Nature

Copyright 1997 Macmillan Magazines Ltd.
Volume 386(6627)             April 24, 1997             pp 779-787
Nucleocytoplasmic transport: signals, mechanisms and regulation
[Review Article]

Nigg, Erich A.

E. A. Nigg is at the Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland.
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Outline

In eukaryotic organisms, DNA replication and RNA biogenesis occur in the cell nucleus, whereas protein synthesis occurs in the cytoplasm. Integration of these activities depends on selective transport of proteins and ribonucleoprotein particles between the two compartments. Transport across the nuclear envelope occurs through large multiprotein structures, termed nuclear pore complexes. It is signal-mediated and requires both energy and soluble factors, including shuttling carriers. Here I summarize current understanding of nucleocytoplasmic transport and illustrate the importance of regulated transport for signal transduction.

In eukaryotic cells, the nucleus is separated from the cytoplasm by a double membrane system known as the nuclear envelope. The resulting spatial segregation of major cellular activities requires efficient mechanisms for selective transport of proteins and nucleic acids (1-5), and it affords levels of regulation that do not exist in prokaryotes (6,7). In exponentially proliferating cells, hundreds of proteins and ribonucleoprotein particles (RNPs) are translocated through each nuclear pore complex (NPC) every minute. This extensive transport activity concerns proteins and several types of RNAs, hereafter collectively referred to as cargo *(Table 1)*. The most prominent cargo for nuclear import consists of proteins, whereas export concerns mostly messenger RNAs, transfer RNAs, and ribosomal RNAs. Some RNAs, including many uridine-rich small nuclear RNAs (U snRNAs), are transported in both directions as part of their assembly into small nuclear RNP (snRNP) complexes (4). Furthermore, many proteins shuttle continuously back and forth between the cytoplasm and the nucleus, some slowly (8,9), others rapidly (10-12). Nucleocytoplasmic transport of viral genomes is vital for the replication and assembly of many viruses (13). Nuclear transport of human immunodeficiency virus (HIV), for instance, is critical for productive infection of non-dividing cells (14).

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Table 1. Cargo and signals for nuclear import and export
Both proteins and nucleoprotein complexes are transported through NPCs, but the transport of different classes of cargo requires at least partly distinct factors. Several of these factors have now been identified, and both biochemical and genetic studies demonstrate that the basic mechanisms of nucleocytoplasmic transport have been highly conserved during evolution (3,15,16). These exciting new developments set the stage for studying the regulation of nucleocytoplasmic transport and exploring its relevance to cell physiology. My aim here is to review the emerging understanding of nuclear import and export, to identify major unresolved questions, and to illustrate the importance of nucleocytoplasmic transport for intracellular signal transduction.

The nuclear pore complex and nucleoporins To Top

NPCs mediate bidirectional transport between the cytoplasm and the nucleus (17). They provide aqueous channels of about 9 nm in diameter, which allow the diffusion of ions, metabolites and small proteins (relative molecular mass M sub r less than (40-60) x 10 sup 3, 40K-60K), and mediate the selective transport of particles up to 26-28 nm in diameter by energy-dependent mechanisms (5,18). By electron microscopy, NPCs appear as roughly cylindrical structures, with eight-fold rotational symmetry in the plane of the nuclear envelope (*(Figure 1)*) (2,5,19,20). At the available resolution of 60-100 Angstrom, each NPC consists of a basic framework (a spoke complex embracing a central channel), positioned between a cytoplasmic ring and a nuclear ring (*(Figure 1)*a). The cytoplasmic ring is decorated by eight fibrils (*(Figure 1)*a, *(Figure 1)*b), and a basket-like assembly is attached to the nuclear ring (*(Figure 1)*a, *(Figure 1)*c). Vertebrate NPCs have an estimated outer diameter of 120 nm and M sub r values of about 125,000K; they may thus contain approximately 1,000 proteins, with multiple (generally 8 or 16) copies of some 50-100 different proteins (21). Many of these proteins, collectively termed nucleoporins, have now been characterized. Genes encoding vertebrate nucleoporins have been cloned primarily through the use of antibodies, and several nucleoporins have been localized to distinct NPC components by immuno-electron microscopy (5). Most were found on either the cytoplasmic fibrils or the nuclear basket, and comparatively few on the basic framework (*(Figure 1)*d, *(Figure 1)*e and *(Figure 1)*f). Yeast nucleoporins have been characterized by a combination of genetic and biochemical approaches (18,22,23). Extensive analyses of mutant phenotypes indicate a certain degree of functional overlap between individual nucleoporins but, interestingly, mutations in several essential nucleoporins inhibit nuclear import as well as export, consistent with the emerging view that these processes are coupled (18,23).

