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Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p

1998; Springer Nature; Volume: 17; Issue: 8 Linguagem: Inglês

10.1093/emboj/17.8.2196

1460-2075

Bruno Senger, George Simos, F. Ralf Bischoff, Alexandre V. Podtelejnikov, Matthias Mann, Ed Hurt,

RNA and protein synthesis mechanisms

Article15 April 1998free access Mtr10p functions as a nuclear import receptor for the mRNA-binding protein Npl3p Bruno Senger Bruno Senger Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author George Simos George Simos Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author F.Ralf Bischoff F.Ralf Bischoff Abteilung Molekulare Biologie der Mitose, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Alexandre Podtelejnikov Alexandre Podtelejnikov EMBL, Proteins and Peptides, Meyerhofstraße 1, D-6911 Heidelberg, Germany Search for more papers by this author Matthias Mann Matthias Mann EMBL, Proteins and Peptides, Meyerhofstraße 1, D-6911 Heidelberg, Germany Search for more papers by this author Ed Hurt Corresponding Author Ed Hurt Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Bruno Senger Bruno Senger Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author George Simos George Simos Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author F.Ralf Bischoff F.Ralf Bischoff Abteilung Molekulare Biologie der Mitose, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany Search for more papers by this author Alexandre Podtelejnikov Alexandre Podtelejnikov EMBL, Proteins and Peptides, Meyerhofstraße 1, D-6911 Heidelberg, Germany Search for more papers by this author Matthias Mann Matthias Mann EMBL, Proteins and Peptides, Meyerhofstraße 1, D-6911 Heidelberg, Germany Search for more papers by this author Ed Hurt Corresponding Author Ed Hurt Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany Search for more papers by this author Author Information Bruno Senger1, George Simos1, F.Ralf Bischoff2, Alexandre Podtelejnikov3, Matthias Mann3 and Ed Hurt 1 1Biochemie-Zentrum Heidelberg (BZH), Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany 2Abteilung Molekulare Biologie der Mitose, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 3EMBL, Proteins and Peptides, Meyerhofstraße 1, D-6911 Heidelberg, Germany *Corresponding author. E-mail: [email protected] The EMBO Journal (1998)17:2196-2207https://doi.org/10.1093/emboj/17.8.2196 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info MTR10, previously shown to be involved in mRNA export, was found in a synthetic lethal relationship with nucleoporin NUP85. Green fluorescent protein (GFP)-tagged Mtr10p localizes preferentially inside the nucleus, but a nuclear pore and cytoplasmic distribution is also evident. Purified Mtr10p forms a complex with Npl3p, an RNA-binding protein that shuttles in and out of the nucleus. In mtr10 mutants, nuclear uptake of Npl3p is strongly impaired at the restrictive temperature, while import of a classic nuclear localization signal (NLS)-containing protein is not. Accordingly, the NLS within Npl3p is extended and consists of the RGG box plus a short and non-repetitive C-terminal tail. Mtr10p interacts in vitro with Gsp1p-GTP, but with low affinity. Interestingly, Npl3p dissociates from Mtr10p only by incubation with Ran-GTP plus RNA. This suggests that Npl3p follows a distinct nuclear import pathway and that intranuclear release from its specific import receptor Mtr10p requires the cooperative action of both Ran-GTP and newly synthesized mRNA. Introduction In eukaryotic cells, the nuclear interior is separated from the cytoplasm by the double nuclear membrane, and all transport between these two compartments occurs through the nuclear pore complexes (NPCs), large macromolecular assemblies embedded in the nuclear envelope (reviewed in Doye and Hurt, 1997). A number of recent discoveries have led to the development of a model for active nuclear protein import, the principles of which may also apply to active nuclear export of proteins and RNA (reviewed in Görlich and Mattaj, 1996; Corbett and Silver, 1997; Goldfarb, 1997; Nakielny et al., 1997; Nigg, 1997). According to this model, import of proteins