH, D-37073 Göttingen, Germany Present address: Institut für Biochemie, ETH, Universitätsstrasse 16, CH-8092 Zürich, Switzerland Search for more papers by this author Dirk Görlich Corresponding Author Dirk Görlich ZMBH, D-37073 Göttingen, Germany ZMBH, INF 282, 69120 Heidelberg, Germany Search for more papers by this author Gerd Lipowsky Gerd Lipowsky ZMBH, D-37073 Göttingen, Germany Search for more papers by this author F.Ralf Bischoff F.Ralf Bischoff DKFZ, 69120 Heidelberg, Germany Search for more papers by this author Petra Schwarzmaier Petra Schwarzmaier ZMBH, D-37073 Göttingen, Germany Search for more papers by this author Regine Kraft Regine Kraft MDC, 13122 Berlin-Buch, Germany Search for more papers by this author Susanne Kostka Susanne Kostka MDC, 13122 Berlin-Buch, Germany Search for more papers by this author Enno Hartmann Enno Hartmann Universität Göttingen Abteilung Biochemie II, D-37073 Göttingen, Germany Search for more papers by this author Ulrike Kutay Ulrike Kutay ZMBH, D-37073 Göttingen, Germany Present address: Institut für Biochemie, ETH, Universitätsstrasse 16, CH-8092 Zürich, Switzerland Search for more papers by this author Dirk Görlich Corresponding Author Dirk Görlich ZMBH, D-37073 Göttingen, Germany ZMBH, INF 282, 69120 Heidelberg, Germany Search for more papers by this author Author Information Gerd Lipowsky1, F.Ralf Bischoff2, Petra Schwarzmaier1, Regine Kraft3, Susanne Kostka3, Enno Hartmann4, Ulrike Kutay1,5 and Dirk Görlich 1,6 1ZMBH, D-37073 Göttingen, Germany 2DKFZ, 69120 Heidelberg, Germany 3MDC, 13122 Berlin-Buch, Germany 4Universität Göttingen Abteilung Biochemie II, D-37073 Göttingen, Germany 5Present address: Institut für Biochemie, ETH, Universitätsstrasse 16, CH-8092 Zürich, Switzerland 6ZMBH, INF 282, 69120 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:4362-4371https://doi.org/10.1093/emboj/19.16.4362 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Transport receptors of the importin β superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin β family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A. Introduction Importin β-related nuclear transport receptors mediate many of the nucleocytoplasmic transport events (reviewed in Dahlberg and Lund, 1998; Mattaj and Englmeier, 1998; Görlich and Kutay, 1999; Nakielny and Dreyfuss, 1999). These receptors shuttle between nucleus and cytoplasm, interact with nuclear pore complexes (NPCs) and recognize and bind cargo molecules. According to the direction in which they carry a cargo, they can be classified as importins or exportins. The directionality of transport appears to be determined by a RanGTP gradient across the nuclear envelope (NE). Transport receptors are RanGTP-binding proteins that respond to this gradient by loading and unloading their cargo in the appropriate compartment; importins bind their import substrates at low RanGTP levels in the cytoplasm, release them upon RanGTP binding in the nucleus (Rexach and Blobel, 1995; Chi et al., 1996; Görlich et al., 1996b; Izaurralde et al., 1997; Schlenstedt et al., 1997; Siomi et al., 1997; Jäkel and Görlich, 1998) and return as cargo-free RanGTP–importin complexes to the cytoplasm (Izaurralde et al., 1997; Hieda et al., 1999). RanGTP–importin complexes are finally disassembled by the concerted action of cytoplasmic RanGAP and RanBP1 (or RanBP2), releasing the importin to bind and import another substrate molecule (Bischoff and Görlich, 1997; Floer et al., 1997; Lounsbury and Macara, 1997). Exportins are regulated in a precisely converse manner to importins. They bind their export substrates preferentially at high RanGTP concentrations in the nucleus and exit the nucleus as trimeric cargo–exportin–RanGTP complexes (Fornerod et al., 1997a; Kutay et al., 1997a, 1998; Arts et al., 1998a; Kaffman et al., 1998). The cytoplasmic disassembly of such complexes also requires RanGAP and RanBP1 (or RanBP2) (Bischoff and Görlich, 1997; Kutay et al., 1997a), and results in cargo release from the exportins and GTP hydrolysis, and allows the exportins to re-enter the nucleus and to accomplish further rounds of export. The overall sequence similarity between the various transport receptors is low and, in many cases, restricted to the N-terminal RanGTP-binding motif (Fornerod et al., 1997b; Görlich et al., 1997). This can be explained at least in part by the fact that these receptors bind very different cargoes, such as the basic IBB domain in the case of importin β (Görlich et al., 1996a; Weis et al., 1996), tRNA in the case of exportin-t (Arts et al., 1998a; Kutay et al., 1998) or a leucine-rich nuclear export signal (NES) in the case