Structural basis for the nuclear export activity of Importin13
2013; Springer Nature; Volume: 32; Issue: 6 Linguagem: Inglês
10.1038/emboj.2013.29
ISSN1460-2075
AutoresMarlene Grünwald, Daniela Lazzaretti, F. Bono,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle22 February 2013free access Structural basis for the nuclear export activity of Importin13 Marlene Grünwald Marlene Grünwald Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Daniela Lazzaretti Daniela Lazzaretti Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Fulvia Bono Corresponding Author Fulvia Bono Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Marlene Grünwald Marlene Grünwald Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Daniela Lazzaretti Daniela Lazzaretti Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Fulvia Bono Corresponding Author Fulvia Bono Max Planck Institute for Developmental Biology, Tübingen, Germany Search for more papers by this author Author Information Marlene Grünwald1, Daniela Lazzaretti1 and Fulvia Bono 1 1Max Planck Institute for Developmental Biology, Tübingen, Germany *Corresponding author. Research Groups, Max Planck Institute for Developmental Biology, Spemannstrasse 35, Tübingen 72076, Germany. Tel.:+49 7071 6011367; Fax:+49 7071 6011308; E-mail: [email protected] The EMBO Journal (2013)32:899-913https://doi.org/10.1038/emboj.2013.29 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Importin13 (Imp13) is a bidirectional karyopherin that can mediate both import and export of cargoes. Imp13 recognizes several import cargoes, which include the exon junction complex components Mago-Y14 and the E2 SUMO-conjugating enzyme Ubc9, and one known export cargo, the translation initiation factor 1A (eIF1A). To understand how Imp13 can perform double duty, we determined the 3.6-Å crystal structure of Imp13 in complex with RanGTP and with eIF1A. eIF1A binds at the inner surface of the Imp13 C-terminal arch adjacent and concomitantly to RanGTP illustrating how eIF1A can be exported by Imp13. Moreover, the 3.0-Å structure of Imp13 in its unbound state reveals the existence of an open conformation in the cytoplasm that explains export cargo release and completes the export branch of the Imp13 pathway. Finally, we demonstrate that Imp13 is able to bind and export eIF1A in vivo and that its function is essential. Introduction In eukaryotic cells, the nuclear envelope selectively separates the nucleus, where transcription and splicing occur, from the cytoplasm, where translation takes place. Proteins and RNAs can cross this barrier through the nuclear pore complexes (NPCs). Small molecules, up to ∼20–40 kDa, can passively diffuse across the NPCs, while other molecules need to be actively transported (Görlich and Kutay, 1999; Mohr et al, 2009). Active nucleo-cytoplasmic transport is mainly mediated by a superfamily of proteins, known as karyopherins. Karyopherins can be divided into two groups based on their directionality: importins translocate cargoes from the cytoplasm to the nucleus, while exportins transport their cargoes from the nucleus into the cytoplasm. The directionality of both import and export is determined by the same driving force: a gradient of the small GTPase Ran. Importins bind their cargoes in the cytoplasm and release them upon binding to the GTP-bound form of Ran (RanGTP), which is confined to the nucleus. Conversely, exportins require RanGTP to stably bind their cargo in the nucleus; the dissociation of the export complex is then triggered in the cytoplasm by the hydrolysis of Ran-bound GTP to GDP (Görlich and Kutay, 1999; Cook et al, 2007). So far, only a few karyopherins including Importin13 (Imp13), Exportin4 and Msn5, have been characterized as bidirectional transport factors being able to both import and export cargoes from the nucleus (Mingot et al, 2001; Yoshida and Blobel, 2001; Gontan et al, 2009). In Drosophila larvae, Imp13 affects neurotransmitter release at the neuromuscular junctions and homozygous imp13 mutations are lethal (Giagtzoglou et al, 2009). In humans, Imp13 has been involved in the import of the core exon junction complex components Mago-Y14, the E2 SUMO-conjugating