Architecture of CRM1/Exportin1 Suggests How Cooperativity Is Achieved during Formation of a Nuclear Export Complex

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1 Molecular Cell, Vol. 16, , December 3, 2004, Copyright 2004 by Cell Press Architecture of CRM1/Exportin1 Suggests How Cooperativity Is Achieved during Formation of a Nuclear Export Complex Carlo Petosa, 1 Guy Schoehn, 1,2,3 Peter Askjaer, 4 Ulrike Bauer, 4 Martine Moulin, 1 Ulrich Steuerwald, 1 Montserrat Soler-López, 1 Florence Baudin, 1,2,3 Iain W. Mattaj, 4 and Christoph W. Müller 1, * 1 European Molecular Biology Laboratory Grenoble Outstation, B.P Grenoble Cedex 9 France 2 Laboratoire de Virologie Moléculaire et Structurale EA 2939 Université Joseph Fourier Grenoble Cedex 9 France 3 Institut de Biologie Structurale UMR 5075 CEA-CNRS-UJF 41 rue Jules Horowitz Grenoble Cedex 1 France 4 European Molecular Biology Laboratory Meyerhofstrasse 1 D Heidelberg Germany Summary CRM1/Exportin1 mediates the nuclear export of pro- teins bearing a leucine-rich nuclear export signal (NES) by forming a cooperative ternary complex with the NES-bearing substrate and the small GTPase Ran. We present a structural model of human CRM1 based on a combination of X-ray crystallography, homology modeling, and electron microscopy. The architecture of CRM1 resembles that of the import receptor transportin1, with 19 HEAT repeats and a large loop impli- cated in Ran binding. Residues critical for NES recog- nition are identified adjacent to the cysteine residue targeted by leptomycin B (LMB), a specific CRM1 in- hibitor. We present evidence that a conformational change of the Ran binding loop accounts for the co- operativity of Ran- and substrate binding and for the selective enhancement of CRM1-mediated export by the cofactor RanBP3. Our findings indicate that a sin- gle architectural and mechanistic framework can explain the divergent effects of RanGTP on substrate binding by many import and export receptors. Introduction plex (NPC). All karyopherins interact with NPC components (nucleoporins) and with the GTP bound form of Ran, a small GTPase that is predominantly bound to GTP in the nucleus and to GDP in the cytosol. Importins bind their cargo in the cytosol, translocate through the NPC, and release the cargo in the nucleus upon binding RanGTP. Exportins associate cooperatively with their cargo and with RanGTP in the nucleus, forming a ternary complex that translocates to the cytosol and subsequently dissociates when GTP is hydrolyzed by Ran. The molecular basis for this difference in behavior upon binding RanGTP is poorly understood. The karyopherin CRM1/Exportin1 mediates the nuclear export of many cellular and viral proteins and ribonucleoproteins. Cargos directly recognized by CRM1 include proteins bearing a leucine-rich NES, a short motif with a loosely conserved pattern of three or four hydrophobic residues (Fischer et al., 1995; Wen et al., 1995). Over 75 NES-containing proteins have been experimentally identified (la Cour et al., 2003), including HIV Rev, protein kinase A inhibitor (PKI), and MAPKK (Fornerod et al., 1997a; Fukuda et al, 1997; Stade et al., 1997; Ossareh-Nazari et al., 1997). CRM1 also directly exports Snurportin1, a trimethylguanosine (m 3 G)-cap binding protein that lacks a leucine-rich NES (Paraskeva et al., 1999) and indirectly exports certain U snrnas via the adaptor protein PHAX (Ohno et al., 2000). As with all exportins, the intrinsically low cargo-binding affinity of CRM1 is enhanced upon binding RanGTP. For certain NES-bearing cargos, the CRM1/RanGTP/cargo com- plex is stabilized by the cofactor RanBP3, a nuclear member of the Ran binding protein 1 family (Englmeier et al., 2001; Lindsay et al., 2001). Atomic structures are known for two transport recep- tors of the importin superfamily, importin (Imp or Kap 1) and transportin1 (Trn1 or Kap 2) (Cingolani et al., 1999; Chook and Blobel, 1999). Imp imports cargos bearing a classical nuclear localization signal (NLS), as well as cargos with no obvious NLS, either directly or in conjunction with other adaptor proteins or karyopherins. Trn1 mediates the import of mrna binding and ribosomal proteins. Imp and Trn1 are composed of tandem repeats of the HEAT motif, an 50 residue motif that is characterized by two antiparallel helices, designated A and B (Andrade and Bork, 1995; Andrade et al., 2001). The array of repeats defines a spiral-shaped superhelix that is composed of two arches corresponding to the N- and C-terminal halves of the receptors. The concave inner surface of the spiral is formed by the B helices; it binds to RanGTP through the N-terminal arch and to cargo through the C-terminal arch (Cingolani et al., 1999, 2002; Chook and Blobel, 1999; Vetter et al., 1999; Lee et al., 2003). The convex outer surface is formed by the Macromolecular traffic between the nucleus and cytosol is largely mediated by the karyopherin/importin superfamily of transport receptors, also called importins and A helices and interacts with FG-containing motifs of exportins (reviewed in [Fried and Kutay, 2003; Weis, nucleoporins (Bayliss et al., 2000, 2002). 2003]). The 14 karyopherins in yeast and over 20 in CRM1 shares sequence similarity with Imp and other mammals deliver various classes of macromolecular karyopherins over an N-terminal region termed the substrates, or cargo, through the nuclear pore com- *Correspondence: mueller@embl-grenoble.fr CRIME domain (Fornerod et al., 1997b; Görlich et al, 1997). In Imp and Trn1 this domain corresponds to HEAT repeats 1 3, which recognize RanGTP via its

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3 Architecture of CRM1 763 Table 1. Crystallographic Statistics P a 118.9, b 62.9, c Two Molecules/Asymmetric Unit Data Collection and Phasing Native SeMet Resolution (Å) Outer Shell (Å) ESRF Beamline ID14-2 ID14-4 R sym (%) a,b 12.0 (68.1) 9.2 (22.5) Unique reflections 35,395 (2,850) 25,650 (3,886) Observed 135,399 (7,523) 262,347 (41,462) Redundancy 3.8 (2.6) 10.2 (10.6) Completeness (%) 90.8 (73.3) 93.8 (99.7) I/ (I) 9.3 (1.6) 19.2 (9.7) Number of sites 19 R iso (%) c 38.0 (54.4) Phasing power acentric 1.86 (0.913) R cullis acentric (0.847) Overall figure of merit: Centric (0.121) Acentric (0.344) Structure Refinement Number of residues 642 Number of protein atoms 5108 Number of water molecules 192 R cryst (%) d 22.9 R free (%) e 27.7 Rmsd bonds (Å) Rmsd angles (degrees) 1.2 Residues in Ramachandran plot (%): Most favored regions 90.0 Allowed 9.7 Generous 0.3 Disallowed 0.0 a Values in parentheses are those for the outer resolution shell. b R sym I I / I, where I is the observed intensity of a given reflection. c R iso F PH F P / F P, where F PH and F P are the