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Review

The Molecular Mechanism of Transport of Macromolecules Through Nuclear Pore Complexes

Richard Baylissa, Anita H. Corbettb and Murray Stewarta* a

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK b Department of Biochemistry, Emory University School of Medicine, Rollins Research Center, Atlanta, GA 30322 -3050, USA * Corresponding author: M. Stewart, ms@mrc -lmb.cam.ac.uk

Trafficking of macromolecules between nuclear and cytoplasmic compartments takes place through the nuclear pore complexes (NPCs) of the nuclear envelope. Nuclear trafficking involves a complex series of interactions between cargo, soluble transport factors (carriers) and nuclear pore proteins (nucleoporins) that are orchestrated by the Ras-family GTPase Ran. The primary role of Ran is probably to establish directionality and to sort molecules to be transported by controlling the interaction between carriers and cargoes, so that they bind in one compartment but dissociate in the other. Translocation of carriers and cargo-carrier complexes through NPCs requires interactions between the carriers and nucleoporins that contain distinctive tandem sequence repeats based on cores rich in glycine and phenylalanine residues that are separated by hydrophilic linkers. Much recent work has focused on these interactions and, in particular, their specificity, regulation and function. Evidence is accumulating that carriers move through the NPC by distinct but overlapping routes using specific subsets of nucleoporins. Key words: Importin, interactions, NTF2, nuclear transport factors, nucleocytoplasmic transport, nucleoporin, Ran Received and accepted for publication 16 March 2000

The trafficking of macromolecules between the nuclear and cytoplasmic compartments through nuclear pore complexes (NPCs) is fundamental to eukaryotic cells. For example, proteins synthesised in the cytoplasm need to be imported into the nucleus, whereas mRNA needs to be exported to the cytoplasm. Trafficking of these molecules is generally mediated by soluble carrier molecules (transport factors) that bind their cargo macromolecule in one compartment and release it in the other, after which the carrier is recycled to participate in further rounds of transport. These carrier molecules are primarily members of the importin-

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b/karyopherin-b superfamily of transport factors and the interaction with their cargo is orchestrated by the nucleotide state of the Ras-family GTPase Ran. Although there is now considerable information available on the way Ran functions to alter interactions between carrier and cargo molecules in both the nucleus and the cytoplasm, less is known about the precise mechanism by which carrier-cargo complexes are translocated through the NPCs. In this review, we first give an overview of NPC structure and nuclear trafficking and then discuss the roles of Ran, before considering the structure of nuclear pore proteins and how they may interact with transport factors to mediate translocation.

Nuclear Pore Complexes The trafficking of macromolecules, ions and small molecules between the nucleus and cytoplasm is mediated by nuclear pore complexes (NPCs), cylindrical proteinaceous structures, about 1200 A, in diameter and 700 A, thick, that perforate the nuclear envelope (1,2). Vertebrate NPCs have an Mr of the order of 125000000 Da (3), whereas yeast NPCs are smaller with an Mr of about 66000000 Da (4). The morphology of both yeast and vertebrate NPCs has been reviewed extensively elsewhere (2,3). As illustrated schematically in Figure 1, NPCs are constructed from an approximately cylindrical central body sandwiched between nucleoplasmic and cytoplasmic rings. The central body has prominent 8-fold rotational symmetry and reconstructions from electron micrographs indicate that it is constructed from eight spoke-like segments (1 – 3). In addition, there are fibers that extend into both the cytoplasm and the nucleus and, in the case of the nuclear fibers, these form a basket-like structure below the body of the NPC (5). The body of the NPC has a central channel through which macromolecules are transported, although the precise details of this feature are controversial. Threedimensional reconstructions based on vitrified amphibian or yeast NPCs suggest the presence of a distinct cylindrical ‘transporter’ (2,6), but other authors have suggested that this feature might instead represent material in transit (3). Small molecules may also move through channels located at high radius in the spokes of the central cylinder (Figure 1) (3,7). Biochemical fractionation of NPCs has shown that many nucleoporins are associated with one another to form subcomplexes with a defined composition. The rat p62 complex, for example, contains p62, p54 and p58 (8). Moreover, these subcomplexes appear to be functional

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subunits of the NPC (8–10). Some, such as the yeast Nsp1p subcomplex, have been reconstituted from recombinant protein (11) suggesting that subcomplexes might be intermediates in the assembly of NPCs.