of CRM1 (Fischer et al., 1995; Wen et al., 1995; Fornerod et al., 1997a; Stade et al., 1997). The RanGTP-binding motif can thus be considered as a diagnostic feature of importin β-related transport receptors, and in fact it allowed the identification of most of the 14 family members from the yeast Saccharomyces cerevisiae (Fornerod et al., 1997b; Görlich et al., 1997). Higher eukaryotes employ an even larger number of transport receptors; at least 22 in the case of mammals (see Görlich and Kutay, 1999; Nakielny and Dreyfuss, 1999; this study and our unpublished data). It has been a major effort in the field of nuclear transport to allocate functions to each of these receptors and to identify the transport signals that they recognize. While characterizing a novel mammalian exportin (see below), we identified eIF-5A (eukaryotic translation initiation factor 5A) as a potential export substrate. eIF-5A was originally isolated as a candidate translation factor from a polyribosome-bound fraction (Kemper et al., 1976; Benne et al., 1978) and was suggested to be involved in the formation of the first peptide bond. Subsequent studies, however, indicated that translational initiation is not directly affected by a loss of eIF-5A function (Kang and Hershey, 1994; Zuk and Jacobson, 1998) and that eIF-5A is not required to assemble translation initiation complexes. Bona fide translation initiation factors are usually present substoichiometrically to ribosomes and associate with them only during initiation. In contrast, eIF-5A is present in excess over ribosomes (Hershey, 1994) and largely bound to cytoplasmic, puromycin-sensitive structures that might represent (rER-bound) polysomes (Shi et al., 1997). One should therefore assume that if eIF-5A functions in translation, then it is at some step subsequent to initiation. eIF-5A carries a uniquely modified lysine that is referred to as hypusine [Nϵ-(4-amino-2-hydroxybutyl) L-lysine; Shiba et al., 1971; Park et al., 1982; Cooper et al., 1983]. eIF-5A is apparently the only hypusine-containing protein. It is present in eukaryotes (Gordon et al., 1987) and archaebacteria (Bartig et al., 1992) and is in fact one of the best conserved proteins between the two kingdoms. eIF-5A itself and its modification pathway are essential for viability in yeast (Schnier et al., 1991; Wöhl et al., 1993; Sasaki et al., 1996). The X-ray structures of eIF-5A from two archaea species have been solved (Kim et al., 1998; Peat et al., 1998). They show eIF-5A to be composed of two compact domains that are linked by a flexible hinge. N-terminal domain I contains the hypusine modification site in an extended, protruding and highly conserved loop. The modification is unlikely to have a major effect on the eIF-5A structure. Instead, its absolute conservation indicates that the hypusine mediates essential interactions with other macromolecules. Hypusine is a 2-fold positively charged amino acid and resembles nucleic acid-binding polyamines such as spermine and spermidine. Domain II is similar to the RNA-binding motif found in the prokaryotic cold shock protein CspA, which has been suggested to function as an RNA chaperone (Jiang et al., 1997). Domain II and the hypusine-containing loop from domain I might thus constitute a bipartite RNA-binding site (Kim et al., 1998; Peat et al., 1998). An RNA-binding activity of eIF-5A has indeed been detected in vitro and has been found to depend on the hypusine modification (Liu et al., 1997). However, it is still unclear whether this RNA-binding activity reflects a genuine function of eIF-5A and if so what the physiological RNA ligand(s) of eIF-5A might be. Eubacteria lack a hypusine-modified eIF-5A equivalent. However, the sequence similarity between eubacterial EF-P and archaebacterial/eukaryotic eIF-5A is significant enough to assume safely that the two represent homologous proteins (Kyrpides and Woese, 1998). EF-P is essential for viability in Escherichia coli (Aoki et al., 1997) and present in all eubacterial genomes examined so far. eIF-5A/EF-P can thus be considered a universally conserved and essential protein; it is apparently the only known protein that falls into this category whose function has remained elusive. Loss of eIF-5A function is ultimately lethal (Schnier et al., 1991; Kang and Hershey, 1994; Sasaki et al., 1996; Zuk and Jacobson, 1998; Jansson et al., 2000). In their mortal phase the cells show diverse defects. Saccharomyces cerevisiae cells stop cell division, but continue to enlarge in size. One specific defect is an impaired degradation of mRNA, particularly of short-lived messages (Zuk and Jacobson, 1998). The