derivative and native stucture factor amplitudes, respectively. d R cryst F o F c / F o, where F o and F c are the observed and calculated structure factor amplitudes, respectively. e R free is equal to R cryst for a randomly selected 5% subset of reflections not used in the refinement. switch II region (Vetter et al.,1999; Chook and Blobel, 1999). Consistent with a role in Ran binding, deletion of the CRIME domain eliminates the ability of CRM1 to form a ternary export complex (Ossareh-Nazari and Dargemont, 1999). CRM1 possesses a highly conserved central region implicated in RanGTP-dependent NES recognition (Ossareh-Nazari and Dargemont, 1999). A cysteine residue in this region is covalently modified by LMB, a specific inhibitor of CRM1-mediated export (Nishi et al., 1994; Wolff et al., 1997; Kudo et al., 1999). A thorough understanding of the molecular details underlying karyopherin-mediated nuclear export has been hampered by a lack of structural information. Here, we present a structural model of human CRM1 based on the 2.3 Å crystal structure of the C-terminal third, homology models of the N-terminal and middle thirds, Figure 1. Structure of the C-terminal Region of CRM1 (A) Fragment used for structure determination and previously identified regions of CRM1. (B) Ribbon and topology diagrams. CRM1 CTR (50 Å 35 Å 20 Å) is shown with the A and B helices in red and yellow, respectively; the insertion containing helix 19 is in green. Repeat 19 is flipped: the face contacting repeat 18 corresponds to the lower face of the other repeats. (C) Stereo C trace. The six CRM1 CTR HEAT repeats were structurally aligned with the 19 repeats of Imp. The CRM1 CTR repeats are colored from red to magenta as shown; those of Imp are in black. For clarity the interhelical loops in Imp repeats 8, 13, and 17 are omitted. (D) Sequence alignment and secondary structure. The six repeats share little sequence similarity except for a conserved pattern of hydrophobic residues, highlighted in gray. HEAT-IMB is the consensus motif specific for the class of HEAT repeats found in karyopherins; the underlined Leu, Val, and Ala residues designate the seven core positions common to diverse HEAT repeat classes (Andrade et al, 2001). The CRM1 CTR repeats have hydrophobic residues at five of these core positions but lack the conserved Val(24) and Ala(27) residues, as well as the consensus Pro(11), Asp(19), and Arg/Lys(25) residues. Helices are underlined. (E) Surface representation showing the ridge of conserved residues. Residues are colored from white to green according to degree of conservation in a sequence alignment of 17 CRM1 orthologs. Residues in the conserved ridge are primarily charged (R845, D932, H935, R1016, and D1017) or aromatic (F838, W880, and F986). Right-hand view is that of the middle of Figure 1B. A or B helices face the reader as shown.

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5 Architecture of CRM1 765 and an electron microscopic reconstruction of the full- in a right-handed sense over repeats but in the length protein. Our findings provide insights into NES opposite sense over the last repeat. This occurs be- recognition, the cooperativity of Ran and cargo binding, cause helix 19 in the linker following repeat 18 intercalates and the effects of LMB and RanBP3 on CRM1-mediated between helices 18A and 19A, disrupting the norand export. mal stacking arrangement of repeats (Figure 1B). Most of the evolutionarily conserved residues in Results and Discussion CRM1 CTR are buried within or between HEAT repeats. Those that are solvent exposed cluster along one edge Crystal Structure of the C-Terminal Third of CRM1 of the molecular surface, forming a conspicuous ridge Limited proteolysis led us to identify a stable fragment spanning the six repeats (Figure 1E). The conserved of human CRM1 spanning 328 residues in the C-terminal ridge consists primarily of charged and aromatic resi- region (amino acids ) downstream of the centhe dues from the intramotif turns of repeats and from tral conserved region (Figure 1A). We crystallized this turn preceding helix 19A. Evidence below suggests fragment, hereafter denoted CRM1 CTR, and solved its that these residues interact with the Ran binding loop. structure by the method of single-wavelength anomalous dispersion (SAD) with a selenomethionine deriva- HEAT Repeat Map and Modeling of the N-Terminal tive. The structure was refined at 2.3 Å resolution to a and Middle Regions crystallographic R value of 22.9% and a free R value of Sequence similarity to the N-terminal regions of Imp 27.7% with good stereochemistry (Table 1). The asymmetric and Trn1 and secondary structure predictions suggest unit contains two molecules related by dyad sym- that CRM1 consists entirely of HEAT repeats, and the metry, and the final model contains residues CRM1 CTR crystal structure strongly supports this hypoth- from each of these. Residues are disordered. esis. However, apart from repeats 1 3 (the CRIME domain), CRM1 CTR adopts an -helical structure formed by six the identification of repeats located upstream of helical hairpin motifs that can be structurally aligned CRM1 CTR is difficult, as the sequence identity of this onto the HEAT repeats of Imp with good fit (Figures region with Imp or Trn1 is low, and because the HEAT 1B and 1C). The six repeats share low sequence identity motif is highly degenerate. In an attempt to map these with one another and deviate considerably from the repeats, we sought to relate the CRM1 amino acid se- HEAT repeat consensus, explaining why they were un- quence to that of either Imp or Trn1 by searching for detectable by sequence analysis (Figure 1D). As shown suitably intermediate sequences, a strategy used to re- below, residues immediately upstream of CRM1 CTR cor- late distant homologs (Park et al., 1997). We performed respond to HEAT repeat 13 of Trn1, and so we number pairwise sequence comparisons between all available the six repeats from 14 to 19. orthologs of CRM1 and those of Imp or Trn1 (Supplemental Gel filtration and crosslinking experiments indicate Figure S1 available online at that CRM1 CTR is dimeric in solution (not shown). In the org/cgi/content/full/16/5/761/dc1/) with the program crystal, the hydrophobic distal face of repeat 14 packs Blast2p (Tatusova and Madden, 1999). This approach against the equivalent face from the other monomer in revealed that the 250 residues immediately upstream of the asymmetric unit. This dimerization interface presumably CRM1 CTR are significantly similar to residues of mimics the interface between repeats 13 and 14 in Trn1, which comprise helix 8B and repeats The full-length CRM1. Numerous charged and polar residues resulting sequence