into the nucleus is mediated by soluble and mobile receptors which bind to their import substrates through recognition of sequences that function as nuclear localization signals (NLS). These receptors are responsible for targeting of the import complex to the NPC and its subsequent translocation into the nucleoplasm where the complex is dissociated, the import cargo released and the receptor recycled back into the cytoplasm. In the case of the basic-type (classic) NLS which is found in a variety of different nuclear proteins, the receptor is a heterodimer consisting of importin α, which binds the NLS, and importin β, which mediates the interaction with the NPC through its affinity for repeat-containing nucleoporins. Importin β is the founding member of a large protein family which is characterized, despite the overall limited sequence homology, by the presence of an N-terminally located Ran-GTP-binding domain (Fornerod et al., 1997b; Görlich et al., 1997). Other members of this family which have been shown to function as nuclear import receptors include transportin and karyopherin β3. Transportin binds directly to the M9 domain of the hnRNP A1 protein and mediates its nuclear uptake (Pollard et al., 1996; Fridell et al., 1997), while it is also involved in the nuclear import of hnRNP F which lacks a similar domain (Siomi et al., 1997). The yeast homologue of transportin, Kap104p, has also been shown to be required for the nuclear import of mRNA-binding proteins such as Nab2p and Nab4p (Aitchison et al., 1996). Karyopherin β3 binds directly to a subset of ribosomal proteins (Yaseen and Blobel, 1997), and its yeast homologues Kap123p or Yrb4p have indeed been shown to be required for efficient nuclear import of ribosomal proteins (Rout et al., 1997; Schlenstedt et al., 1997). Importin β has also been implicated in the nuclear import of U snRNPs, a process that does not, however, require importin α (Palacios et al., 1997). These results demonstrate the existence of distinct nuclear import pathways, as defined by different types of NLSs and their cognate transport receptors of the importin β family. However, other members of the importin β family recently have been implicated in nuclear export processes and therefore termed exportins (reviewed in Ullman et al., 1997). CAS mediates the export of importin α from the nucleus (Kutay et al., 1997), while CRM1 functions as an export receptor for the leucine-rich nuclear export signals (NES) (Fornerod et al., 1997a; Neville et al., 1997; Ossareh-Nazari et al., 1997; Stade et al., 1997). This type of NES mediates nuclear export of proteins as well as RNA–protein complexes, as is the case for the human immunodeficency virus (HIV) protein Rev which binds to unspliced or partially spliced viral transcripts (for review, see Gerace, 1995). Nuclear export of cellular RNA may proceed by a similar mechanism, as NES-containing RNA-binding proteins could facilitate nuclear export of the bound RNA. In fact, export of U snRNAs, which requires the cap-binding protein complex (CBC) (Izaurralde et al., 1995), has been suggested to follow the same export route as Rev (Fischer et al., 1995). Similar NESs have also been found in other putative transport factors such as Kap95p (Iovine and Wente, 1997), RanBP1 (Richards et al., 1996), Gle1p (Murphy and Wente, 1996) and Mex67p (Segref et al., 1997). Another type of NES is represented by the M9 domain of hnRNP A1 (Michael et al., 1995). This protein and other hnRNP proteins shuttle between the nucleus and the cytoplasm and are suggested to play a role in mRNA export from the nucleus (Izaurralde et al., 1997a). It should be borne in mind, however, that a clear division of the members of the importin β family into importins and exportins may not be straightforward as, for example, the yeast proteins Kap123p and Pse1p have been implicated in both nuclear uptake of ribosomal proteins and nuclear export of mRNA (Seedorf and Silver, 1997). Apart from importins and exportins, a central role in nucleocytoplasmic transport is played by the small GTPase Ran and its effectors (reviewed in Moore and Blobel, 1994; Koepp and Silver, 1996; Goldfarb, 1997). Hydrolysis of