defect apparently occurs between mRNA decapping and degradation by the Xrn1p exonuclease and could be consistent with the assumed RNA-binding activity of eIF-5A. However, it is unclear whether RNA turnover is the primary function that makes eIF-5A indispensable for viability in all organisms. Upon eIF-5A inactivation in yeast, the overall rate of translation is reduced by ∼30%, but is not immediately abolished (Kang and Hershey, 1994; Zuk and Jacobson, 1998), suggesting that translational initiation and elongation can proceed in the absence or at very low concentrations of eIF-5A. The intermediate effect on the overall translation rate and the absolute requirement of eIF-5A for viability could possibly be explained by a failure of eIF-5A-deficient cells to synthesize a subset of proteins or to synthesize them in a biologically active form. Here we report the identification of a novel mammalian member of the importin β superfamily, which we refer to as exportin 4 (Exp4). We show that Exp4 functions as a nuclear export receptor for eIF-5A and perhaps for other substrates as well. As eIF-5A appears to be an RNA-binding protein, it might function as an export adapter in RNA export. Alternatively, Exp4-mediated eIF-5A export might be used to restrict eIF-5A activity to the cytoplasm. Results Identification and molecular cloning of Exp4 Many of the nuclear transport pathways are mediated by RanGTP-binding proteins of the importin β superfamily. To identify further mammalian family members, we used affinity chromatography on immobilized RanGTP to enrich them from a HeLa cell extract (see Figure 1). The RanGTP-bound fractions were separated by SDS–PAGE and bands between 90 and 140 kDa were cut out. Peptide sequencing identified not only the already known transport receptors, but also a novel protein, which for reasons detailed below we refer to as Exp4. Figure 1.Identification of exportin 4 (Exp4) amongst RanGTP-binding proteins from HeLa cells. A HeLa cell extract was subjected to binding to either immobilized RanGDP (wild-type protein) or RanGTP (Q69L mutant). Starting material and bound fractions were analysed by SDS–PAGE followed by Coomassie Blue staining. Protein bands specifically recovered on the RanGTP beads were analysed by microsequencing. Identified proteins are indicated. Exp4 is a novel protein. Download figure Download PowerPoint We then used this partial sequence information to isolate a full-length Exp4 cDNA from a mouse cell line (see Materials and methods). It codes for a 129.9 kDa protein (Figure 2) with an isoelectric point of 4.9. Exp4 shares distant, but still significant similarity with other members of the importin β superfamily. The region of homology is restricted to the N-terminal RanGTP-binding motif and gives the best matches to exportin 1 (CRM1) and to the tRNA export receptor exportin-t. Other parts of the proteins do not show significant homologies to any other known protein. The multiple alignment of the known importin β-like factors (based on full-length sequences) indicates that Exp4 is the most distant member of this superfamily identified so far (not shown). Figure 2.Primary sequence of Exp4. The partial sequence information from human Exp4 was used to clone a full-length cDNA coding for mouse Exp4. The figure shows the deduced amino acid sequence of the open reading frame. Peptides obtained by protein sequencing are underlined. Download figure Download PowerPoint Identification of an Exp4-specific export substrate At this stage it was unclear whether Exp4 would mediate import, export or indeed nucleocytoplasmic transport at all. We therefore expressed Exp4 in E.coli and first established that the recombinant protein interacts with RanGTP in a manner that closely resembles other importin β-related factors (see below). As detailed in the Introduction, these transport receptors use RanGTP binding to regulate the interactions with their respective cargoes. To test whether Exp4 would behave in an analogous way, and to identify potential transport substrates, we tested which components from a HeLa extract it would bind to. The binding was performed with or without addition of the GTPase-deficient RanQ69L mutant (loaded with GTP). This mutant remains in the GTP-bound form even in the presence of cytoplasmic RanGAP (Bischoff et al., 1994; Klebe et al., 1995) and can thus be used to mimic a nuclear environment. As seen from Figure 3A, two proteins bound to the immobilized Exp4 in a Ran-regulated manner. These were identified by peptide sequencing as thymidylate synthase and eIF-5A. Thymidylate synthase appeared preferentially bound to the Ran-free