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Figure 1. The nuclear pore complex (adapted from (5,33,136,137)). a, Schematic representation of the three-dimensional architecture of the NPC. The major structural components include the 52,000K basic framework (the spoke complex; adapted from (19)) in pink, the 12,000K central channel complex in translucent light blue, the 32,000K cytoplasmic ring with eight short, kinky fibrils in blue, and the 21,000K nuclear ring with attached nuclear basket in red. The cytoplasmic (b) and nuclear (c) periphery of the NPC are revealed by quick freezing/freeze drying/rotary metal evaporation of spread Xenopus oocyte nuclear envelopes. b, On the cytoplasmic face of the NPC 'short cylinders' or 'collapsed filaments' are protruding from the massive cytoplasmic ring. c, On the nuclear face of the NPC eight thin filaments emanate from the more tenuous nuclear ring to join distally into a terminal ring, thereby forming a nuclear basket. d-f, Immunolocalization of Nups within the three-dimensional architecture of the NPC. Gallery of selected examples of colloidal gold labelled NPC cross-sections revealing the localization of distinct Nup epitopes; d, Nup214/CAN near the tips of the cytoplasmic fibrils; e, p62 at both the cytoplasmic and nuclear periphery of the central gated channel complex; and f, Nup 153 close to or at the terminal ring of the nuclear basket. Scale bars, 100 nm (b,c) and (d-f).
The primary structures of the known nucleoporins, as well as the phenotypes resulting from mutations of the corresponding genes, have been discussed in detail in several recent reviews (5,21,23). Here, only the most salient structural features of nucleoporins are summarized briefly. Many nucleoporins display highly repetitive motifs conforming to either the consensus FXFG and/or GLFG (amino acids are described using the single-letter code, with X indicating any amino acid). These FG-repeats interact in vitro with transport factors (24-27), but their precise functions in vivo remain to be clarified. It is attractive to speculate that different FG-repeats might differentially interact with distinct classes of cargo and thereby define separate transport pathways. Many nucleoporins also contain heptad repeats favouring alpha-helical coiled-coil formation, and these may promote the assembly of nucleoporin subcomplexes (28-31). Some nucleoporins (for example, mammalian nucleoporin (Nup)153 and Nup358) exhibit cysteine-rich zinc-binding motifs that are likely to mediate protein-protein or protein-nucleic acid interactions (32-35), whereas others (for example, yeast Nup 100p, 116p and 145p) display octapeptide motifs also found in RNA-binding proteins (36). Whether these latter motifs reflect a function for the corresponding nucleoporins in RNA transport, or, alternatively, the presence of structural RNAs within the NPC, remains unknown. Similarly unclear is the significance of the attachment of O-linked N-acetylglucosamine to many vertebrate nucleoporins (18).

Intriguingly, two nucleoporins (Nup98, Nup214) have been identified as fusion partners in distinct chromosomal translocations associated with myeloid leukaemia (37,38), and the N terminus of a 265K nucleoporin, termed Tpr (for translocated promoter region), has been found in oncogenic fusions with the kinase proto-oncogene products Met, Trk and Raf (39). This suggests that the fusion of certain nucleoporins to particular proteins might contribute to tumori-genesis. However, the molecular mechanisms by which this occurs remain to be clarified.