enzyme Ubc9 (Mingot et al, 2001), histone fold-containing and paired-type homeodomain transcription factors (Ploski et al, 2004; Kahle et al, 2005; Walker et al, 2009), the glucocorticoid receptor (Tao et al, 2006) and the actin-binding protein myopodin (Liang et al, 2008). The only export cargo of Imp13 known so far is the eukaryotic initiation factor 1A (eIF1A) (Mingot et al, 2001). However, the function and importance of this export pathway have not yet been addressed in vivo. eIF1A is a protein conserved across eukaryotes with multiple functions in translation initiation (Jackson et al, 2010; Hinnebusch, 2011). Together with another translation initiation factor, eIF1, it directly associates to the small ribosomal subunit and is required for the assembly of the pre-initiation complex (Jackson et al, 2010; Aitken and Lorsch, 2012). The two initiation factors cooperate in promoting an ‘open’, scanning-competent conformation of the 40S subunit (Passmore et al, 2007). eIF1A consists of an oligonucleotide-binding (OB) β-barrel fold followed by an extended helix, and two unstructured tails at the N- and C-termini (Battiste et al, 2000). Due to its small size (17 kDa), eIF1A is thought to passively diffuse through the NPCs; its active export might therefore be required both to deplete eIF1A from the nucleus and to maintain sufficient cytoplasmic levels (Mingot et al, 2001). Despite the importance of eIF1A in translation, limited information is available on its localization. Several structures of karyopherins have been determined to date, comprising two nuclear import factors (Importin β (Impβ) and Transportin (Tpn)) (Cingolani et al, 1999; Vetter et al, 1999a; Bayliss et al, 2000; Lee et al, 2003, 2005; Cansizoglu and Chook, 2007; Cansizoglu et al, 2007; Cook et al, 2007; Imasaki et al, 2007; Wohlwend et al, 2007; Mitrousis et al, 2008; Bhardwaj and Cingolani, 2010; Forwood et al, 2010; Xu et al, 2010; Zhang and Chook, 2012) and four nuclear export factors (Cse1, Crm1, Expo5 and Xpo-t) (Matsuura and Stewart, 2004; Cook et al, 2005, 2009; Monecke et al, 2009; Okada et al, 2009; Dong et al, 2009a, 2009b; Güttler et al, 2010) but it is still unclear how a transport factor, such as Imp13, can have a mixed transport competence. How RanGTP binding to bidirectional karyopherins can cause opposite effects, dissociation of the cargo to be imported and association of the cargo to be exported, is an open question in the nuclear transport field. Our previous work on the structures of the Imp13-RanGTP intermediate state and the Imp13-Mago-Y14 and Imp13-Ubc9 cargo complexes have provided key insights into import cargo recognition and dissociation by RanGTP (Bono et al, 2010; Grünwald and Bono, 2011). Well-characterized import factors such as Impβ and Tpn normally recognize only a small portion of their cargos as an import signal (Cingolani et al, 1999, 2002; Cansizoglu and Chook, 2007; Imasaki et al, 2007; Wohlwend et al, 2007; Mitrousis et al, 2008; Bhardwaj and Cingolani, 2010; Forwood et al, 2010; Xu et al, 2010; Zhang and Chook, 2012). Remarkably, Imp13 recognizes the folded domains of the import cargoes Ubc9 and Mago-Y14, predominantly via charged and polar residues distributed over the entire proteins. RanGTP binding to Imp13 is similar to RanGTP binding by Impβ and Tpn although Imp13 lacks the acidic loop that is found in the canonical import factors (Cook et al, 2007). Imp13 uses non-overlapping surfaces for the recognition of different import cargoes. As a consequence, the release mechanism of these cargoes is different: Mago-Y14 is released via a steric hindrance mechanism, while Ubc9 and RanGTP directly compete for the same binding surface on Imp13 (Bono et al, 2010; Grünwald and Bono, 2011). Our structural and biochemical studies of Imp13 in its unbound state and the Imp13-RanGTP-eIF1A trimeric complex shed light on how bidirectional karyopherins can perform double duty and also be regulated in opposite ways by RanGTP. We further address the functional basis by which the nuclear transport factor Imp13 is able to recognize both import and export cargoes and deliver them in the appropriate cellular compartment and