form of Exp4 and could thus represent an import substrate for this transport receptor. However, subsequent tests could not detect any import activity of Exp4 towards recombinant thymidylate synthase (data not shown). We therefore did not characterize this interaction further. Figure 3.Identification of eIF-5A as a putative export substrate for Exp4. (A) zz-tagged fusions of Exp4, exportin-t and importin β were immobilized on IgG Sepharose and used for binding from a HeLa cell extract. Analysis was as in Figure 1. Load in the bound fractions corresponds to 35 times the starting material. Note, eIF-5A was specifically recovered with immobilized Exp4 in the presence of the RanQ69L mutant (3.2 μM GTP form). eIF-5A binding was not detectable when RanQ69L was omitted or Exp4 replaced by another nuclear transport receptor. ‘*’ indicates thymidylate synthase; ‘**’ indicates actin that also bound non-specifically in the presence of RanQ69L to the control beads that did not contain any zz fusion protein. (B) Binding from HeLa extract to immobilized zzRanQ69L (GTP). When a saturating concentration (1 μM) of exogenous Exp4 had been added, both Exp4 and a prominent eIF-5A band were recovered in the bound fraction, indicating the formation of a trimeric eIF-5A–Exp4–zzRanGTP complex. (C) Binding from HeLa extract to immobilized RanGTP after addition of 1 μM exogenous Exp4, CRM1, CAS or exportin-t. eIF-5A present in the bound fractions was detected by western blotting. Note, eIF-5A assembled into an export complex with Exp4 but not with CRM1, CAS or exportin-t. Download figure Download PowerPoint eIF-5A was selectively recovered with the RanGTP-bound form of Exp4, but not with any other transport receptor when tested under identical conditions (Figure 3A). This suggests that eIF-5A can specifically assemble into a trimeric eIF-5A–Exp4–RanGTP export complex and might thus represent an export substrate of Exp4. To confirm these results, we performed the binding from the HeLa extract the other way around and immobilized RanQ69LGTP instead of Exp4 (Figure 3B and C). Without further addition, very little eIF-5A was recovered in the bound fraction, probably because concentration of endogenous Exp4 in the extract is low. However, when 1 μM Exp4 was added to the incubation mixture, a prominent, Coomassie-stainable eIF-5A band, along with Exp4 itself, was recovered in the RanGTP-bound fraction, indicating the formation of a trimeric eIF-5A–Exp4–Ran complex (Figure 3B). The identity of the eIF-5A band was verified by immunoblotting (Figure 3C). The same panel also shows that eIF-5A specifically assembles into complexes only with Exp4, but not with other exportins such as CAS, exportin-t or CRM1. Figure 3A demonstrated that the eIF-5A–Exp4 interaction is greatly enhanced by the presence of RanGTP. To test whether this represents a truly co-operative binding between these three components, we employed a more quantitative assay. This assay is based on the observation that binding of an importin β-like factor to RanGTP prevents GTPase activation by RanGAP (Floer and Blobel, 1996; Görlich et al., 1996b). GTP hydrolysis can easily be quantified and used to calculate the proportion of Ran that is associated with the respective factor. From the dose dependence of the effects one can estimate dissociation constants of the complexes. We first varied the concentration of Exp4 in the absence of eIF-5A (Figure 4A) and observed a half-maximum RanGTP binding at an Exp4 concentration of ∼40 nM, which corresponds to the apparent KD (equilibrium dissociation constant) for the RanGTP–Exp4 interaction. The KD was lowered to ∼1.5 nM in the presence of a saturating concentration of native, fully modified eIF-5A, indicating co-operative binding. This co-operative effect was highly specific for the eIF-5A–Exp4 interaction and was not observed for combinations of Exp4 with other export substrates, such as importin α, tRNA or the CRM1 substrate snurportin 1. Our data also indicate that eIF-5A is bound by Exp4 at least 1000 times better than by human CRM1 (compare Figure 4A with B), exportin-t or CAS (Figure 3C and data not shown). Figure 4.Quantitative characterization of the eIF-5A–Exp4–RanGTP interaction. (A) The assay exploits the observation that binding of RanGTP to an importin β-like factor prevents GTPase activation by RanGAP. Ran-[γ-32P]GTP (50 pM) was pre-incubated at 15°C with the Exp4 concentrations indicated in the absence or presence of either eIF-5A (fully hypusinated), snurportin 1, importin α (imp α) or tRNA (2 μM each). After 30 min, a 30 s GTPase reaction was started by addition of 40 nM S.pombe RanGAP. Hydrolysis