GTP by Ran is probably used as an energy source for the translocation of import complexes into the nucleus through the NPCs. However, recent data suggest that nuclear export of various substrates does not require Ran-dependent GTP hydrolysis, but rather the presence of nuclear Ran-GTP (Izaurralde et al., 1997b; Richards et al., 1997). Indeed, Ran-GTP can trigger the dissociation of the importin–import substrate complex (Rexach and Blobel, 1995; Görlich et al., 1996; Izaurralde et al., 1997b), while it promotes the association of an exportin with the corresponding export cargo (Fornerod et al., 1997a; Kutay et al., 1997). The presence of Ran-GTP in the nucleoplasm is ensured by the nuclear localization of the Ran nucleotide exchange factor RCC1 and the exclusion from the nucleus of the GTPase-activating protein RanGAP1. Ran-GDP generated in the cytoplasm or in the vicinity of the NPC by the concerted action of RanGAP1 and RanBP1/RanBP2 is required both for nuclear import and for the last step of an export reaction, the release of the export substrate (Bischoff and Görlich, 1997). Therefore, the asymmetric distribution of the components of the Ran system may determine the directionality of the nuclear transport processes. However, Ran or energy may not be required for the nuclear uptake of importins in the absence of an import substrate (Kose et al., 1997). Genetic screens in yeast for mutants defective in poly(A)+ RNA export (Amberg et al., 1992; Kadowaki et al., 1992) and synthetic lethal screens starting with nucleoporin mutants (Doye and Hurt, 1997) have led to the identification of many factors required for poly(A)+ RNA nuclear export, which are neither exportins (i.e. not belonging to the importin β-like family) nor components of the Ran system. Among these, Nup159p (Gorsch et al., 1995), Mtr2p (Kadowaki et al., 1994b), Gle1p (Murphy and Wente, 1996) and Mex67p (Segref et al., 1997) could play a direct role in the mRNA export process, because conditionally lethal mutants exhibit a fast and strong onset of the mRNA export defect. Furthermore, Npl3p, a yeast hnRNP protein, which shuttles between the nucleus and cytoplasm, was also suggested, in analogy to hnRNP A1, to be involved in mRNA export (Lee et al., 1996). These proteins could function as adaptors between exportins and RNA molecules, as elements of the NPC required for RNP docking and translocation, or they may define RNA export pathways which are not exportin-dependent. Finally, as shuttling proteins have to be re-imported into the nucleus, they should also associate with import receptors. Yeast Nup84p was identified through its genetic interaction with the essential nucleoporin Nsp1p. It subsequently was shown that Nup84p forms a complex with five additional proteins, Nup120p, Nup85p, Sec13p, Seh1p and the C-terminal domain of Nup145p (Siniossoglou et al., 1996; Teixeira et al., 1997). The Nup84p complex is required for both nuclear pore distribution within the nuclear membrane and poly(A)+ RNA export. We report here the identification of MTR10 as a component that genetically interacts with NUP85. Mutations in the MTR10 gene previously have been reported to cause accumulation of poly(A)+ RNA in the nucleus (Kadowaki et al., 1994a). Mtr10p associates with the NPC and mediates the nuclear import of Npl3p with which it also physically interacts. Taking into account the homology of Mtr10p to members of the importin β family, we suggest that Mtr10p is a nuclear import receptor specialized in the transport of Npl3p and possibly other RNA-binding proteins. Results Identification of MTR10 in a synthetic lethal screen with the nup85Δ mutant Several members of the Nup84p nucleoporin complex including Nup85p are involved in mRNA export (Siniossoglou et al., 1996). To find components which functionally interact with Nup85p and thus could belong to the mRNA transport machinery, we isolated synthetic lethal (sl) mutants of the nup85Δ allele (see Materials and methods). An uncharacterized sl mutant (sl125) from this screen was found to be complemented by the MTR10 gene (DDBJ/EMBL/GenBank accession number Q99189). MTR10 initially was isolated in another genetic screen for poly(A)+ RNA export mutants (Kadowaki et al., 1994a) and encodes a 972 amino acid protein with a predicted mol. wt of 110 kDa. It has been reported recently that Mtr10p belongs to a protein family characterized by a sequence motif related to the Ran-binding site of importin β (Fornerod et al., 1997b; Görlich et al., 1997). Members of this family have been shown to function either as nuclear import receptors (importins) or as nuclear export receptors (exportins) (reviewed in Ullman et al., 1997). To study further the in vivo role of Mtr10p in nucleocytoplasmic transport, we generated mtr10 mutants. Deletion of the MTR10 gene causes a strong growth defect of cells at physiological temperatures (e.g. 30°C) and a complete growth arrest at the non-permissive temperature (37°C) (Figure 1A and B). By in vitro random mutagenesis of the isolated MTR10 gene, we could isolate several thermosensitive (ts) mutants, which revealed slightly reduced growth rates at 30°C, but still arrested at 37°C. One of these ts mutants, Ømtr10-7, was used for further analysis (Figure 1B). Figure 1.Generation and characterization of mtr10 mutants. (A) Tetrad analysis of a sporulated yeast diploid strain RS453 (Table I) disrupted for the MTR10 gene. Haploid progeny carrying the mtr10::HIS3 gene disruption exhibit a very slow growing phenotype at 30°C, so that colonies only become visible after 5 days of incubation. The fast growing colonies are MTR10+ progeny. (B) Growth dot-spot analysis of the mtr10::HIS3 null strain, ts mtr10-7 cells and mtr10::HIS3 cells complemented by ProtA–Mtr10p and GFP-Mtr10p. Pre-cultures were diluted in growth medium and equivalent amounts of cells (diluted in 10−1 steps) were spotted onto YPD plates. Plates were incubated for 3 days at the indicated temperatures. Download figure Download PowerPoint GFP-tagged Mtr10p exhibits a nuclear pore, intranuclear and cytoplasmic location To determine the subcellular location of Mtr10p, a green fluorescent protein (GFP)-tagged version of it (GFP-Mtr10p) was expressed in the mtr10::HIS3 disruption mutant. GFP-Mtr10p was functional and complemented the thermosensitive growth defect of the mtr10 mutant (Figure 1B). When living cells were analysed by fluorescence microscopy, GFP-Mtr10p accumulates inside the nucleus, but a nuclear envelope and distinct cytoplasmic staining was also noticed (Figure 2A). By digital confocal imaging which uses mathematical algorithms to deconvolve the digital image (see Materials and methods), the nuclear envelope location of GFP-Mtr10p becomes more evident (Figure 2B). To determine whether the nuclear envelope location reflects an association with the NPCs, GFP-Mtr10p location was analysed in nup133− cells, in which NPCs are clustered in one or few foci. Indeed, GFP-Mtr10p was apparently localized in these clusters (Figure 2C). This shows that a pool of Mtr10p physically associates with the NPCs under steady-state conditions. This association may be dynamic as Mtr10p is present in both the cytoplasm and the nucleoplasm, and therefore may shuttle between these two compartments (see also Discussion). Figure 2.Localization of GFP-tagged Mtr10p in living cells. (A) Fluorescence microscopy of mtr10::HIS3 cells expressing GFP-Mtr10p. GFP-Mtr10p is located predominantly inside the nucleus, with the tendency to be more concentrated around the nuclear envelope. Less GFP-Mtr10p signal with vacuolar exclusion is seen in the cytoplasm. (B) Same as (A), but the digital picture was processed further by digital confocal imaging (see Materials and methods). (C) Fluorescence microscopy of GFP-Mtr10p in nup133− cells. GFP-Mtr10p clusters together with NPCs in one or a few spots. Download figure Download PowerPoint Purification of Mtr10p reveals association with Npl3p and another putative RNA-binding protein For its biochemical purification, Mtr10p was tagged at its N-terminal end with two IgG-binding domains derived from Staphylococcus aureus protein A. Furthermore, a cleavage site for the TEV protease comprising seven amino acids was inserted between the ProtA tag and Mtr10p (see Materials and methods). This ProtA–TEV–Mtr10p fusion protein was functional since it could complement the ts