the in vivo relevance of Imp13 function. Results and Discussion Structure determination and quality We determined the crystal structures of Imp13 in its nuclear export complex with RanGTP and eIF1A and also in the unbound form that corresponds to a cytosolic state. For the crystallization of the ternary export complex, human (Hs) Imp13, Ran and eIF1A were expressed separately in E. coli. While Imp13 was used as a full-length construct, the small GTPase Ran was truncated to contain the residues 1–180 and a Gln69Leu mutation to inhibit GTP hydrolysis, as previously described (Bischoff et al, 1994; Matsuura and Stewart, 2004; Lee et al, 2005; Cook et al, 2009; Monecke et al, 2009; Bono et al, 2010) (Figure 1A; Supplementary Figure 1A). Attempts to crystallize the trimeric complex in presence of full-length eIF1A were not successful probably due to the two large unstructured regions at the N- and C-termini (Battiste et al, 2000) (NTT and CTT, respectively). Therefore, a truncated version of eIF1A was used that includes the residues from 1 to 112 and lacks the CTT portion of the protein (eIF1AΔC; Figure 1A and Supplementary Figure 1A). The complex was reconstituted in vitro with an excess of RanGTP and eIF1A and purified to homogeneity by size exclusion chromatography (SEC). The export complex crystallized in a centred monoclinic space group (C2). Initial phases for the structure were obtained by molecular replacement (MR) with the Imp13-RanGTP structure as a search model (Bono et al, 2010; pdb id.: 2x19). Although density for eIF1A could be observed, a reliable MR solution could not be obtained when using available structures of eIF1A as search models (Battiste et al, 2000; pdb ids: 2oqk and 2dgy). We manually placed the crystal structure of eIF1A from Cryptosporidium parvum (pdb id.: 2oqk; 75% sequence identity over the region encompassed by our construct) into the electron density using an anomalous Fourier map of a seleno-methionine (SeMet) substituted eIF1A as a guide (Supplementary Figure 1A). This eIF1A was mutated to include a third methionine residue so that the structure could be positioned accurately by using three anomalous difference peaks (Supplementary Figures 1A and 2A). A view of the quality of the electron density of eIF1A is shown in Supplementary Figure 2B. Model building of the complex was also verified by single-wavelength anomalous diffraction (SAD) structure solution of a trimeric complex reconstituted with a SeMet substituted Imp13 (unpublished observation). Figure 1.Structure of the ternary export complex Imp13-RanGTP-eIF1A and constructs used. (A) Schematic representation of the architecture of the proteins used in this study. Colour-filled areas in the scheme identify structural domains, which include the Ran core (in yellow) and the OB-fold of eIF1A (in brown). The residue numbers corresponding to the constructs used are indicated. The portions of the polypeptides ordered in the three-dimensional structure are shaded in grey. Cartoon view (B) and view rotated 60° along the x axis (C) of the export complex. Imp13 is shown in a colour gradient from grey (N-terminus) to green (C-terminus). Ran is in yellow with GTP as a stick model in black and eIF1A is coloured in brown. Secondary structure elements and HEAT repeats are labelled. (D) Imp13 apo structure in a similar colour code and view as in (A). (E) Surface rendering representation of Imp13 (top) and of eIF1A (bottom) in the export complex. The surface is coloured according to the electrostatic potential. Top, eIF1A and Ran, in the same colour code as above, are shown as loops with GTP as stick model in black. For the electrostatic potential calculation of eIF1A, the backbone was used as a template to include all the side chains with MODELLER (Sali and Blundell, 1993) (r.m.s.d. of 0.277 Å over 1121 atoms). These and all other protein structure figures were generated using PyMOL (http://www.pymol.org). Download figure Download PowerPoint The asymmetric unit is composed of three distinct complexes. Complexes 1 and 2 present the ternary export complex and are very similar (r.m.s.d. of 0.634 Å over 868 Cα atoms). Complex 3 does not show density