of Ran-bound GTP was determined as released [32P]phosphate. Note that the presence of eIF-5A increased the affinity of Exp4 for RanGTP ∼30-fold, while snurportin 1, importin α and tRNA had no effect. (B) Measurements were performed exactly as in (A) except that CRM1 was added instead of Exp4. Note that snurportin 1 bound selectively to CRM1, while eIF-5A showed no binding. Download figure Download PowerPoint The hypusine modification in eIF-5A apparently contributes to Exp4 binding The maturation of eIF-5A to the fully hypusinated form occurs in two steps. First, deoxyhypusine synthase transfers a 4-amino-butyl group from spermidine to the ϵ-amino group of lysine 50 (in the human sequence) to form deoxyhypusine (Park et al., 1982; Dou and Chen, 1990; Wolff et al., 1990). This reaction is evident in all eukaryotes and archaea species examined (Gordon et al., 1987; Bartig et al., 1990). The deoxyhypusine is then hydroxylated by an as yet unidentified enzyme to yield the mature hypusine. This second step occurs in all eukaryotes and crenarchaea species tested, but not in euryarchaea (Bartig et al., 1990), suggesting that deoxyhypusine can substitute for hypusine at least in some organisms. The hypusine/deoxyhypusine modification of eIF-5A is essential for viability (Sasaki et al., 1996) and thus for eIF-5A function. We therefore wanted to test whether the hypusine residue is also involved in the interaction with Exp4. To this end we had to generate eIF-5A at various stages of modification. The fully modified eIF-5A was enriched by a multi-step procedure from HeLa cells to a purity of >95% (see Materials and methods and Supplementary material, available at The EMBO Journal Online). Unmodified eIF-5A was obtained by recombinant expression of the human protein in E.coli (note that eubacteria lack hypusination). For best comparability, eIF-5A was expressed with its authentic N- and C-termini (i.e. untagged) and purified by conventional chromatography. The recombinant protein was properly folded as judged by the following criteria. First, it was soluble when expressed in E.coli. Secondly, it showed similar chromatographic properties to the native protein and eluted in sharp peaks from the ion exchange and gel filtration columns (note, these chromatographic procedures probe protein shape and charge distribution). Thirdly, it was an efficient substrate for deoxyhypusination. To obtain deoxyhypusinated eIF-5A, we expressed human deoxyhypusine synthase in E.coli and used the purified enzyme to modify recombinant eIF-5A in the presence of NAD and spermidine (see Materials and methods). We next wanted to determine apparent KDs for the interactions of the Exp4–RanGTP complex with the various forms of eIF-5A. For this purpose, we used the assay described for Figure 4A, but kept the concentration of Exp4 constant at 25 nM and instead varied that of eIF-5A in the assay. Increasing concentrations of eIF-5A promoted the RanGTP binding to Exp4. The half-maximum effect for the E.coli-expressed, unmodified eIF-5A was observed at a concentration of ∼75 nM, which can be taken as a rough estimate for the KD for dissociation of eIF-5A from the trimeric complex (see Table I). Deoxyhypusination of recombinant eIF-5A improved Exp4 binding ∼3-fold. The native, hypusine-modified eIF-5A bound the Exp4–RanGTP complex with a KD of ∼2 nM, i.e. ∼35 times more strongly than the unmodified protein. The hypusination site is located within eIF-5A at the middle of a flexible, highly exposed loop that appears not to be involved in secondary structures or other interactions with the rest of the eIF-5A molecule. This strongly suggests that the hypusine improves Exp4 binding not by changing eIF-5A conformation but instead by making a direct contact to Exp4. Our data also indicate that deoxyhypusine can only partially substitute for hypusine in the interaction with Exp4. Table 1. Apparent affinities of eIF-5A derivatives for Exp4–RanGTP complex eIF-5A species Apparent KD for dissociation from eIF-5A–Exp4–RanGTP complex Relative affinity for Exp4 (native eIF-5A = 100) Purified from HeLa cells, fully hypusinated 2 nM 100 Recombinant, non-modified 75 nM 2.5 Recombinant, deoxyhypusinated 25 nM 8 Recombinant domain I (residues 1–83), deoxyhypusinated ∼1–2 μM 0.1 Recombinant domain II >10 μM <0.01 As detailed in the Introduction, eIF-5A is composed of two domains. The N-terminal domain I (aa 1–83) harbours the hypusination site and was indeed an efficient substrate for deoxyhypusine synthase. Surprisingly, the isolated, deoxyhypusinated domain I bound Exp4 with only 1% affinity as compared with the identically modified