growth defect of mtr10::HIS3 cells (Figure 1B). ProtA–TEV–Mtr10p was affinity-purified from this strain under non-denaturing conditions by IgG–Sepharose chromatography (Figure 3A). The Mtr10p protein was released from the column upon incubation with recombinant TEV protease. SDS–PAGE analysis of the released proteins revealed the presence of Mtr10p (migrating at ∼100 kDa), a prominent band of 58 kDa and a weaker band of 75 kDa (Figure 3A, lane 1). Whereas the presence of the 75 kDa band varied from preparation to preparation, the 58 kDa band was always present in a similar stoichiometric ratio. Both bands were analysed by mass spectrometry in order to identify the proteins that co-purify with Mtr10p (see Materials and methods). The 75 kDa protein corresponds to Hsp70 (Ssa1p). The prominent 58 kDa band is composed of two proteins that co-migrate, Npl3p (DDBJ/EMBL/GenBank accession number Q01560) and Hrb1p (DDBJ/EMBL/GenBank accession number P38922). Npl3p is an RNA-binding protein that shuttles between the nucleus and the cytoplasm and is required for nuclear export of mRNA (Lee et al., 1996). Hrb1p is an uncharacterized protein, but it appears to belong to the family of RNA-binding proteins as it contains three RNP motifs and a domain in the N-terminal part which resembles the RGG box of Npl3p (Figure 3B). Npl3p seems to be the predominant protein within the 58 kDa band, because the mass spectrometric data always gave higher intensity peptide peaks corresponding to Npl3p than for the Hrb1p peptides. The presence of Npl3p in the Mtr10p preparation was verified independently by Western blot analysis using anti-Npl3p antibodies (data not shown). We conclude that Npl3p and an uncharacterized putative RNA-binding protein are physically associated with Mtr10p. Figure 3.Affinity purification of ProtA–TEV–Mtr10p reveals interaction with Npl3p and Hrb1p. (A) Affinity purification of ProtA–TEV–Mtr10p by IgG–Sepharose chromatography and release of the Mtr10p by TEV-mediated proteolytic cleavage was performed as described in Materials and methods. Shown is a Coomassie-stained SDS–polyacrylamide gel which contains: lane 1, the purified Mtr10p preparation (consisting of Mtr10p plus a prominent co-purifying 58 kDa band composed of Npl3p and Hrb1p, and a weaker staining 75 kDa band which corresponds to Ssa1p); lane 2, mock control eluate derived from a strain not expressing ProtA–TEV–Mtr10p; lane 3, protein standard with marker proteins that have a stepwise 10 kDa increase in molecular weight (the strongly stained band corresponds to 50 kDa). (B) Comparison of the domain organization of Npl3p and Hrb1p. The amino acid sequences of Npl3p and the other Mtr10p-interacting protein Hrb1p are shown. For Npl3p, the proline-rich domain, RNA-binding domain and the RGG box are drawn. The C-terminally located RGG domain within Npl3p is compared with a related domain found in the N-terminal part of Hrb1p. Underlined are the RNP-I motifs within Npl3p and Hrb1p. In bold and underlined is a short sequence at the end of the RGG box of Npl3p which also occurs in the corresponding RGG box of Hrb1p. Download figure Download PowerPoint Nuclear import of GFP-tagged Npl3p is inhibited in the thermosensitive mtr10-7 mutant Since Npl3p binds to Mtr10p, we tested whether mtr10 mutants are impaired in either nuclear import or export of Npl3p. Therefore, a GFP-tagged Npl3p construct under the control of an inducible GAL promoter was expressed in the ts mtr10-7 and mtr10 null strains. Since the mtr10-7 mutant is already impaired in cell growth at 30°C (semi-permissive temperature; see also Figure 1B), cells were incubated at 18°C. GFP-Npl3p expression was then induced by shifting the cells from raffinose- to galactose-containing medium for 2 h. After this induction period, GFP-Npl3p expression was repressed by growing the cells in glucose-containing medium for a further 2 h. The culture was finally split and one half was left at the permissive temperature (18°C), whereas the other half was shifted for 2 h to the non-permissive temperature (37°C). The intracellular location of GFP-Npl3p was analysed by fluorescence microscopy (Figure 4). Wild-type MTR10+ cells exibit an