for eIF1A and superposes with an r.m.s.d. of 2.0 Å over 855 Cα atoms to complex 1. Complex 1 shows the best quality electron density and therefore, throughout the paper, we will refer to complex 1, unless explicitly stated. The model was refined to 3.6 Å resolution with an Rfree and Rwork of 29.9 and 26.8%, respectively, and good stereochemistry (Table I). Disordered regions are at the very N- and C-termini of Imp13 as well as a long inter-repeat loop at residues 655–672. The NTT of eIF1A is not observed (residues 1–27) together with several loop regions (residues 54–56; 97–100; 109–112) (Figure 1A and B). In complex 2, additional regions of the C-terminal arch of Imp13 and eIF1A are disordered. Table 1. Crystallographic statistics Data collection Data set Imp13-RanGTP-eIF1A Imp13-RanGTP-eIF1A SAD Imp13 unbound Beamline SLS PXII SLS PXII SLS PXII Space group C2 C2 P43212 Unit cell (Å) a=186.5, b=100.4, c=274.7, α=γ=90°, β=90.9 a=185.7, b=100.9, c=273.3, α=γ=90°, β=90.5 a=b=167.7, c=95.6, α=β=γ=90° Wavelength (Å) 0.97919 0.97952 1.0409 Resolution range (Å)a 50–3.6 (3.7–3.6) 50–3.8 (3.9–3.8) 100–3.0 (3.1–3.0) Total no. of observations 195 637 686 292 415 721 Unique reflections 57 267 (4046) 97 665 (7340) 27 903 (2557) Redundancy 3.4 (3.2) 7.0 (7.3) 14.9 (12.5) Completeness (%)a 96.6 (87.4) 99.9 (100.0) 99.9 (100.0) I/σ(I)a 11.35 (2.08) 11.96 (2.37) 16.36 (2.28) Rsym(%)a 8.2 (94.1) 10.7 (74.1) 10.7 (116.7) Refinement Resolution range (Å) 50–3.6 50–3.0 No. of reflections 57266 27853 No. of reflections in test set 2843 1394 Rfree (%)a 29.92 31.09 Rwork (%)a 26.84 26.82 No. of atoms Protein 21782 6106 Ligand/ion 99 — Water — — B factors Protein 139.3 106.2 Ligand/ion 166.4 — Water — — R.m.s.d. bond (Å) 0.003 0.003 R.m.s.d. angle (deg) 0.775 0.737 Ramachandran valuesb Favoured (%) 98.2 (outliers 0.1) 97.2 (outliers 0.0) Allowed (%) 99.9 100 Molprobity score (with H) 1.83 2.18 a Data collection and refinement statistics of the crystal structure of Imp13-RanGTP-eIF1A complex and of Imp13 apo. a Values in parentheses correspond to the highest resolution shell. b Molprobity (Chen et al, 2010). The Drosophila (Dm) Imp13 apo form crystallized in spacegroup P43212 and the crystals diffracted to 3.0 Å (Figure 1D). The structure was solved by MR using Dm Imp13 as a search model (Bono et al, 2010; pdb id.: 2x1g) fragmented into three portions. The refined model has an Rfree of 31% with good stereochemistry (Table I). For a sample of the electron density map, see Supplementary Figure 2C. In the unbound Imp13 structure, some N-terminal regions (residues 1–53; 78–117; 100–121), intermediate loops as well as the very C-terminus (residues 968–971) are not well defined and could not be modelled (Figures 1D and 6A). Architecture of Imp13 in the export complex and in the unbound state As previously shown, Imp13 folds into 20 consecutive HEAT repeats (Bono et al, 2010; Grünwald and Bono, 2011). HEAT repeats are motifs consisting of two helices (A and B) connected by a short loop (intra-repeat). Each HEAT repeat stacks against the following one, via an inter-repeat loop, to generate a superhelical arrangement (Cook et al, 2007). The A helices form the outer surface of the superhelix, while the B helices form the inner concave surface. The C-terminus of Imp13 is stabilized by a HEAT-like repeat motif composed of three helices (A–C). A hinge region around HEAT10 (H10) divides Imp13 into two arches, the N- and C-terminal arches. Two further hinge regions at H4 and H14 increase the protein's conformational flexibility (Bono et al, 2010; Grünwald and Bono, 2011). In complex with RanGTP and eIF1A, Imp13 adopts a toroidal conformation with the N- and C-terminal edges offset by 8.2 Å and an inner diameter of ∼58 Å (Figure 1B and C). The C helix of H20 approaches the N-terminal tip of H5 (Figures 1B and 6D). Both arches of Imp13 are engaged in complex formation. As in the Imp13-RanGTP structure, RanGTP binds to the inner concave surface of the N-terminal arch shifted towards the intra-repeat loops (Bono et al, 2010). However, when in complex with the export cargo, the toroidal conformation of Hs Imp13 is less compact than in the Imp13-RanGTP