Overview of Nuclear Trafficking Proteins and nucleic acids are transported actively between the nucleus and cytoplasm along the central axis of the NPC (12,13). For example, proteins containing a nuclear localization sequence (NLS) are imported into the nucleus, whereas proteins containing a nuclear export sequence (NES) are exported to the cytoplasm. Although there are several different nuclear trafficking pathways, they share a number of common features (14–17). In each, the substrate or cargo does not interact directly with the NPC, but instead is transported bound to a soluble carrier molecule, which then has to be recycled back to the original compartment (Figure 2).

Protein trafficking Nuclear protein import is perhaps the best understood example of nuclear trafficking (see Figure 2). Assays based on digitonin-permeabilized cells (18), in which the plasma membrane is perforated but the nuclear envelope remains intact, have identified a number of soluble transport factors that act in conjunction with the NPCs (14 – 16,18). Protein import to the nucleus is a multi-step process that begins with an NLS-containing protein binding to a soluble carrier molecule of the importin-b family. In some cases this attachment is direct (20), whereas in other cases an adapter molecule such as importin-a is employed (21). The importin-cargo complex then docks at the cytoplasmic face of the NPC, before being translocated into the nucleus. Translocation is followed by displacement of cargo from the carrier molecule by Ran in its GTP-bound state within the nucleus (19,22), after which the carrier, complexed with RanGTP, is recycled back through the NPCs to the cytoplasm where RanGTP is hydrolyzed and the carrier is released (23 – 25). Nuclear protein export uses a similar pathway. For example, cargo bearing a leucine-rich nuclear export signal (NES) is recognized by the importin-b family member CRM1 in a RanGTP-dependent manner (26,27). The cargo-CRM1-RanGTP complex is then translocated through the NPC to the cytoplasm where it is dissociated in the same way as the importin-b-RanGTP complex. Importin-a is recycled to the cytoplasm by an analogous pathway using the importin-b family member CAS/CSE1 (28).

RNA Trafficking Multiple classes of RNAs, including mRNAs, tRNAs, and U snRNAs, are transcribed and processed within the nucleus and then transported to their sites of action in the cytoplasm. Competition experiments have shown that the export mechanisms of different classes of RNA are likely to be distinct from one another (16,29). A great deal of information has emerged regarding factors that are essential for RNA export (30), but, like protein trafficking, the exact mechanism of translocation through the NPC remains elusive. As compared with proteins, RNAs require extensive processing before they reach their mature form and are ready to exit the nucleus, and so it has sometimes been difficult to separate activities required for RNA processing (such as splicing) from bona fide mediators of export.

Figure 1: Exploded diagram of a nuclear pore complex (NPC) showing its overall morphology (see Figure 3). NPCs are constructed from a central cylinder composed of eight spoke-like units, sandwiched between nucleoplasmic and cytoplasmic rings from which fibers emanate. There is a central channel (red) through which macromolecules are transported.

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The export of tRNA, U snRNA and rRNA follow pathways analogous to nuclear protein export. For example, tRNA is recognized directly by the importin-b family carrier exportin-t (31,32) and is exported as a complex with Ran-GTP, which is disassembled in the cytoplasm where the Ran-GTP is hydrolyzed to Ran-GDP. Preferential export of mature tRNAs seems to be achieved at least in part by the specificity of exportin-t for the mature processed, modified, and appropriately aminoacylated tRNAs (33). U snRNAs are synthesized in the nucleus, transported to the cytoplasm where they associate with protein components of mature

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Figure 2: Schematic model for nuclear protein import. Carrier binds to cargo in the cytoplasm, docks at the NPC and is translocated to the nucleus where RanGTP binds to the carrier dissociating the cargo-carrier complex. The carrier-RanGTP complex is then exported to the cytoplasm where RanGAP and RanBP1 activate the RanGTPase activity and dissociate the carrier-Ran complex, thus freeing the carrier for a further cycle of nuclear import.