alignment between CRM1 and Trn1 on the distal face of repeat 19 allow this repeat to termi- over this region is convincing because it has several nate the array of repeats as a hydrophilic cap. Repeat statistically significant blocks of conserved residues (as 19 may be further stabilized by the high proportion judged by Macaw analysis; Schuler et al., 1991), because ( 45%) of charged residues in the C-terminal tail region the assignment of A and B helices agrees well missing from our construct (residues ). This with secondary structure predictions, and because the tail region comprises only 15 residues in certain orthologs, predicted HEAT-repeat register is in phase with that too few to constitute a proper repeat; its struc- observed from the CRM1 CTR crystal structure: helix A of ture might resemble that formed by the C-terminal 25 the sixth-to-last repeat (repeat 14) of Trn1 aligns with residues of Trn1, which comprise two small helices helix A of the sixth-to-last repeat of CRM1 CTR (Figure 2A). (sometimes designated HEAT repeat 20). A similar approach with paralogs instead of orthologs As in other HEAT repeat proteins, the array of repeats allowed us to extend the HEAT repeat assignment from in CRM1 CTR defines a superhelix, or solenoid, in which residue 150 at the end of repeat 3 to residue 290, ac- the A- and B helices are oriented nearly perpendicular to counting for repeats 4 6, and more tentatively to residue the superhelical axis. The solenoid winds continuously 380 at the end of helix 8A (Supplemental Figures S2 and Figure 2. HEAT Repeats in CRM1 (A) Sequence alignment between the middle regions of CRM1 and Trn1. Identically and highly conserved residues are in inverse font and highlighted in gray, respectively. The chymotrypsin cleavage site marks the beginning of the C-terminal 70 kda fragment. Colored bars indicate helices observed from crystal structures; predicted helices are in gray. The LMB site and residues implicated in RanBP3 binding are indicated. Sequence numbering is that of human Trn1 (top) and human CRM1 (bottom). (B) HEAT-repeat maps. CRM1 is predicted to contain the same number of repeats as Trn1 and Imp. The difference in sequence length is due to CRM1 having larger individual repeats (primarily repeats 1, 3, 7, 13, 15, and 19) and a long tail region. The C-terminal region of Trn1 sometimes designated repeat 20 is labeled C. The hashed bar shows where the mapping is uncertain.

6 Molecular Cell 766 S3). The complete HEAT repeat map for CRM1 shows well into region 3. Automatic docking with SITUS (Chacón a one-to-one correspondence with the repeats of Imp and Wriggers, 2002) confirmed this assignment and and Trn1 (Figure 2B). A common architecture for such enabled the model to be positioned more precisely. The distantly related paralogs suggests that all karyopherins homology models of the N-terminal and middle regions may share the same number of HEAT repeats despite of CRM1 were fit as rigid bodies into regions 1 and 2. their considerable variability in length. The 19 HEAT repeats fit snugly into the EM map, with few We used the sequence alignments of Figures 2A and residues out of density and little density unaccounted for S3 to construct homology models for the N-terminal and (Figure 3C). middle regions of CRM1 by the spatial-restraints method The topology of our final model matches that of Imp described by Sali and Blundell (1993) (see Experimental and Trn1, with A helices on the outside and B helices Procedures). The validity of the two models was verified on the inside. The toroidal appearance is partly due to by a number of stereochemical quality criteria, as well the curvature of C-terminal repeats, which deviates from as by the ability of point mutants designed on the basis that of the two importins. This can be seen by superposing of these models to fold properly. The largest errors in the CRM1 CTR crystal structure with the C-terminal these models are likely to arise from inaccuracies in the repeats of Imp : the Imp repeats spiral around the interrepeat packing angles, as the relative orientations central axis, whereas the CRM1 repeats curl away (Fig- of repeats in CRM1 may differ from those of the Imp ure 3D). The toroidal shape also implies a different curvature and Trn1 template structures. However, we expect the for the N-terminal repeats, which we modeled as a topology of residues and the structure of individual re- single 50 rotation between the N-terminal and middle peats to be quite accurate, with the possible exception regions (relative to their orientations in Trn1), although of residues , where the sequence alignment is a set of smaller rotations distributed over several repeats poor. is more likely. In our structural model, HEAT repeat 1 lies next to repeats The occlusion of putative Electron Microscopy and Pseudoatomic Ran binding residues in this repeat might contribute to Model of CRM1 the low intrinsic affinity of CRM1 for RanGTP. Because full-length CRM1 did not crystallize, we pursued a low-resolution structure by using electron mi- Evidence for a Large Ran Binding Loop croscopy (EM) and single particle analysis. This is feasible within HEAT Repeat 8 because CRM1 (123 kda) is comparable in size to In the HEAT-repeat map of CRM1, predicted helices 8A aquaporin-0 (118 kda), for which an EM reconstruction and 8B are separated by a stretch of approximately 65 has been determined at better than 20 Å resolution residues (residues ; Figure 2B). The corresponding (Zampighi et al., 2003). We used the negative stain technique residues in Trn1 form a large acidic loop that inter- rather than cryo-em, as the low image contrast acts with the basic patch ( 4 5 region) of Ran (Chook of the latter precludes its use for particles of mass less and Blobel, 1999). Considerable evidence indicates that than 250 kda. Visual inspection of raw images revealed CRM1 residues also form such a loop. This several distinct shapes (rods, hooks, rings, and lariats), region is highly sensitive to a number of proteases, in- and the enhanced appearance of these shapes in the cluding chymotrypsin, subtilisin, and V8 protease (Figures class averages confirmed that particles were correctly 4A and 4B). The main cleavage products, a 45 kda aligned and classified (Figure 3A). The resolution of the and a 70 kda fragment, migrate as a single band on a final three-dimensional reconstruction was estimated by native gel and copurify by gel filtration at the same elu- Fourier shell correlation to be 22 Å. tion volume as the uncleaved protein, consistent with The CRM1 reconstruction has a worm-like appearance proteases merely nicking exposed loop residues (Figfrom clearly related to that of density maps calculated