complex, with the C-terminal arch more open to accommodate eIF1A (Figure 1B). In the complex with Imp13 and eIF1A, the structure and conformation of Hs RanGTP is very similar to the structure of RanGTP in complex with RanBD1 (r.m.s.d. of 0.48 Å over 168 Cα atoms) and of RanGTP in the binary complex with Imp13 (r.m.s.d. of 0.34 Å over 147 Cα atoms) and has been extensively described elsewhere (Vetter et al, 1999b; Bono et al, 2010). In the structure of Dm Imp13 in the unbound state, the karyopherin toroidal structure is in an open conformation (Figure 1D). H1 and the external helices of H2 and 3 are disordered in the structure, suggesting flexibility at the very N-terminus of the protein. Flexibility in this region might be required for the docking of RanGTP in the nucleus to displace the import cargoes (Grünwald and Bono, 2011) (Figures 1D, 6A and B) and to recognize cargoes that bind at the N-terminal arch of Imp13, such as Ubc9. Export cargo recognition by Imp13 and RanGTP The OB-fold of eIF1A is composed of five β strands (β1–β5 strands of eIF1A: β1E–β5E) forming an elliptic cavity interrupted by a loop between β3E and β4E (L3E) that folds into a small helix (α1E). C-terminal to the β-barrel eIF1A features an additional helix (α2E). Although the NTT of eIF1A was present in the construct that was crystallized, no density for this part of the structure was observed in the trimeric export complex, likely due to conformational flexibility in this region (Battiste et al, 2000). When the full-length solution structure is superposed on the export complex, the NTT points into solvent (Supplementary Figure 6B). To rule out the possibility that the NTT is required for binding and the possibility that the NTT was not observed due to the relatively low resolution map, we performed SEC with truncated versions of eIF1A. These experiments show that the folded part of eIF1A (residues 26–115; ΔNΔC) is sufficient for binding to Imp13 (Figure 2D and E). Figure 2.Detailed view of the interaction between Imp13 and eIF1A. Top right, the export complex is rendered with Imp13 and RanGTP as surface and eIF1A as cartoon in the same colour coding as in Figure 1B. The interaction surface of eIF1A on Imp13-RanGTP is in pink. (A) Close-up view of the eIF1A interaction with Imp13 at BS 1 in a similar view as in Figure 1B. Residues involved in the interaction on eIF1A and Imp13 are shown as sand and pink sticks, respectively. HEAT repeats are labelled in black and secondary structure elements of eIF1A are marked in brown or white. (B) Zoomed in view of the BS 2 interaction site. (C) Protein co-precipitation by GST-tagged Imp13 incubated with RanGTP and eIF1A with either wt or mutant proteins. In this experiment, Ran was His tagged for better separation from eIF1A on gel. For the input control, 1/6 of the samples were kept (upper panel) and the rest was co-precipitated with glutathione sepharose beads (lower panel) and analysed on Coomassie-stained 15% SDS–PAGE. The far left lane was loaded with a molecular weight marker. Some contaminations likely from degradation of GSTImp13 are visible between 25 and 35 kDa. GST controls for this experiment are in Supplementary Figure 6A. (D) Overlay of SEC plots of Imp13 alone or together with different eIF1A constructs with or without RanGTP. (E) Samples of the peaks fractions were analysed on Coomassie-stained SDS–PAGE (Any kD, BIORAD). The far left lane was loaded with a molecular weight marker. Download figure Download PowerPoint eIF1A can be approximated to a triangle that is recognized at its vertices on three main interaction surfaces, two on Imp13 and one on RanGTP. The two corners at the base of the triangle β2E and L3E, and α2E interact at two opposite surface areas (BS1 and BS2, respectively) that bridge the inner central part of Imp13 and its very C-terminus (Figures 1B, C, 2A, B and 3A). The interaction surface of eIF1A to Imp13 contains several conserved, positively charged residues, which complement the negatively charged inner binding surface of Imp13 (Figure 1E). Figure 3.Structural basis of Imp13 bidirectionality and KD affinities in the Imp13 cycle. (A) RanGTP and eIF1A concomitant