Signals for nuclear import and export To Top

Localization signals have been identified for several classes of cargo, but by no means all (*(Table 1)*). Best understood are nuclear localization signals (NLS) responsible for targeting proteins to the nucleus. These were defined by systematic deletion and transfer experiments and in general are characterized by the presence of basic residues in either one or two clusters (40). Accordingly, these NLSs are referred to as mono- or bipartite (*(Table 1)*). However, proteins may contain similar clusters of basic residues for reasons unrelated to nuclear transport, and, conversely, additional classes of (non-basic) NLSs may still await discovery (41). Also, not every nuclear protein should necessarily be expected to possess its own NLS, as in some cases nuclear entry may be afforded by interactions with NLS-containing partners. On the other hand, histone proteins enter the nucleus by a NLS-mediated route, even though they would be small enough to traverse the NPC by diffusion (42).

Considering that all proteins are made in the cytoplasm, yet many need to be imported into the nucleus, the existence of NLSs is easy to rationalize. It is perhaps less obvious why proteins should need nuclear export signals (NESs), as it would seem sufficient to prevent their import in the first place. Yet, efficient mechanisms for translocating proteins from the nucleus to the cytoplasm are expected to be important for the export of RNPs (see below), and in situations where the presence of a particular protein in the nucleus may be harmful to the cell. Notably in signal transduction, a protein's presence in the nucleus may be important at some times but deleterious at others. In line with these views, the first NESs were identified in HIV-1 Rev protein (43), and in a polypeptide inhibitor (PKI) of the cAMP-dependent protein kinase (PKA) (44). Rev is implicated in the export of viral RNA, whereas PKI most probably functions in the termination of signalling. The prototypic NESs of Rev and PKI are both short and hydrophobic, with a high leucine content (*(Table 1)*). Structurally related motifs are currently being identified in many distinct proteins. However, not every sequence motif functioning as a NES in a reporter construct should necessarily be expected to perform such a function in the context of the corresponding native protein.

The signals specifying transport of RNAs are not well defined, but are generally believed to reside with RNA-associated proteins. Binding of such proteins may depend on specific RNA secondary structures or modification (*(Table 1)*). In the case of mRNA, export signals appear to be associated with abundant heterogeneous nuclear RNA-binding proteins (hnRNP proteins). In vertebrates, particular attention has been focused on A1, a hnRNP protein that shuttles rapidly between the nucleus and the cytoplasm (10). Its transport in both directions depends on a 38 amino-acid motif, termed M9, indicating that import and export signals are either identical or interdigitated (45-47). When transferred to reporter proteins, M9 functions as both an NLS and an NES, yet its sequence bears no obvious similarity to either mono- or bipartite NLSs or to the NES of the Rev/PKI-type (Type 1). Export of 5S RNA in Xenopus oocytes depends on binding to either the ribosomal protein L5 or the transcription factor TFIIIA (48). Interestingly, TFIIIA displays a NES closely resembling those of Rev and PKI (49), but whether this NES is important for 5S RNA export is not known. In somatic cells, 5S RNA as well as rRNAs are exported in the form of preribosomal particles. Signals specifying export of rRNAs remain poorly understood, and the same is true for tRNAs (4,13,50,51).

A model emerges To Top

Current models for transport through the NPC rest on three major premises: first, that distinct types of cargo contain specific molecular signals, as described above. Second, that signal-dependent translocation of cargo through the NPC is mediated by mobile (shuttling) carriers. And third, that most (albeit probably not all) transport processes require the small GTPase Ran. In essence, it is thought that soluble carriers bind cargo in one compartment, escort it through the NPC, release it in the other compartment, and shuttle back to the first compartment (*(Figure 2)*). This general model is well supported for nuclear import; whether it also applies to export remains to be proved. In the case of protein import, carriers may be viewed as adaptors for mediating interactions between a large number of structurally distinct cargoes and a common, NPC-associated translocation machinery. In the case of RNP export, it is possible that similar adaptor functions might be provided by RNA-binding proteins escorting entire classes of RNAs. The shuttling carrier model is attractive for its simplicity, and it has the merit of focusing attention on discrete steps in the transport process. It is based on information obtained primarily from vertebrates and the budding yeast Saccharomyces cerevisiae. In the following, I will use a terminology applying to vertebrate homologues wherever possible, and, for the sake of clarity, each protein will be referred to by a single name, although many synonyms continue to be in use (for a guide to nomenclature, see *(Table 2)*).