ures 4B and 4C). Also consistent with a flexible loop the Imp and Trn1 crystal structures at an equivalent structure, residues are highly variable among resolution (Figure 3B). The resemblance is not due CRM1 orthologs. Moreover, a conserved cluster of acidic to model bias, as the EM reconstruction was obtained residues (residues ) is located before helix 8B without reference to the atomic structures. The dimen- and could feasibly mediate Ran binding (Figure 4A). sions of the tubular density further confirm that CRM1 We confirmed the presence of a loop structure by is entirely made of HEAT repeats. Unlike the spiral replacing residues by a ten residue linker sequence, shapes of the two importins, the CRM1 density presents ASSGSGGSGS (mutant Loop; Figure 4A). This a more toroidal or ring-like appearance, which is consis- mutant is soluble and elutes by gel filtration like wildtype tent with the shape deduced for the yeast ortholog Xpo1 (wt) CRM1, suggesting that the protein is properly in a small-angle scattering study (Fukuhara et al., 2004). folded. The mutated residues are therefore unlikely to However, some flattening of the sample due to the negative belong to a HEAT repeat, because the replacement of stain procedure cannot be excluded and could con- an entire or a partial repeat by a hydrophilic sequence tribute to a more compact appearance. is expected to disrupt the hydrophobic packing interac- The CRM1 reconstruction has two large lobes (regions tions with the flanking repeats and to compromise 1 and 3) connected by a narrower tube of density (region proper folding. Indeed, the three-dimensional structure 2), suggesting that terminal repeats occupy the lobes of the mutant appears identical to that of the wt by and central repeats occupy region 2. Manual docking electron miscropy (Figure 3A), showing that the mutated of the CRM1 CTR structure into the EM map allowed the region forms an excursion that is dispensable for the correct hand to be determined and showed that the structural integrity of the solenoid. crystal structure is incompatible with region 1 but fits The Ran binding function of this loop was verified by

7 Architecture of CRM1 767 Figure 3. Electron Microscopy and Pseudoatomic Model of CRM1 (A) Negative-stain electron microscopy and single particle analysis. Shown are five of the 196 class averages used to calculate the threedimensional (3D) structure and reprojections from the final 3D reconstruction. From left to right are the hook, ring (two views), rod, and lariat orientations. Each square is 224 Å 224 Å. (B) 3D reconstruction of CRM1 and calculated density maps of Imp and Trn1. Maps were contoured to enclose the volume corresponding to the molecular mass by using a partial specific volume of 0.84 Da/Å 3. The arrow between regions 1 and 3 indicates where the ring of density first becomes discontinuous as the cut-off threshold is raised. Maps were calculated at 22 Å from the crystal structures of Imp (Cingolani et al., 1999) and Trn1 (Chook and Blobel, 1999) with the bound ligand removed. (C) Stereoview of the pseudoatomic model in the EM density. Helices are colored as in Figure 1B. (D) Curvature of C-terminal repeats. Aligning CRM1 CTR with Imp shows that the two structures follow similar courses over repeats but then diverge, with Imp curling inward and CRM1 CTR curling outward, giving CRM1 a less spiral appearance. The difference is due to larger side chains causing the B helices of CRM1 repeats to be spaced farther apart and to the intercalation of helix 19, which displaces repeat 19 from the mean superhelical axis. An alignment over all six repeats yields rmsd 210C 3.3 Å and over repeats yields rmsd 109C 1.5 Å.

8 Molecular Cell 768 Figure 4. Evidence for a Ran Binding Loop (A) Sequence alignment and loop mutants. Protease cleavage sites were identified by N-terminal sequencing and mass spectrometry. The hypervariable region varies in length from 4 to 103 residues; a 66 residue insertion is indicated in parentheses. Predicted helices are shown as gray bars. (B) Chymotryptic time course. CRM1 was incubated with chymotrypsin (100:1 w/w) and the reaction stopped at the indicated times by the addition of PMSF. The 45 and 70 kda fragments migrate as two bands on a denaturing polyacylamide gel (top), but only as one on a native gel (bottom), suggesting that they remain noncovalently associated. Digestion also produces the CRM1 CTR fragment but with slower kinetics. Experiments with elastase, subtilisin, and proteinase K gave nearly identical results.

9 Architecture of CRM1 769 using a RanGTPase-activating protein (RanGAP) inhibi- CRM1 and comprises the acidic cluster region, helix 8B, tion assay. In this assay, GTP hydrolysis by Ran is stimulated and repeats Plotting the sequence conservation by RanGAP but inhibited by the association of onto the surface of our CRM1 structural model reveals a RanGTP with CRM1 (Askjaer et al., 1999). The concentration strip of invariant residues on the B helices and intramotif of CRM1 required to inhibit GTP hydrolysis by turns of repeats 10 and 11 and smaller patches on the 50% provides a measure of intrinsic Ran binding affinity; A helices (Figure 5A). A remarkably similar pattern is the enhanced affinity in the presence of a cargo further observed in the corresponding plot for Trn1, apart from inhibits hydrolysis, allowing one to assess the degree of the small patches (Figure 5B). The conserved Trn1 resi- ternary CRM1/RanGTP/cargo complex formation and, dues interact with the acidic loop, which traces a circuitous indirectly, cargo binding activity. We tested an NES peptide path over the intramotif turns and B helices of the derived from the NS2 protein of minute virus of mice central repeats. A corresponding path and set of interactions (mvm), which binds CRM1 particularly strongly (Askjaer seems likely for the Ran binding loop of CRM1. et al., 1999), and Snurportin1 as export cargos. In this Consistent with a sizable intramolecular interface, this assay, CRM1 was estimated to bind Ran with an apparent loop is cleaved at both ends by chymotrypsin but re- K d of 8.2 M in the absence of cargo (not shown) mains noncovalently associated with the larger 45 kda and an apparent K d of nm in the presence of either and 70 kda fragments (see Supplemental Data). The the NES peptide (Figure 4D) or Snurportin1 (not shown; acidic loop of Trn1 mediates the Ran-induced dissociation results are summarized in Figure 4E). Disruption of the of bound cargo. When Ran binds to the N-terminal loop by chymotrypsin treatment led to a loss of detect- arch of Trn1, the acidic loop changes conformation and able RanGTP binding activity in the absence of cargo displaces the cargo from