exclusive intranuclear location of GFP-Npl3p, both at 18 and 37°C (Figure 4A). Similarily, mtr10-7 cells accumulate GFP-Npl3p inside the nucleus at the permissive temperature (Figure 4C). However, shifting mtr10-7 cells to 37°C causes a strong cytoplasmic mislocation of GFP-Npl3p (Figure 4D). Strikingly, the mtr10::HIS3 null mutant which grows very slowly at 18°C (data not shown), completely mislocalizes GFP-Npl3p to the cytoplasm under these conditions (Figure 4B). These results show that nuclear import of Npl3p is inhibited in mtr10 mutants. To test whether this reflects a general nuclear import defect in the mtr10 mutants, we analysed the localization of a classic NLS-containing reporter protein (NLS-GFP-lacZ). However, no inhibition of nuclear protein import of this protein was observed in the mtr10 null mutant at the permissive or restrictive temperature (Figure 4F). The mislocalization of Npl3p in the cytoplasm may be a pleiotropic effect of impaired cell growth and inhibition of mRNA export. To exclude this possibility, the GFP-Npl3p reporter was also introduced into the mex67-5 ts mutant, which is strongly impaired in mRNA export at the restrictive temperature (Segref et al., 1997). In contrast to the mtr10 mutants, nuclear accumulation of GFP-Npl3p was normal in the ts mex67-5 cells at the restrictive temperature (Figure 4E). We conclude, therefore, that Mtr10p specifically mediates the nuclear import of Npl3p. Figure 4.Analysis of nuclear accumulation of GFP-Npl3 in mtr10 mutants. MTR10+ cells (A), mtr10::HIS3 cells (B), mtr10-7 cells (C and D) and mex67-5 cells (E) were transformed with plasmid pPS811 containing the GAL::GFP-NPL3 reporter construct; mtr10::HIS3 cells were also transformed with a plasmid containing the NLS-GFP-lacZ reporter gene (F). Cells expressing GFP-Npl3p or NLS-GFP-lacZ were grown at the indicated temperatures and then analysed in the fluorescence microscope for the GFP fluorescence signal as described in Lee et al. (1996). Download figure Download PowerPoint To identify the sequence within Npl3p which mediates nuclear localization, various domains of Npl3p were fused to GFP, and the in vivo location of the corresponding fusion proteins was determined by fluorescence microscopy. Since it has already been shown that the RGG box domain of Npl3p (Figure 3B) is necessary for nuclear location (Flach et al., 1994; Lee et al., 1996), we tested whether this domain alone constitutes the NLS. We could show that the RGG box (residues 283–396) plus the last 18 C-terminal amino acids (residues 397–414) of Npl3p [called RGG(283–414)-GFP] are indeed necessary and sufficient to mediate nuclear accumulation of attached GFP (Figure 5A and B) and to bind efficiently to Mtr10p (Figure 5M, lane 3). In contrast, the N-terminal and middle domains of Npl3p, which contain the proline-rich sequence and the RNA-binding motifs, respectively, cannot mediate nuclear accumulation (Figure 5G–I) and association with Mtr10p (Figure 5M, lane 2). As anticipated, nuclear import of RGG(283–414)-GFP is inhibited in the mtr10-7 mutant at the non-permissive temperature (Figure 5B and C). However, deletion of the last 18 C-terminal residues from the RGG box domain (Figure 5J–L) or expressing only the C-terminal tail (Figure 5D–F) renders the NLS function very inefficient and no, or only very little, nuclear accumulation takes place. Furthermore, the RGG box alone lacking the C-terminal tail no longer stably interacts with Mtr10p (data not shown). Thus, the RGG box domain requires the presence of the short non-repetitive C-terminal tail to mediate nuclear accumulation of Npl3p and to interact with its specific import receptor Mtr10p (see Discussion). Figure 5.The RGG domain plus the short non-repetitive C-terminal tail constitute the NLS within Npl3p. In the upper part, a schematic drawing of the Npl3p deletion constructs with the corresponding amino acid boundaries (see also Figure 3B) is shown. The constructs were fused N-terminally to GFP, expressed in NPL3+ cells and their subcellular location was analysed by fluorescence microscopy (A–L). The asterisks indicate which GFP fusion proteins were used for ProtA–TEV–Mtr10p purification