snRNPs, and are then re-imported to the nucleus where they function in mRNA splicing (34). The mono-methylated G cap of the initial export substrate is recognized by the cap binding complex (CBC) (34) and then exported from the nucleus in a CRM1-dependent manner (26). As there is no evidence that the CBC binds directly to CRM1, it seems likely that an adaptor protein mediates this interaction. rRNAs are exported in the context of large assembled RNP complexes. Although export depends on Ran (35,36), it is controversial whether Ran plays a direct role in export or whether instead its activity may be essential for the import of components required for RNP assembly (37). Export of poly (A) + RNA remains the least well understood of the RNA export mechanisms. mRNAs are not exported to the cytoplasm as naked nucleic acids, but rather as ribonucleoprotein complexes and it is generally agreed that the export machinery recognizes signals within the proteins of these complexes rather than the RNA itself (30). For example, export of unspliced HIV transcripts from the nucleus is mediated by Rev through its export by CRM1 (38). While this mechanism is exploited by HIV, it is not clear whether CRM1, any other member of the importin-b family, or indeed Ran itself plays a central role in mRNA export. Numerous candidate mRNA export factors have been identified (30). Many of these are hnRNP proteins that interact with poly (A) + RNA in vivo. A number of these hnRNP proteins shuttle between the cytoplasm and the nucleus

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and are thought to escort the RNAs as they are exported through the NPC (29,30). However, the mammalian protein TAP and its functional yeast homologue Mex67p are promising candidates for direct mediators of mRNA export (38,39,41). TAP was originally identified as a factor necessary for the export of simian type D viral RNAs that contain a CTE (constitutive transport element). Subsequent experiments showed that TAP is also required for the export of endogenous mRNAs (39,40) and that mutations in the MEX67 gene cause a rapid onset defect in the export of poly (A) + RNA (41). Attractive features of the TAP/Mex67 proteins are that they shuttle between the cytoplasm and the nucleus (39), and bind both to RNAs and to nucleoporins (42). Thus, they could potentially target bound RNAs directly to the NPC for export. Other candidate mRNA carriers include hnRNPA1 and yeast Npl3p and Hrp1p, which also shuttle between cytoplasm and nucleus most likely in concert with RNA (29,43). Future work should test whether hnRNP proteins contribute to the export pathway mediated by the TAP/Mex67 protein.

Function of Ran The nucleotide state of Ran is important in determining its interactions with other proteins, as for other Ras-family GTPases, and is an important determinant of nuclear trafficking (14,19). However, GTP hydrolysis by Ran does not appear to be linked directly to transport (44 – 47) and instead could be used, for example, to specify directionality (14,54) or to sort molecules prior to transport (17). The intrinsic rates of nucleotide exchange and hydrolysis on Ran are low (48), and so the nucleotide state of Ran is determined primarily by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Thus, the Ran GTPase activity is stimulated by RanGAP1 (49), which is located primarily in the cytoplasm with a proportion attached to fibers that emanate from the cytoplasmic face of the NPCs (50,51). The Ran GEF, RCC1 (52), by contrast is located primarily in the nucleus (53). This spatial separation of GAP and GEF activities is thought to result in cytoplasmic Ran being primarily in the GDP-bound state, whereas nuclear Ran is primarily in the GTP-bound state. This gradient of Ran nucleotide state has been proposed to be an important determinant of the directionality of transport (14,19,54). RanGTP binds to a common region of transport receptors of the importin-b family and also to other soluble proteins such as RanBP1 (19). In contrast to Ras, where only the GTP-bound form is active, both nucleotide states of Ran appear to be important in nuclear trafficking: RanGDP is associated with nuclear protein import, whereas RanGTP is associated with several export pathways (19,54). A principal function of RanGTP binding to transport receptors is in changing the affinity of the receptor for its cargo. On the nucleoplasmic face of the NPC, RanGTP dissociates import receptors from their cargo thereby terminating the import process (14). In addition, RanGTP is also required for for-

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mation of export cargo-carrier complexes (20). In the cytoplasm, RanGAP and RanBP1 promote hydrolysis of RanGTP bound to a transport receptor (23). This releases Ran from cargo receptors, thus preparing import receptors for another round of nuclear import and terminating a round of nuclear export (see Figure 2). Cycles of export by complexes containing RanGTP together with import by complexes, which do not contain Ran, deplete Ran from the nucleus. Although Ran is small enough to diffuse through the NPC, this appears not to be fast enough for efficient nuclear trafficking in vivo. Recent work has indicated that a key role of NTF2/p10 is to mediate the nuclear import of Ran (55,56), and so may function in a manner analogous to the guanine nucleotide dissociation inhibitor, RabGDI (57) to direct RanGDP to the nucleus for nucleotide exchange.