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Figure 2. A unifying model for nucleocytoplasmic transport. This model emphasizes the role of shuttling carriers. It is well supported for protein import but largely speculative for RNP export. The NPC (in black) is shown schematically, embedded in the nuclear envelope (grey). Cytoplasmic fibrils and baskets are omitted and, as a consequence, cytoplasmic and nuclear sides are not defined.

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Table 2. A nomenclature navigator
A prominent role in nucleocytoplasmic transport has been attributed to the protein Ran, a member of the ras superfamily of small GTPases (16,52,53). Ran is highly abundant (10 sup 7 copies per mammalian cell) and cycles between two forms, Ran-GTP and Ran-GDP (*(Figure 3)*). The GTP- and GDP-forms of Ran bind selectively to distinct components of the transport machinery, and thereby control multiple protein-protein interactions important for cargo translocation (16). Moreover, much of the energy requirement for nucleocytoplasmic transport can be attributed to GTP hydrolysis by Ran. This conclusion is based on the demonstration that a Ran mutant with a nucleotide specificity altered from GTP to XTP (xanthosine-5'-triphosphate) can support protein import in an in vitro system supplied with XTP even in the presence of non-hydrolysable analogues of ATP and GTP (54). However, an independent study using a similar approach reported evidence for an involvement of a second GTPase distinct from Ran (55). Thus, although protein import in vitro can apparently be driven by Ran only, it is possible that other GTPases may enhance transport efficiency or provide parallel import pathways. Also, the energy requirements for nucleocytoplasmic transport in vivo remain unknown, and it would be premature to exclude rigorously a role for ATPases.

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Figure 3. The Ran-GTP/GDP cycle. The model emphasizes the distinct nucleocytoplasmic localizations of major Ran regulators (RanGAP1 and RCC1), and the consequent expected asymmetry in the distributions of Ran-GTP and Ran-GDP. However, additional regulators of Ran may exist and the precise subcellular distributions of Ran-GTP and Ran-GDP have not been determined directly. NPCs are drawn with cytoplasmic fibrils and nuclear baskets.
The low intrinsic GTPase activity of Ran is drastically stimulated by a GTPase-activating protein, termed RanGAP1. Conversely, the replacement of Ran-bound GDP by GTP is stimulated by a guanine-nucleotide exchange factor (GEF) termed RCC1 (3,16). As illustrated in *(Figure 3)*, these two regulators of Ran display a striking compartmentalization: the bulk of RanGAP1 is located in the cytoplasm, whereas RCC1 is predominantly nuclear. As a consequence, cytoplasmic Ran is expected to exist primarily in the GDP-bound state, whereas Ran-GTP is thought to be the prevailing form in the nucleus. However, the precise temporal and spatial distribution of the two forms of Ran is likely to be determined by selective interactions with several Ran-binding proteins (RanBPs) (56-60). Both genetic and biochemical data demonstrate that Ran and its regulators are essential for nuclear import (52,53,61-63), and genetic studies suggest a role also in export (64-67). However, as import and export processes are intimately linked (see below), in vitro studies will be required to prove an unequivocal role for Ran in export (68). Also, the functions of Ran and its regulators may not be limited to nucleocytoplasmic transport (16,69).

Mechanisms of nuclear import To Top

A major breakthrough for the study of nuclear import was the development of efficient in vitro systems (70,71). One particularly powerful assay takes advantage of the fact that digitonin can be used to permeabilize cells while leaving the nuclear envelope intact (71). In vitro import systems have allowed the characterization of several essential transport factors, the importance of which is corroborated by genetic studies in yeast (3,15,16). In brief, the docking of a NLS-protein to the NPC requires a heterodimeric carrier, whereas translocation through the NPC depends on the Ran-GTP/GDP cycle, and the small protein NTF2 (nuclear transport factor 2). This inventory is unlikely to be complete and additional transport factors probably await discovery. Permeabilized cells in fact still represent a complex system and factors will appear limiting only if they are efficiently removed during cell permeabilization. *(Figure 4)* summarizes the major events involved in the nuclear import of a NLS-protein; these are discussed in some detail below.