its binding site in the C-ter- and a 20- to 40-fold decrease in the presence of the minal arch (Chook et al., 2002). We hypothesized that NES peptide or of Snurportin1 (apparent K d values of the Ran binding loop of CRM1 mediates the cooperative 600 nm and 360 nm, respectively; Figures 4D and 4E). binding of Ran and cargo in a similar fashion: the loop Thus, the integrity of the loop appears critical for proper adopts a conformation whereby it simultaneously masks Ran binding and ternary complex formation. the cargo binding site and hinders Ran from binding We verified the importance of the acidic cluster in stably. When Ran or cargo binds, the loop changes Ran recognition by constructing two mutants: Acid, in conformation and either exposes the cargo binding site which the acidic region was replaced by the ASSG or allows Ran to bind stably, respectively (see Fig- SGGSGS linker sequence, and NQ7, in which seven ure 6D). acidic residues were mutated to the corresponding Gln Support for this hypothesis comes from mutational or Asn residue (Figure 4A). For both mutants, Ran bind- analysis of the conserved CRM1 surface. We made 13 ing activity was undetectable in the absence of cargo alanine point mutations involving 11 invariant exposed and reduced by approximately four orders of magnitude residues, including double and triple mutations at spatially in their presence (Figures 4D and 4E), strongly supporting clustered positions. Mutants behaved like the wt the hypothesis that Ran recognition occurs via the during purification, indicating that their structure was acidic region. To see whether Ran binding could be not greatly perturbed. We used the RanGAP assay to enhanced by simply increasing the number of acidic measure intrinsic Ran binding activity and the ability to residues in this region, we mutated four hydrophobic form a ternary export complex with either the NES pep- residues to Asp or Glu residues (mutant DE4). However, tide or Snurportin1. The latter provides a useful indirect this led to a severe loss of Ran binding activity, both measure of cargo binding affinity, which in the absence with and without cargo, suggesting that the mutated of RanGTP is too weak to estimate reliably. The results residues play an important structural or functional role. are summarized in Figure 5C. Ran binding affinity in the presence of cargo could be Only one mutant, Y454A, behaved like the wt. The partly restored by mutating six additional residues to rest were either altered for intrinsic Ran binding activity Asp or Glu (mutant DE10), consistent with Ran recogni- (class 1), for ternary complex formation (class 2), or for tion involving a significant electrostatic component. both (class 3). Remarkably, four of the class 1 mutants showed an increase in intrinsic Ran binding affinity Role of the Central Conserved Region (class 1a). The largest increase was seen for mutant and Model of Cooperativity M583A/K590A, with an intrinsic affinity over 30 times The central conserved region of CRM1 implicated in higher than that of the wt (apparent K d 250 nm versus Ran-dependent NES recognition (residues ) 8.2 M); in contrast, mutant F554A showed a large decrease falls nearly entirely within the Trn1-like middle region of (class 1b). Except for Leu525, all class 1 residues (C) Gel filtration of chymotrypsin-treated CRM1. Untreated CRM1 (0 min) and CRM1 digested with chymotrypsin (30 min; 100:1 w/w) elute at the same volume. Denaturing gel electrophoresis of fractions from the upper profile shows that the 45 and 70 kda fragments remain associated, and coelute with residual uncleaved CRM1 (inset). (D) RanGAP assay of loop mutants in the presence of an NES. CRM1 was treated with chymotrypsin ( Cleaved ) until completely converted to the 45 and 70 kda fragments and subsequently purified from the protease by gel filtration. Data shown are the mean values and standard errors from three series of measurements. (E) Ran binding affinities of loop mutants. RanGAP assays were performed in the absence of cargo or in the presence of 2.5 M NES or 0.6 M Snurportin1. K d values shown are the mean values and standard errors determined from three to five measurement series. Values are listed as large if no detectable inhibition was observed at the highest tested concentration, which was 10 M except for DE10 (1.5 M), NQ7 (8.3 M), and Acid (28 M). A minor amount of inhibition was detected at 10 M DE4 with Snurportin1.

10 Molecular Cell 770 Figure 5. Functional Analysis of the Central Conserved Region (A) Surface representation of repeats 8 14 of the CRM1 structural model. Color scheme is that of Figure 1E. Alanine point mutations are indicated. (B) Surface representation of Trn1 repeats Residues are colored according to degree of conservation in a sequence alignment of 12 Trn1 orthologs. (C) Ran binding affinities of point mutants. RanGAP assays were performed as in Figure 4E. Apparent K d values significantly higher or lower than those of the wt are highlighted in yellow and green, respectively. The value boxed is that of the triple mutant severely compromised for NES binding. of the class 1a mutants, interacts with the acidic cluster region so as to hinder the interaction with Ran; replace- ment by alanine eliminates this constraint, allowing the loop to bind Ran more easily. correspond to Trn1 residues lying directly underneath the acidic loop. A change in Ran binding affinity due to a perturbed loop interaction with the conserved surface is consistent with our cooperativity model: class 1a mutations destabilize the (cargo-excluding) loop conformation characterized by low Ran binding affinity (hence increasing intrinsic affinity), and the class 1b mutation stabilizes it (decreasing affinity) (Figure 6D). One plausible scenario is that residue Lys590, common to three NES and Snurportin1 Recognition In over half of the mutants tested, Ran binding activity in the presence of the NES was significantly decreased (classes 2 and 3; Figure 5C). The mutated residues local-