Nuclear Pore Proteins (Nucleoporins) Although NPCs are huge macromolecular assemblies, they appear to be constructed from multiple copies of a relatively small number of proteins. In fact, MALDI-TOF mass spectroscopy (58) indicates that isolated yeast NPCs contain 40 different proteins present mainly in either eight or multiples of eight copies per NPC. Vertebrate NPCs, being somewhat larger, probably contain additional proteins, but probably are constructed from the order of 50–70 different proteins. The NPC proteins, collectively called nucleoporins, frequently contain characteristic phenylalanine glycine (FG) sequence motifs (see below) and many have similar biochemical properties. Redundancy among the motif-containing nucleoporins is evident in that many nucleoporin nulls of Saccharomyces cerevisiae are viable, although they frequently show synthetic lethality with other nucleoporins (59). In addition to FG repeats, some nucleoporins contain other sequence motifs, such as those for coiled-coils, which appear to perform a structural role or are important in forming complexes (8). Other motifs, such as the hydrophobic segments found in Pom152p, appear to be integral membrane domains (60). Two vertebrate nucleoporins Nup153 and Nup358 also contain Zn-finger motifs, which bind to both DNA and to Ran (61,82). A striking feature of many nucleoporins is the presence of FG-repeats, large regions consisting of tandem repeats of a hydrophobic core and a hydrophilic linker (8,58,60). FG-repeats are based on highly conserved cores, containing one or two phenylalanines linked by hydrophilic spacers of variable sequence, but which are rich in charged and polar residues. The two most common repeats are based on GLFG or FxFG cores (where x is usually a small residue such as serine, glycine or alanine). Domains often contain many copies of these repeats, for example 19 FxFG repeats in Nsp1p and 33 GLFG repeats in Nup116p (Figure 3). Several key pieces of evidence suggest that these repeats are directly involved in nuclear trafficking. In vertebrates, many FxFG repeat nucleoporins bind wheat germ agglutinin (WGA). Nuclei assembled from Xenopus egg ex-

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tract depleted of WGA-binding proteins do not retain the ability to import NLS-containing substrates, but this ability is restored by adding back material eluted from WGA-sepharose (10), indicating that either the WGA-binding FxFG nucleoporins, or proteins bound to them, are required for transport. Nuclear trafficking is blocked by antibodies that recognize FG repeats or by added nucleoporin repeat constructs. For example, Mab414 (66), which recognizes FxFG repeats, blocks nuclear protein import and anti-Nup98 blocks RNA export (62). In addition, an RNA trafficking defect was observed upon overexpression of Nup153 FxFG repeats in BHK cells (63). Thus, different transport substrates appear to be transported via different routes using particular subsets of nucleoporins. In support of this model, FxFG-repeats from Nup153 inhibit NLS nuclear import but not transportin-mediated import (64), and nuclear protein import is not inhibited by anti-Nup98, which blocks RNA export (62). Overall, these data are consistent with the idea that although many nucleoporins are common to several trafficking pathways, others are only required for a specific pathway (65). The location of many repeat-containing nucleoporins has been investigated by electron microscopy, using gold-conjugated antibodies such as Mab414 against FxFG repeats (66) or by tagging yeast proteins at their C-terminus with protein-A (3,58). Because of the size of the antibodies used, such methods usually have an uncertainty of the order of 5 – 10 nm, which, although substantial, is much less than the dimensions of a NPC. Pre-embedding labeling shows the majority of yeast nucleoporins located symmetrically on nuclear and cytoplasmic faces of NPC with only a few being restricted to one side (Nup60p and Nup1p being

Figure 3: Sequences of nucleoporin FG repeats. A) Ten FxFG repeats from rat p62. Each repeat is composed of a more conserved hydrophobic core and a hydrophilic linker, which varies both in length and sequence between repeats. B) Seventeen FxFG repeats from Nsp1p. This repeat domain has an unusually high consensus between hydrophilic linkers. C) Thirty GLFG repeats from Nup116p. The linkers also contain multiple FG dipeptides.