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Figure 4. Protein import into the nucleus. a, NLS protein recognition by an importin-alpha/beta heterodimer and docking to the NPC in a two-step reaction (80). b, Translocation of cargo-carrier complex; this step is poorly understood, but requires cytoplasmic Ran-GDP, NTF2, GTP hydrolysis by Ran, and perhaps additional energy (at least in vivo). c, Termination of translocation by binding of Ran-GTP to importin-beta, resulting in the release of importin-alpha and NLS protein into the nucleus. The mechanisms involved in the recycling of importin-alpha and -beta to the cytoplasm are unknown. For further explanation see text.
Recognition of cargo by shuttling carriers and docking to the NPC. Proteins carrying either a mono- or a bipartite NLS are recognized by a soluble, heterodimeric carrier that consists of proteins of about 60 and 90K; in vertebrate species, these proteins have been given many names, including importin-alpha/beta (72,73), karyopherin-alpha/beta (74), or pore targeting complex (PTAC) 58/97 (75,76) (for additional synonyms and names of yeast homologues see *(Table 2)*); hereafter they will be referred to as importin-alpha and importin-beta. Both importins contain several hydrophobic 42 amino acid repeats (known as 'arm' repeats, named after the Drosophila gene armadillo). Importin-alpha binds to NLS-proteins, whereas importin-beta strengthens the affinity of the complex for the NLS and mediates the docking of the cargo-carrier complex to the NPC (25,73-75,77-79). The cargo-carrier complex initially contacts the NPC at the distal end of cytoplasmic fibrils, from where it is transferred to the cytoplasmic entry of the central channel (80). Intriguingly, electron microscopy suggests that this transfer may involve bending (perhaps by brownian motion) of cytoplasmic fibrils (80).

Recent studies have revealed that the importin-alpha/beta complex is not the only carrier involved in protein import (81,82). Specifically, nuclear import of the shuttling hnRNP protein A1 is mediated by an importin-beta-related protein, termed transportin in mammals (81) and Kap104p in yeast (82). Transportin recognizes the M9 signal in protein A1, and presumably structurally related signals in other proteins, but does not interact with mono- or bipartite NLSs (81). Most remarkably, transportin functions independently of an alpha subunit, although it is structurally related to importin-beta. Thus, it is attractive to propose that importin-beta might represent the archetype of a carrier protein, whereas importin-alpha might have evolved as an adaptor, favouring the recognition of a vast multitude of different NLS proteins.

Competition studies clearly demonstrate that transportin defines a second pathway for protein import (81). In quantitative terms, the transportin and importin-alpha/beta carriers may contribute to a comparable extent to the total flux of proteins to the nucleus. NLS proteins, as recognized by the importin-alpha/beta carrier, may collectively be considered as a high-complexity class of cargo, with most members being present at comparatively low abundance. In contrast, mRNA-associated proteins are present in very large numbers (10 sup 6-10 sup 7 copies per cell), and thus represent a high abundance, low-complexity cargo. It is perhaps not surprising, therefore, that specialized carriers have evolved for the nucleocytoplasmic transport of such high-abundance cargoes. The genome of S. cerevisiae harbours only one gene for importin-alpha (SRP1/KAP60), but four distinct genes distantly related to importin-beta. Of these, Kap95 and Kap104 are the likely homologues of mammalian importin-beta and transportin, respectively (77,82,83). The other genes related to importin-beta are expected to encode carriers for as yet unidentified cargoes. Metazoan organisms express not only multiple proteins related to importin-beta, but also several importin-alpha isoforms (3). Mammalian importin-alpha subunits show tissue-specific expression patterns and many function as adaptors with at least partly distinct specificities for NLS proteins (84). In line with these views, mutational inactivation of one importin-alpha homologue of Drosophila, termed OHO31/pendulin, does not seem to impair protein import in all cells, but for some reason causes malignant transformation of haematopoietic cells (85,86).