and SDS–PAGE (M). WT, wild-type; N, nuclear; C, cytoplasmic; N/C, nuclear/cytoplasmic. (M) SDS–PAGE analysis of a ProtA–TEV–Mtr10p purification in a NPL3+ strain which co-expresses GFP-tagged Npl3p truncation proteins. Lane 1, protein standard with marker proteins that have a stepwise 10 kDa increase in molecular weight (the strongly stained band corresponds to 50 kDa); lane 2, fusion protein consisting of GFP and Npl3p(1–283); lane 3, fusion protein consisting of GFP and Npl3p(284–414). Download figure Download PowerPoint Interaction of recombinant Mtr10p with Ran-GTP Since the N-terminal part of Mtr10p has similarity to the Ran-binding motif found in importin β-related proteins (Fornerod et al., 1997b; Görlich et al., 1997), we tested for the ability of Mtr10p, expressed and purified from Escherichia coli, to interact with Gsp1p (the yeast homologue of Ran). We first tested for inhibition of nucleotide exchange on Gsp1p by Mtr10p as an assay for a physical interaction. GTP exchange on Gsp1p was induced by addition of the chelating agent EDTA which removes the magnesium ions required for tight binding of the nucleotide to Gsp1p. Employing such an in vitro assay, the dissociation constant for the Mtr10p–Gsp1p-GTP complex was determined to be ∼200 nM (Figure 6A, Mtr10p). This would indicate that Gsp1p-GTP binds Mtr10p with an ∼200-fold lower affinity than yeast importin β (Figure 6A, yImp β). Deletion of the C-terminal domain from Mtr10p (amino acids 421–972) does not change the affinity for Gsp1p-GTP (Figure 6A, Mtr10p-N), indicating that the first 420 amino acid residues of Mtr10p are sufficient for Gsp1p binding. Similar results were obtained when Mtr10p binding was monitored by inhibition of GTPase activation of Gsp1p by Rna1p (Figure 6B). Since the affinity of Mtr10p for Gsp1p is relatively low compared with other nuclear import factors of the importin β type, we tested whether binding of Npl3p to Mtr10p increases the affinity for Gsp1p-GTP. Such a mode of cooperative binding of Ran-GTP to a transport receptor in the presence of the transport substrate was seen in the case of human CAS which functions as a nuclear export receptor for importin α (Kutay et al., 1997). However, the affinity of Gsp1p-GTP for Mtr10p was not changed when the purified yeast Mtr10p–Npl3p–Hrb1p complex was tested in the in vitro assay (Figure 6A), suggesting that Npl3p is an import rather than an export substrate for Mtr10p (see also Discussion). Figure 6.Interaction of recombinant Mtr10p with Ran-GTP. Purification of recombinant full-length and the N-terminal domain of Mtr10p, importin β and Rna1p from E.coli, and the Mtr10p–Npl3p–Hrb1p complex from yeast is described in Materials and methods. (A) Inhibition of EDTA-induced GTP exchange on Gsp1p. Gsp1p-[γ-32P]GTP (50 pM) was pre-incubated with either bacterially expressed Mtr10p, Mtr10p-N (residue 1–420) and yeast importin β (yImp β), or the purified yeast Mtr10p–Npl3p–Hrb1p complex, at the final concentrations indicated or with incubation buffer. Then, 40 mM EDTA and 200 nM GDP were added for another 15 min. Gsp1p-bound radioactivity finally was determined by the filter-binding assay. (B) Inhibition of Rna1p-induced GTP hydrolysis on Gsp1p. Gsp1p-[γ-32P]GTP (50 pM) was incubated for 15 min with Mtr10p-N and yeast importin β (yImp β) at the final concentrations indicated or with incubation buffer. Rna1p (20 nM) was added and the reaction allowed to proceed for 2 min. Hydrolysis of Gsp1p-bound GTP was determined as released [32P]phosphate. Download figure Download PowerPoint Release of Npl3p from Mtr10p is mediated by the cooperative action of Ran-GTP and RNA Since Mtr10p exhibits a low affinity for Ran-GTP, which is not stimulated by Npl3p, we tested whether RNA as the nuclear target of Npl3p increases this affinity. Therefore, ProtA–TEV–Mtr10p with bound Npl3p was first immobilized on IgG–Sepharose beads as described before (see also Figure 3A). However, it was not eluted by TEV protease, but incubated with buffer, human Ran-GDP or Ran-GTP, and Ran-GDP or Ran-GTP plus yeast total RNA, respectively. Release of Npl3p was analysed finally by SDS–PAGE and Coomassie staining (Figure 7). Buffer, Ran-GDP