full-length protein, while the complementary domain II (residues 83–154) alone showed <0.1% binding (see Table I). This suggests that Exp4 requires large parts of the eIF-5A molecule for recognition and does not just bind a short, contiguous export signal sequence. Exp4 mediates export of eIF-5A from the nucleus The way Exp4 interacts with eIF-5A closely resembles the interaction of other exportins with their respective export substrates in that binding is of high affinity in the presence of RanGTP, i.e. in a nuclear environment and weak without RanGTP, i.e. under cytoplasmic conditions (see Introduction). Also, just as is the case for other transport receptors, the trimeric eIF-5A–Exp4–RanGTP complex is disassembled in the simultaneous presence of cytoplasmic RanBP1 and RanGAP (data not shown). Taken together, this strongly suggests that Exp4 is the eIF-5A-specific exportin. However, we wanted to test this directly. To target eIF-5A to the nucleus in the first place, we fused it to the IBB domain, a potent importin β-dependent nuclear import signal (Görlich et al., 1996a; Weis et al., 1996). The fusion also contained the green fluorescent protein (GFP) for detection by fluorescence microscopy. As an internal control, a second export substrate, Texas Red-labelled importin α, was also added and simultaneously detected in a separate channel. Import into nuclei of permeabilized cells was performed in the presence of a Xenopus egg extract that had been depleted by immobilized RanGTP of endogenous importins and exportins. Importin β was re-added to allow import of importin α and the IBB–eIF-5A fusion. After 15 min incubation, importin α and the IBB–GFP–eIF-5A fusion had clearly accumulated in the nuclei (Figure 5). The mixture was then split into four, and either buffer, Exp4, CAS or CRM1 was added. Fifteen minutes later, each of the incubations was analysed by confocal microscopy. Exp4 specifically promoted export of the eIF-5A fusion, but had no effect on importin α export (Figure 5). Conversely, CAS promoted export of importin α as reported before (Kutay et al., 1997a), but left the eIF-5A fusion unaffected. The very small effect of CRM1 on nuclear localization of both importin α and eIF-5A is probably due to its more efficient NPC binding and competition of importin β-mediated import as compared with the other two exportins. Figure 5.Exp4 mediates nuclear export of an eIF-5A fusion protein. An IBB–GFP–eIF-5A fusion (0.4 μM) and Texas-Red-labelled Xenopus importin α (0.4 μM) were first allowed to accumulate in nuclei of permeabilized cells. Import was in the presence of an energy-regenerating system and a Xenopus egg extract that had been depleted of importin β-like transport receptors and replenished with importin β and RanBP1. Fifteen minutes later, the mixture was split into four and either buffer, 2 μM Exp4, CAS or CRM1 was added. After another 15 min, the distributions of the eIF-5A fusion and of importin α were recorded by confocal microscopy in the fluorescein and Texas Red channels, respectively. Note, Exp4 specifically promoted export of the eIF-5A fusion, but had no effect on importin α. Conversely, CAS promoted export of importin α but had no effect on eIF-5A localization. Download figure Download PowerPoint In Figure 5 we used the IBB domain to target eIF-5A to the nucleus artificially. We next wanted to know whether eIF-5A can also enter nuclei on its own and so we labelled untagged, recombinant eIF-5A at a 1:1 molar ratio with Alexa-maleimide to allow detection by fluorescence microscopy. When incubated with permeabilized cells, eIF-5A gave some cytoplasmic staining and also readily entered nuclei and accumulated in the nucleoli (Figure 6A). Consistent with the small size of eIF-5A (18 kDa), this accumulation occurred by passive diffusion as it was insensitive to dominant-negative importin β mutants (not shown) that are known to block facilitated NPC passage (Kutay et al., 1997b). When Exp4 was added along with Ran and an energy-regenerating system, the nucleolar and nuclear signals completely disappeared, indicating efficient nuclear export (Figure 6A). The recombinant eIF-5A lacks the hypusine modification and binds Exp4 ∼35 times more weakly than the fully modified, native protein. Export was nevertheless efficient, probably because Exp4 was added at 0.5 μM, a concentration above the KD (75 nM) for binding of unmodified eIF-5A. However, it was crucial to show that Exp4 would also export hypusinated eIF-5A and so we also performed in parallel the same assay with native eIF-5A. Native eIF-5A accumulated in the absence of Exp4 more strongly in the nucleoli than non-