11 Architecture of CRM1 771 ize to widely separated sites, implicating nearly the en- Location of the LMB Binding Site tire conserved surface in Ran-mediated NES recogni- LMB, an unsaturated branched-chain fatty acid, inhibits tion. In most cases, the decreased Ran binding affinity CRM1 export by covalently binding residue Cys528 was still 50 times higher than that measured in the (Kudo et al., 1999). The much smaller heterocyclic comabsence of cargo, indicating that mutants could still bind pound N-ethylmaleimide also inhibits export by modithe NES. In contrast, the NES-mediated enhancement of fying Cys528, implying that perturbations to the immedi- Ran binding activity was completely abolished for the ate environment of Cys528 account for inhibition (Holaska triple mutant L525A/K568A/F572A (K d 9.7 M with and Paschal, 1998; Kudo et al., 1999). Our structural NES and 5.0 M without). This effect was NES specific, model places Cys528 in the intramotif turn of repeat 10, as Ran binding affinity was still substantially enhanced in direct contact with the Leu525, Lys568, and Phe572 by Snurportin1. Moreover, the intrinsic Ran binding aftional residues implicated in NES recognition by our mutafinity of this mutant did not significantly differ from that analysis (Figure 6A). This suggests that LMB steri- of the wt, implying a decrease in NES binding affinity cally hinders access to the NES binding site. Our model consistent with the loss of a direct interaction with the also accounts for the LMB resistance of S. pombe strain NES peptide. A smaller effect on NES binding was detations crm1-n1, which has a CRM1 variant with two point muduced for mutants Y454A/M583A and L525A/F561A, predicted to perturb the local structure (Nishi et which involve residues located close to, or overlapping al., 1994; Figure 6B). with, those altered in the triple mutant (Figure 5A). The intramotif turn containing Cys528 is shifted by The Leu525, Lys568, and Phe572 residues implicated only two repeats from the base of the Ran binding loop, in direct NES binding localize to the A helices of repeats and the corresponding turn in Trn1 intimately contacts 10 and 11 (Figure 6A). This is unexpected, because Imp the acidic loop (Figures 6B and 6C). Thus, the binding and Trn1 recognize cargo through B helices, and raises of LMB to Cys528 could feasibly influence the conforma- the interesting possibility that nucleoporins might dibinding induces a significant conformational change in tion of the loop and vice versa. Consistent with this, LMB rectly interact with the NES binding site. Of the three CRM1 (Fornerod et al., 1997a), and a RanGAP assay residues altered in the triple mutant, Lys568 and Phe572 shows that LMB-treated CRM1 has significantly remay be more important for NES recognition, because duced intrinsic Ran binding activity (not shown). Conmutant L525A binds the NES with nearly wt affinity (Figversely, an influence of loop conformation on LMB bindure 5C); however, a degenerate binding mode could also ing would account for the behavior of two other tolerate such a mutation. NES motifs are recognized S. pombe strains. Strain crm1-809 is hypersensitive to through their hydrophobic residues, and thus an interac- LMB and has a single point mutation, E430K (Nishi et tion with the hydrophobic Leu525 and Phe572 side al., 1994). Glu430 (human Glu429) lies within the acidic chains is reasonable. The aliphatic moiety of the Lys568 cluster region of the loop (Figure 4A), and the charge side chain could also interact with hydrophobic groups, reversal could feasibly alter the loop conformation so and its flexible nature might help accommodate the varias to facilitate LMB binding. The LMB-resistant strain ety of spacings observed among NES motifs. A docking crm1-119 has an additional mutation, F992S, which supstudy with the known atomic structures of NES motifs presses the E430K phenotype (Nishi et al., 1994). The showed that the Leu525, Lys568, and Phe572 side corresponding Phe986 residue of our structural model chains could potentially engage three of the hydrophois located in repeat 19, too far from Cys528 to interact bic NES residues (see Supplemental Data). directly with the LMB binding site (Figure 6B). However, Our hypothetical model postulates that the NES bind- the mutation could conceivably affect the LMB binding ing site is masked by the Ran binding loop. Residues site indirectly via the Ran binding loop. An interaction Lys568 and Phe572 correspond to Trn1 residues located between Phe986 and the loop is feasible, given that in within 12 and 5 Å, respectively, of the Trn1 acidic loop; Trn1 the tip of the acidic loop contacts repeat 18 and a similarly positioned CRM1 loop could feasibly interfere is only 7 Å away from repeat 19. Phe986 belongs to the with NES binding. A direct contact would require the conserved ridge of CRM1 CTR (Figure 1E), which is on the CRM1 loop to extend significantly further onto the same side of CRM1 as the LMB binding intramotif turn A-helices than does the Trn1 loop, as indeed is sug- and the base of the Ran binding loop (Figure 6B). An gested by the more highly conserved surface of CRM1 interaction with the loop would neatly account for the here (Figures 5A and 5B, right). Consistent with a direct conserved nature of this ridge. Thus, LMB may exert its loop contact, mutant L525A shows a 6-fold increase in inhibitory effects both by sterically blocking NES binding intrinsic Ran binding affinity (Figure 5C), putatively due and by interfering with the function of the Ran binding to the decreased stability of the cargo-excluding loop loop. conformation (see above). The L525A/K568A/F572A mutation that abolished How RanBP3 Likely Stabilizes NES recognition also severely reduced Ran binding ac- a CRM1 Export Complex tivity in the presence of Snurportin1, implying that the RanBP3 greatly increases the intrinsic affinity of CRM1 two cargo binding sites overlap significantly (Figure 5C). for Ran and for certain cargos (Englmeier et al., 2001; However, no mutations abolished Snurportin1 binding Lindsay et al., 2001). An obvious way to do this is to completely, suggesting that recognition involves multi- stabilize the Ran binding loop in its unmasking conforple sites widely separated on the CRM1 surface. Indeed mation, which simultaneously exposes the cargo bindthe two triple mutants with the largest decrease in ap- ing site and permits the stable binding of RanGTP. parent Snurportin1 binding activity involve clusters of Strong evidence for a direct interaction with the Ran residues spaced far apart. binding loop comes from a recent mutagenesis study.

12 Molecular Cell 772 Figure 6. Predicted Structure of the Central Conserved Region and Proposed Cooperativity Model (A) Close-up of the LMB and putative NES binding sites. Cys528 is predicted to pack against residues Leu525 and Phe572 and against the aliphatic moiety of Lys568. The acidic loop of Trn1 is superposed to give an approximate idea of the path of the Ran binding loop in CRM1. A helices are in red, B in yellow. (B) CRM1 structural model showing repeats 8 19 and summary of mutations. Residues mentioned in the text are indicated. B helices are in yellow; A helices and helix 19 are in gray. Strain crm1-n1 has two point mutations, G503D and M546I (G502 and M545 in human CRM1). Met545 is adjacent to Cys528 and is half a helical turn away from Ile547, which packs against Gly502.