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exclusively nucleoplasmic, Nup159p and Nup42p being exclusively cytoplasmic (58)). The localization of most labeled nucleoporins is somewhat diffuse, although it is clear that some are further away from the plane of the nuclear envelope than others and some are clearly on the nuclear basket or cytoplasmic fibrils rather than in the body of the NPC. Also, some labels are seen at higher radii than others. However, although the use of introduced tags gives a high level of specificity, pre-embedding will only label proteins that are accessible to the gold-conjugated antibody. Although epitopes on the surface of NPCs may be sufficiently exposed, those in the interior may be masked if gold-antibody conjugate does not enter the NPC. In principle, post-embedding labeling should overcome some of the accessibility problems, but protein-A tags are not detected effectively by this method. Moreover, protein-A tags are usually attached to the C-terminus of the nucleoporin and so, if the nucleoporin is very elongated or the C-terminus is flexible, these tags may not give a completely accurate reflection of the location of the bulk of the protein. Postembedding labeling has been used with vertebrate NPCs and Mab414 which recognizes FxFG-repeat nucleoporins (66) and indicates that these repeats line the central channel as well as covering the nuclear and cytoplasmic faces of the NPCs (67,68). NTF2/p10, a soluble factor which binds to FxFG repeats, shows a similar pattern of localization when microinjected into Xenopus oocytes (69) or when visualized in HL60 cells using immunogold labeling (68) whereas gold conjugates of NTF2 mutants that bind FxFG repeats less strongly show reduced NPC binding and are only rarely found in the central channel (70). Overall, electron microscopy appears to be consistent with the idea that FxFG repeats line the central transport channel as well as coating both faces of NPCs.

Interactions Between Nucleoporins and Transport Factors Several transport factors, for example importin-b and NTF2, show a distinctive accumulation at the nuclear envelope (71,72) and indeed at the NPC (69), consistent with evidence both in vitro and in vivo that those factors bind nucleoporins. Different transport factors preferentially bind specific nucleoporins and/or classes of nucleoporin. Importin-b, for example has been shown to bind both FxFG and GLFG nucleoporins by several methods (64,73,74). NTF2 binds FxFG nucleoporins (70,75–77), but although an interaction of NTF2 with GLFG repeats was reported using blot overlays (76), this interaction has not been confirmed by other methods (78). Recent work has highlighted that different members of the importin-b family bind to different, sometimes overlapping, subsets of nucleoporins (65,79,80). For example, transportin and importin-b bind to different domains of Nup153 (64). It has also been suggested that the multiple Ran-binding domains, which form part of the cytoplasmic fibril protein RanBP2/Nup358 and the nuclear basket nucleoporin Nup153, may have a role in improving the efficiency of nuclear trafficking. These do-

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mains may concentrate Ran at the periphery of the NPC (81), or may participate more actively by targeting importinb and transportin to the NPC (64,82), or by recycling importin-b after its export from the nucleus (83). Direct interactions have been observed between importin-a and Nup2p and Nup1p (84), and the interaction is required for efficient export of the yeast importin-a homologue Srp1p (85). Examples such as these illustrate putative roles for specialized nucleoporin domains at the periphery of the NPC in concentrating transport factors at the mouth of the NPC and, perhaps, catalyzing the turnover of transport complexes. Although it has long been known that the binding of importin-b to FxFG repeats is inhibited in the presence of RanGTP (73), recent work indicates that the influence of Ran on nucleoporin – importin-b interactions is more general. For example, CRM1 only binds to the p62 complex or Can/Nup214 in the presence of RanGTP (80,86) and RanGTP inhibits Pse1p binding to Nsp1p, Nup159p and Nup116p (79). These observations raise the possibility that the RanGTP-bound form of importin-b family carriers could be targeted to an export pathway via a specific set of nucleoporins dedicated to export, while the Ran-free form is targeted to a different set dedicated to import. Such a system might explain how NPCs are seemingly able to both import and export substrates simultaneously (13).

In vitro binding assays, antibodies, and dominant negative repeat domains suggest that different transporters follow distinctive pathways through the NPC. Support to this hypothesis has been obtained recently in vivo using F6 luorescence R6 esonance E6 nergy T6 ransfer (FRET) between spectral variants of the green fluorescent protein to monitor contacts made within the yeast NPC by both an import receptor, Pse1p, and an export receptor, Msn5p (65). The advantage of this experimental approach is that measurements are carried out in living cells (and thus fully native NPCs) in the course of actual transport reactions. Results indicated that the two transporters interacted with a number of identical nucleoporins but that each also made specific contacts with different nucleoporins. For example, Pse1p interacted with specifically with Nup53p, and Msn5p interacted specifically with Nup82p and Nup84p. Novel interactions detected in this study were confirmed by biochemical methods to demonstrate the validity of the FRET interactions (65). In the future this type of in vivo analysis could be used in combination with biochemical methods to map specific pathways through the NPC for a large variety of transporters. Little is known about the structural basis of carrier-nucleoporin interactions. Although the crystal structures of importin-b and transportin are known (87 – 90), it has not been possible to define the precise binding site of FxFG repeats on these molecules. Deletion studies suggest the binding site is between residues 152 and 352 in human importin-b