Translocation through the NPC. Transfer of the cargo-carrier complex to the nucleoplasmic side of the NPC requires Ran-GDP on the cytoplasmic side, free GTP and NTF2. On the basis of in vitro protein-protein binding studies, it seems plausible that NTF2 may promote an interaction between cytoplasmic Ran-GDP, importin-cargo complexes and a subset of nucleoporins (87-89), and that nucleotide exchange and GTP hydrolysis may occur on NPC-bound Ran (54,58,61,90,91). Another abundant, cytoplasmic protein implicated in linking the docking and translocation steps in Ran-binding protein 1 (RanBP1): this protein promotes the association of Ran with importin-beta and enhances RanGAP1 activity (57,60,66,92), but its precise function remains unknown.

Translocation of the cargo-carrier complex through the NPC requires that a distance of about 100 nm be covered, and thus probably involves multiple steps. Several of these might require energy; alternatively, the actual energy-dependent translocation step could occur at a central structure of the NPC, and might require only a relatively small movement (80). A priori, it might have seemed attractive to invoke a participation of NPC-associated mechanochemical motor proteins, akin to myosin or kinesin. However, none of the known NPC genes codes for a recognizable motor, and most (if not all) energy consumption during protein import appears to reflect the GTPase activity of Ran, at least in vitro (54) (but see (55)). Thus, alternative models have been considered (3,16,93). In particular, it has been proposed that translocation might occur according to the 'brownian ratchet' hypothesis (94), which states that directionality may be conferred upon random thermal motions by chemical asymmetries. In the case of protein import through the NPC, one striking asymmetry stems from the distribution of Ran-GDP and Ran-GTP. Additional asymmetries may result from the disposition of individual nucleoporins. Furthermore, the affinity of importin-alpha for NLSs may be down-regulated selectively within the nucleus, and cargo may be recruited to non-diffusible sites, preventing its re-export. According to one specific model, translocation of NLS proteins might proceed by a series of docking and undocking reactions, involving multiple interactions between cargo-carrier complexes and FXFG-repeats in nucleoporins, and repeated rounds of Ran-GTP hydrolysis (24-26). To assess the merits of the currently prevailing models, it will be important to determine to what extent cargo-carrier complexes dissociate during translocation, and how many molecules of GTP are hydrolysed per cargo translocated. At the heart of the problem, it remains to be uncovered how the conceptual gating function of the NPC is reflected at the structural level. This may well require the development of new technologies, capable of monitoring motion with a high resolution in both time and space.

Dissociation of cargo-carrier complexes and carrier recycling. Once the cargo-carrier complex has reached the nucleoplasmic side of the NPC, the NLS protein is released, most probably through the action of nuclear Ran-GTP. In fact, the GTP form of Ran binds to importin-beta, and thereby displaces importin-alpha as well as the NLS cargo (25,92,95). Moreover, an importin-beta mutant which lacks the Ran-binding site is unable to discharge cargo into the nucleus (61). Thus, the role of Ran is not limited to its GTPase activity. Instead, cytoplasmic Ran-GDP appears to be required at an early stage of NLS protein import, whereas nuclear Ran-GTP is implicated in terminating the translocation. In this fashion, the asymmetric distribution of Ran-GDP and Ran-GTP is likely to play an important role in determining the directionality of protein import. Following cargo-carrier complex dissociation, importin-beta returns to the cytoplasm by unknown mechanisms, apparently without ever dissociating from the NPC (61,73,96). In contrast, both the NLS protein and importin-alpha are released into the nucleus. What mechanisms, other than dissociation from importin-beta, promote the separation of importin-alpha from cargo is unknown, but phosphorylation might be involved (97). Also, the recycling of importin-alpha to the cytoplasm remains poorly understood, but clearly requires determinants distinct from those specifying nuclear import (84,98).

Nuclear import of other cargoes. In addition to proteins, nuclear import also concerns RNPs. Well studied examples include U snRNPs involved in splicing. Shortly after transcription, snRNAs U1, U2, U4 and U5 are in fact exported to the cytoplasm, where they acquire a set of specific proteins (known as Sm core proteins) and undergo additional methylations of the 5' cap, before they are imported back into the nucleus (4). The binding of Sm core proteins is essential for nuclear import, whereas the formation of the trimethyl-guanosine cap (m sub 3 G-cap) increases import efficiency, depending on the U snRNA or the cell type (99). How these two signals cooperate is not understood. Competition studies show that nuclear import of U snRNPs involves components that are partly distinct from those mediating NLS protein import, and these may include specialized, as yet unidentified carriers (100). Other requirements for transport, such as the Ran-GTP/GDP cycle, may be similar (101).