interaction in the ternary export complex. Interacting residues on Ran and eIF1A are rendered as spheres in pink and sand, respectively. (B) Imp13-Mago-Y14 structure oriented as in Figure 3A (pdb ids.: 2x1g). Imp13 is represented as loop trace. A dashed line in black marks the boundary between compatible and hindered binding. (C) Protein co-precipitations by GST-tagged Imp13 incubated together with His-RanGTP and eIF1A with either wt or mutant proteins performed as in the previous figure. (D) Table representing KD values determined by DSF from at least three independent experiments. Download figure Download PowerPoint The larger area of interactions (BS1) is centred around H9 of Imp13 and stretches across the hinge between the N- and C-terminal arches involving H7-11 on the side of the inter-repeat loops. On eIF1A the loop L1E points to the groove between H8 and 9 (Figure 2A); β3E and the N-terminal stretch of L3E contact H8 and H10-11, respectively, while the N-terminal part of β5E and L4E interacts with H7-8. A series of salt bridges are likely to stabilize this interface: Asp369 on H8 of Imp13 (Asp369I) points to Lys88 of eIF1A (Lys88E) on β5E; on H9, Glu436I approaches Arg46E on L1E; Asp481I at the inter-repeat loop between H10-11 is in proximity to Arg66E and Lys67E on L3E. At the smaller interaction surface, helix α2E packs end-on to the C-terminal helix of Imp13. At this site, Arg938I approaches helix α2E as well as the N-terminal stretch of eIF1A (Figure 2B; Supplementary Figure 1). Consistent with the structural analysis, reverse charge mutations of Asp369I, Asp370I, Glu436I and Asp481I to arginine in BS1 or of Arg938I to glutamate in BS2 resulted in reduction or loss of binding to eIF1A in pull-down assays (Figure 2C; Supplementary Figure 1). Although structurally less well defined (many side chains of eIF1A do not have visible electron density), the contribution of positively charged residues of eIF1A to Imp13 binding could also be confirmed using reverse charge mutations. Residues Lys29E, Arg46E, Arg66/Lys67E and Lys88E are conserved (Supplementary Figure 1A) and mutations to negatively charged residues impeded binding to Imp13, as confirmed by pull-down assays (Figure 2C; Supplementary Figure 6A). Similarly to the import cargoes Ubc9 and Mago-Y14, eIF1A is primarily recognized by Imp13 as a folded domain. This indicates that both import and export signals recognized by Imp13 are complex ones, therefore different from the linear and semi-linear canonical NLSs and NESs recognized by Impβ, Tpn and Crm1 (Xu et al, 2010; Zhang and Chook, 2012). The structural and mutational analyses show that Imp13 recognizes its cargoes by a combination of shape and charge together with specific contacts at conserved positions on eIF1A. Imp13 uses shape complementarity of cargoes to perform bidirectional transport In the export complex, the β-barrel and the very tip of helix α2E of eIF1A bind adjacent to RanGTP at the inner surface of Imp13. The structure strongly suggests that eIF1A is in direct contact with RanGTP in the complex with Imp13. L4 of Ran (L4R, between β5R-6R) is adjacent to L4E and to the N-terminus of eIF1A (Figure 3A). Here, Glu30E and Asp31E point towards the L4R and towards Lys130 of Ran (Lys130R) while L4E approaches Arg95R at helix α2R (Figure 3A; Supplementary Figure 1A). Consistently, mutants of Glu30E and Asp31E to arginine and of Arg95R and Lys130R to glutamate impair binding of Imp13-RanGTP to eIF1A (Figure 3C). To further understand Imp13 bidirectionality, we compared the mode of binding of Imp13 in the export complex with the structure of Imp13 bound to the import cargo Mago-Y14 (Bono et al, 2010). As we have previously shown, when the latter structures are superposed, RanGTP and Mago-Y14 assume an adjacent but overlapping position that precludes concomitant binding of Mago-Y14 with RanGTP due to steric hindrance (Bono et al, 2010). In the complex of Imp13 with RanGTP and eIF1A, the export cargo is much smaller than Mago-Y14 so that it fits neatly into the hole created by the C-terminal arch. In this case there is not a clash with RanGTP, instead the complementary charge interactions with RanGTP