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(72,91,92). The structure of NTF2 (93), however, shows a clear hydrophobic patch centered on Trp7, which has been shown to be the binding site for FxFG repeats on NTF2 (70). A point-mutation at this site on NTF2 reduces its affinity for FxFG repeats and has been shown to reduce its binding to NPCs. Although this point mutant retained wild-type binding to RanGDP, the mutant showed reduced efficiency in NTF2-mediated nuclear import of RanGDP, demonstrating directly the functional importance of the NTF2-FxFG interaction in mediating RanGDP import (70). Import carriers probably interact with a range of different FxFG nucleoporins both during nuclear protein import and recycling to the cytoplasm. Because FxFG repeat nucleoporins are located at both faces of NPCs as well as the central transport channel (3,58,67), they could facilitate the translocation of carrier-cargo complexes through the NPC by enabling them to hop from one repeat to another as illustrated in Figure 4. Alternatively, the repeats could contribute to a Brownian affinity gating (58). It may be that directionality in trafficking could be generated by a gradient of affinity between importin-b family carrier molecules and different nucleoporins (14) and indeed it does appear that importin-b binds more strongly to Nup153p, located on the nucleoplasmic face of the NPC, than to other nucleoporins (64). The most straightforward way such a gradient of affinity could be generated is by the linkers between the FxFG cores making a contribution to the strength of the binding to importin-b. However, because the FxFG repeat nucleoporins function as a multidentate ligand, an affinity gradient could also be generated if the local concentration of FxFG repeats was elevated. The release of importin-b from the nucleoplasmic face of the NPC by RanGTP would then terminate translocation.

Questions Outstanding:

Figure 4: A schematic model illustrating how carrier-cargo complexes could be translocated through NPCs by hopping between FxFG repeats. EM has localized FxFG repeats to the central channel of the NPC as well as to the nuclear and cytoplasmic faces (3,58,67). FxFG repeats have been implicated in the translocation step of nuclear trafficking (65,73). Nucleoporins typically contain 10–30 FxFG repeats and, due in part to the 8-fold symmetry of the NPC, the concentration of FxFG repeats in the NPC is predicted to be high (of the order of 50 mM (70)). The carrier-cargo complex is first directed into the central channel of the NPC by interactions with FxFG repeats on the cytoplasmic face of the NPC. Interactions between the carrier and FxFG repeats mediate the translocation step by allowing the carrier to hop from one FxFG repeat to the next. At the nuclear basket, RanGTP disrupts the binding between the carrier and FxFG repeats leading to release of the carrier and its cargo.

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Many of the molecular components of the nuclear trafficking machinery have now been characterized and their interaction partners identified. Therefore, emphasis is now shifting to control and mechanism of translocation where there are a number of key questions outstanding: “ “

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What is the structural basis for the interaction between transport factors and nucleoporins? Who interacts with who? Are some types of nucleoporin repeats used for import and some for export, or are the rules more complicated? Are there defined routes through the pore for different carriers? How does Ran orchestrate interactions involved in translocation through NPCs? Does Ran binding alter the conformation of importin-b carrier molecules? How is the directionality of carrier movement through pore governed? Is there a gradient of affinity? Does Ran modulate directionality by changing affinity of carriers for nucleoporin repeats? If so, how?

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Can each NPC allow bi-directional transport simultaneously (13), or is each pore specialized for import or export (68)? Does the NPC change its conformation during either trafficking or to control the transport of different molecules?

Answers to many of these questions should start to appear in the near future and open the prospect of finally understanding the entire nuclear trafficking machinery at the molecular level.

Acknowledgments We are indebted to Rosanna Baker and Boots Quimby for their critical reading of the manuscript and to our colleagues in Atlanta and Cambridge for their may helpful suggestions. We also thank Graham Lingley for help with illustrations. Limitations of space prevented our referencing many of the primary papers in the field and we apologize to our many colleagues whose work was regrettably referred to by way of reviews. This work was supported in part by grant RG0270/ 1998 from the Human Frontiers Science Program.

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