13 Architecture of CRM1 773 Single point mutations at residues 411, 414, 474, or common ancestor and the loss of such features in the 481 greatly reduce the ability of CRM1 to bind RanBP3; Imp lineage. Hence, Trn1 and CRM1 are likely to better mutation at 478 also shows a partial phenotype (Hakata represent the karyopherin family than Imp. Indeed, et al., 2003). Residues Arg474, Glu478, and His481 are Imp 3 is predicted to contain a large acidic loop (Chook on helix 9A, close to the base of the Ran binding loop; et al., 2002), and Exportin-t aligns well with CRM1 over residues Pro411 and Phe414 are located within the loop HEAT repeats 1 13, including the Ran binding loop and itself (Figures 4A, 6A, and 6B). An altered loop conforma- central region critical for cooperativity. tion would explain the ability of RanBP3 to protect CRM1 from the inhibitory effect of LMB (Englmeier et al., 2001; Concluding Remarks Lindsay et al., 2001), as LMB binding is putatively sensi- The unequal distribution of RanGTP across the nuclear tive to loop conformation. The major binding site for envelope and the different effects of RanGTP on import CRM1 localizes to the FxFG-repeat containing domain and export receptors fundamentally determine the diof RanBP3 (Lindsay et al., 2001). FxFG repeats are rec- rectionality of nucleocytoplasmic transport. Our findings ognized by Imp via A helices (Bayliss et al., 2000), and indicate that the opposite behavior of these receptors so a role for helix 9A in RanBP3 binding makes sense. can be accounted for by variations in a common underlying molecular mechanism. We anticipate that CRM1 will A Common Mechanism for the Assembly and be a useful paradigm for the study of other export Disassembly of Import and Export Complexes? pathways. The mechanism of Ran-mediated cargo dissociation for Trn1 is compared to our cooperativity model for CRM1 Experimental Procedures in Figure 6D. In the cytoplasm, a Trn1/cargo complex Purification and Crystallization of CRM1 can form because the cargo binding site is exposed. In CTR N-terminally His-tagged CRM1 CTR was bacterially expressed and the nucleus, RanGTP triggers a conformational change purified by affinity chromatography and gel filtration, as detailed in in the Ran binding loop that displaces the cargo from the Supplemental Data. Crystals of CRM1 CTR were obtained by the its binding site. Conversely, in our hypothetical model, hanging drop vapor diffusion method with 1 M Na/K phosphate (ph a CRM1/RanGTP/cargo complex can assemble in the 8.2). Crystals were soaked in 30% glycerol and flash cooled in liquid nucleus, as RanGTP maintains the loop in a conformation nitrogen at 100 K. that exposes the cargo binding site. In the cytosol, Data Collection and Structure Determination GTP hydrolysis and the release of Ran lead to a switch Diffraction data collected at ESRF beamlines ID14-2 ( Å) in loop conformation that displaces the cargo from its and ID14-4 ( Å) on an ADSC Q4R CCD detector were binding site. The two mechanisms are formally similar: processed with XDS (Kabsch, 1993) and programs of the CCP4 suite the Ran binding loop adopts two alternate conforma- (CCP4, 1994). Selenium atoms were located by SHELXD (Schneider tions, either masking or unmasking the cargo binding and Sheldrick, 2002) and by a difference Fourier synthesis. SAD site, and a switch in conformation is triggered when phases calculated with SHARP (de La Fortelle and Bricogne, 1997) and modified by RESOLVE (Terwilliger, 2000) gave a readily inter- Ran either binds or dissociates. The two proteins have pretable map. The atomic model was built with O (Jones et al., 1991) opposite activities because the preferred loop confor- and refined with CNS (Brünger et al., 1998). Strict NCS symmetry mations in the Ran bound and Ran unbound states are contraints applied in the early stages of refinement were released reversed, rendering ligand binding mutually exclusive in later stages, as validated by monitoring the R free. by Trn1 and cooperative by CRM1. The Ran binding loop of CRM1 is more hydrophobic than that of Trn1, Homology Modeling and a preference for the masking conformation in the Homology models for the N-terminal and middle regions of CRM1 were built with Modeller (Sali and Blundell, 1993) and their stereo- Ran unbound state may reflect the stability gained by chemistry validated by Procheck (Laskowski et al., 1993) and Whatif sequestering hydrophobic residues from the solvent, (Vriend, 1990). The modeling problem was greatly simplified by the whereas the preference of the highly charged Trn1 loop HEAT-repeat fold of CRM1. Almost all HEAT repeats of known for the unmasking conformation may reflect the tena atomic structure have highly similar backbone structures, despite dency to maintain charged residues solvent exposed. low level of sequence similarity (e.g., see Figure 1C). Thus, the The Ran-mediated dissociation of importin by Imp modeled structures of the individual repeats are expected to be reasonably accurate, with the possible exception of repeat 7 and occurs through a distinct mechanism, which involves helix 8A, for which the sequence alignment with Imp and Trn1 is Ran and cargo competing for overlapping binding sites less reliable (Supplemental Figure S3). In contrast, there is some and a Ran-induced conformational change in the HEAT uncertainty in the angles between successive repeats. However, the repeats (Chook and Blobel, 2001); indeed, the 11-resitures range of interrepeat angles observed in other HEAT repeat struc- due acidic loop of Imp is likely too short to play a is relatively limited ( 40 in Imp ). Assuming the maximal masking role. Phylogenetic analysis shows that CRM1 error in this angle, mutated residues that cluster on the surface of the CRM1 homology model (Figure 5A) would still be closely located diverged from Imp and Trn1 before the two importins on the surface of the true structure. The homology model of the diverged from each other. Thus, a large loop structure middle region of CRM1 is expected to be more accurate than that of and masking mechanism common to CRM1 and Trn1 the N-terminal region because of the higher quality of the sequence strongly suggests the presence of such features in their alignment (compare Figure 2A to Supplemental Figure S3). The po- (C) C-terminal arch of Trn1 showing the path of the acidic loop. (D) Comparison of Ran-mediated cargo dissociation by Trn1 with proposed cooperativity model of CRM1. Only the conformational changes within the Ran binding loop are shown; the HEAT repeats might also change conformation upon ligand binding. The masking conformation of CRM1 (top of scheme) is putatively stabilized or destabilized in the class 1b and 1a mutants, respectively.