stabilize the binding and increase the binding affinity (Figure 3D). Consistently, SEC experiments of Imp13-eIF1A complexes show only a partial binding of eIF1A in the absence of RanGTP (Figure 2D and E). Imp13 is therefore able to discriminate between cargoes based on their structural features and can therefore promote either cooperative binding (for export) or antagonistic binding (for import). Comparison of the Imp13-RanGTP-eIF1A complex with the Imp13-Mago-Y14 structure also shows that the binding of Mago-Y14 and eIF1A is mutually exclusive. The release of Mago-Y14 by RanGTP is less efficient in vitro as compared to that of another import cargo, Ubc9, which shows a different mode of binding. We have previously shown that addition of eIF1A together with RanGTP enhances the release of Mago-Y14 from Imp13 (Grünwald and Bono, 2011; Figure 3A and B). In contrast, a comparison of the Imp13 export complex with Imp13-Ubc9 import complex shows no major clashes between the cargoes (Figure 6D and E). However, a simultaneous binding of eIF1A and Ubc9 to Imp13 in vitro is not possible (Grünwald and Bono, 2011). The structural and biochemical data suggest that the shape complementarity of eIF1A and its overlap with the Mago-Y14 binding site is critical for achieving the directionality of Mago-Y14 import. RanGTP likely destabilizes Mago-Y14 binding by preventing optimal association with Imp13. Further association of eIF1A with Imp13 would lock the importin in an export conformation, preventing re-association of Mago-Y14 and imparting directionality to this nuclear import step. To better understand the mechanism of the Imp13 import/export cycle, we measured the KD values by differential scanning fluorimetry (DSF) (Niesen et al, 2007) (Figure 3D; Supplementary Figure 3). The estimated KD for the two import cargoes Mago-Y14 and Ubc9 is in a similar range with a value of 235±30 nM and 370±20 nM, respectively. The KD value for RanGTP binding to Imp13, although not accurately calculated due to the upper detection limit of the method, falls in the low nanomolar or picomolar range (<100 nM) and is consistent with values measured for other karyopherins (Bischoff and Görlich, 1997; Deane et al, 1997; Görlich et al, 1997; Lipowsky et al, 2000). The dissociation constant of the export cargo eIF1A to a preformed Imp13-RanGTP complex is 3±0.38 μM, 10-fold higher than the KD values of the import cargoes (Figure 3D). The rather weak affinity of Imp13-RanGTP for its export cargo eIF1A is comparable to that displayed by Crm1 for most NES, such as HIV Rev (KD of about 0.5 μM) (Askjaer et al, 1999; Paraskeva et al, 1999). One exception is Snurportin that binds to CRM1 with much higher affinity because it is recognized through additional interactions (KD∼10 nM) (Paraskeva et al, 1999). Xpot also binds tRNAs with high affinity (KD∼3 nM) (Kutay et al, 1998; Lipowsky et al, 1999). The binding of CAS/Cse1 to Impα and RanGTP is highly cooperative and cargo binding cannot be disjointed from RanGTP binding (Kutay et al, 1997). Imp13 exports eIF1A in vivo and its function is required for cell viability eIF1A is able to passively diffuse through the NPCs and thus reach the nuclear compartment and it has previously been proposed that active export is necessary to deplete eIF1A from the nucleus and contribute in preventing nuclear translation (Mingot et al, 2001). Surprisingly, we observed that endogenous eIF1A is enriched in nucleoli in HeLa cell lines (Supplementary Figure 4A and D). An overexpressed, GFP-tagged version of the protein shows a similar localization in fixed cells (Supplementary Figure 4C); however, the same construct displays a significantly higher cytoplasmic fraction in living cells (Figure 4B), suggesting that fixation affects eIF1A localization to some extent. All together, our results indicate that endogenous eIF1A is localized to both nucleoli and cytoplasm. Figure 4.Imp13 binds and exports eIF1A in vivo. (A–F) Fluorescence microscopy images and the corresponding differential interference contrast (DIC) images of living HeLa cells expressing GFP-tagged eIF1A (eIF1A-GFP) wt or mut
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