14 Molecular Cell 774 tentially lower accuracy of the N-terminal homology model does not affect the interpretation of the mutants shown in Figures 5 and 6, which exclusively concern the middle and C-terminal regions. P. (2001). Comparison of ARM and HEAT protein repeats. J. Mol. Biol. 309, Askjaer, P., Bachi, A., Wilm, M., Bischoff, F.R., Weeks, D.L., Ogniewski, V., Ohno, M., Niehrs, C., Kjems, J., Mattaj, I.W., and Fornerod, Electron Microscopy and Image Processing M. (1999). RanGTP-regulated interactions of CRM1 with nucleopor- CRM1 (0.1 mg/ml) was applied to the clean side of carbon on mica ins and a shuttling DEAD-box helicase. Mol. Cell. Biol. 19, 6276 (carbon/mica interface) and negatively stained with 1% (w/v) sodium silicotungstate (ph 7). Micrographs were taken under low-dose con- Bayliss, R., Littlewood, T., and Stewart, M. (2000). Structural basis ditions with a JEOL 1200 EX II microscope at 100 kv and a calibrated for the interaction between FxFG nucleoporin repeats and importinmagnification of The six best micrographs were selected beta in nuclear trafficking. Cell 102, by using an optical bench and scanned on a Zeiss scanner with a Bayliss, R., Leung, S.W., Baker, R.P., Quimby, B.B., Corbett, A.H., step size of 14 m (3.5 Å on the sample scale). The image analysis and Stewart, M. (2002). Structural basis for the interaction between procedure was begun by selecting 2900 particles in pixel NTF2 and nucleoporin FxFG repeats. EMBO J. 21, squares from three micrographs with X3d (Conway and Steven, 1999). The selected images were band-pass filtered between Brünger, A.T., Adams, P.D., Clore, G.M., Gros, P., Grosse-Juntsleve, Å without CTF correction and normalized to the same mean R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., and standard deviation with SPIDER (Frank et al., 1996). To create et al. (1998). Crystallography and NMR system: a new software a starting model, a single image showing a clear ring of density was system for macromolecular structure determination. Acta Crys- selected, and the density within 50 Å of the center was projected tallogr. D 54, in a pixel box to a height of 40 Å, the thickness CCP4 (Collaborative Computational Project, Number 4) (1994). The of particles observed in raw images. This model was used in the CCP4 suite: programs for protein crystallography. Acta Crystallogr. projection matching method implemented in SPIDER using 46 D Biol. Cyrstallogr. 50, equally spaced projection directions (Roseman et al., 1996; Schoehn Chacón, P., and Wriggers, W. (2002). Multi-resolution contour-based et al., 2000). After 80 cycles of refinement, no further changes in fitting of macromolecular structures. J. Mol. Biol. 317, the map were visible, yielding an intermediate model roughly similar to that shown in Figure 3B. Chook, Y.M., and Blobel, G. (1999). Structure of the nuclear transport To improve the reconstruction, 2100 additional particles were complex karyopherin-beta2-rangppnhp. Nature 399, selected from the three remaining micrographs and images were Chook, Y.M., and Blobel, G. (2001). Karyopherins and nuclear import. corrected for CTF effects. The defocus of each micrograph was Curr. Opin. Struct. Biol. 11, determined as described (Conway and Steven, 1999). A full search Chook, Y.M., Jung, A., Rosen, M.K., and Blobel, G. (2002). Uncouwas performed by a root-mean-square method (James Conway, pling Kapbeta2 substrate dissociation and Ran binding. Biochemispersonal communication) to determine the best amplitude contrast try 41, (25%) and defocus values in the CTF ( m in all micrographs). Cingolani, G., Petosa, C., Weis, K., and Müller, C.W. (1999). Structure Data were corrected by using these values either by simple phase of importin-beta bound to the IBB domain of importin-alpha. Nature flip or by full CTF deconvolution. The information was low-pass 399, filtered to 15 Å, and the phase flipped set of 5000 particles was used to continue the refinement of the intermediate model, by using Cingolani, G., Bednenko, J., Gillespie, M.T., and Gerace, L. (2002). 196 equally spaced projection directions. The best 75% of images Molecular basis for the recognition of a nonclassical nuclear local- (those giving the highest correlation coefficient with the model reization signal by importin beta. Mol. Cell 10, projection) were included in the final reconstruction, which was Conway, J.F., and Steven, A.C. (1999). Methods for reconstructing calculated with the fully deconvoluted image set. The resolution density maps of single particles from cryoelectron micrographs was estimated by dividing images randomly into two equally populated to subnanometer resolution. J. Struct. Biol. 128, sets, reconstructing separately and determining the Fourier de La Fortelle, E., and Bricogne, G. (1997). Maximum-likelihood shell correlation in SPIDER, which gave a value of 22 Å at a threshold heavy-atom parameter refinement in the MIR and MAD methods. of 0.5. Fitting of the EM density is described in Supplemental Data. Meth. Enzymol. 276, Englmeier, L., Fornerod, M., Bischoff, F.R., Petosa, C., Mattaj, I.W., RanGTPase Inhibition Assays of CRM1 Mutants and Kutay, U. (2001). RanBP3 influences interactions between CRM1 RanGAP inhibition assays were performed essentially as described and its nuclear protein export substrates. EMBO Rep. 2, (Askjaer et al., 1999). Further details are given in Supplemental Data. Fischer, U., Huber, J., Boelens, W.C., Mattaj, I.W., and Luhrmann, R. (1995). The HIV-1 Rev activation domain is a nuclear export signal Acknowledgments Received: August 13, 2004 Revised: October 4, 2004 Accepted: October 19, 2004 Published: December 2, 2004 that accesses an export pathway used by specific cellular RNAs. Cell 82, We thank Ludwig Englmeier, Maarten Fornerod, and Gino Cingolani for early work, the European Molecular Biology Laboratory/The Eu- ropean Synchrotron Radiation Facility Joint Structural Biology Group for access and support at The European Synchrotron Radiation Facility beamlines, Willy Wriggers for advice on EM docking, Miguel Andrade for discussion about HEAT repeat detection, Darren Hart and Winfried Weissenhorn for technical advice, Frank Kozielski for access to a crystallization robot, and Rob Ruigrok for support. Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I.W. (1997a). CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 90, Fornerod, M., van Deursen, J., van Baal, S., Reynolds, A., Davis, D., Murti, K.G., Fransen, J., and Grosveld, G. (1997b). The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/ Nup214 and a novel nuclear pore component Nup88. EMBO J. 16, Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996). SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, Fried, H., and Kutay, U. (2003). Nucleocytoplasmic transport: taking an inventory. Cell. Mol. Life Sci. 60, References Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. (1997). CRM1 is responsible for intracel- Andrade, M.A., and Bork, P. (1995). HEAT repeats in the Huntington s lular transport mediated by the nuclear export signal. Nature 390, disease protein. Nat. Genet. 11, Andrade, M.A., Petosa, C., O Donoghue, S.I., Müller, C.W., and Bork, Fukuhara, N., Fernandez, E., Ebert, J., Conti, E., and Svergun, D.