Mechanisms of nuclear export To Top

Progress towards understanding nuclear export has been hampered by the lack of efficient, well characterized in vitro systems. Furthermore, the study of RNA export is complicated by its tight coupling, both spatially and functionally, to pre-RNA processing (3,4,13). It is difficult to isolate homogenous populations of fully processed nuclear RNPs, and the precise protein compositions of many RNP export substrates are unknown. Nascent pre-mRNAs, for instance, associate with about 20 abundant hnRNP proteins, of which several (including A1) accompany the mRNA to the cytoplasm, but others are specifically retained in the nucleus (13,102). Biochemical and genetic data suggest that hnRNP proteins play active roles in mRNP export (46,103). However, deciphering the precise roles of individual proteins will be difficult, because signals for RNA export may be additive, cooperative or partly redundant, and their importance may vary depending on the cargo. Also, multiple signals may be necessary for the translocation of large mRNPs.

In the absence of powerful in vitro systems, nuclear export has been studied by three complementary approaches. First, the large Balbiani ring transcripts in the salivary glands of the insect Chironomus tentans represent favourable objects for morphological studies on the processing and nuclear export of messenger RNPs (104-106). These studies have led to the important conclusion that hnRNPs unravel substantially while they transit through the NPC *(Figure 5)*. It is difficult to envision how such profound structural changes could be brought about without exertion of force within the NPC, although one could argue that RNPs might be pulled into the cytoplasm through the recruitment of their 5' caps onto ribosomes. Second, much information on the signals and factors involved in nuclear export has been obtained through microinjection of proteins or RNAs (or plasmids allowing transcription of RNAs) into the nucleus of Xenopus oocytes. This system has been used to demonstrate that RNA export is energy-dependent and saturable (50,107), and that export of distinct classes of RNAs (mRNAs, U snRNAs, tRNAs and rRNAs) involves at least partly class-specific factors, that is, perhaps distinct carriers (51,108). Third, valuable information about potentially relevant genes is emerging from the analysis of yeast mutant strains displaying defects in RNA export (109,110). At present, no detailed mechanistic models can be proposed for the export of any type of RNA but, as described below, significant progress has been made in a few specific areas.

Graphic Available

Figure 5. Transit of an RNP particle through the NPC. Three-dimensional reconstruction, based on electron micrographs, showing a Balbiani ring transcript RNP in transit through the NPC. The translocating particle (in light blue) is compared with a nucleoplasmic reference particle (yellow and brown). The surrounding material, including the NPC, is shown in white. The positions of the nuclear ring (marked NR) and the spokes (marked S) are indicated. The brown part of the reference particle corresponds to the portion of the translocating RNP particle that has unravelled and entered the NPC, and the yellow part corresponds to the remaining globular portion. The contact regions between the translocating particle and the nuclear ring of the NPC are indicated on the reference particle as white patches (red arrows). Numbers indicate four domains of the RNP particle, as described previously (104). Adapted from (104).
Rev-mediated export of viral RNAs: hijacking of a cellular export pathway. Important insight into export mechanisms has come from studies on the viral Rev protein encoded by HIV-1 (43-111), and its functional homologue Rex of human T-cell leukaemia viruses (112). In HIV-infected cells, Rev is essential for the cytoplasmic accumulation of incompletely spliced viral pre-mRNAs, and this function requires Rev binding to a highly structured RNA motif termed RRE (Rev response element). Rev is a rapidly shuttling protein (11,12) and it directs the nuclear export of RRE-containing RNAs, irrespective of whether or not these RNAs undergo splicing (111). Within its C-terminal domain, the viral Rev protein contains a NES (43) which structurally resembles the NESs identified in cellular proteins that are rapidly exported from the nucleus (*(Table 1)*). Introduction of the NES of Rev, coupled to bovine serum albumin, into Xenopus oocyte nuclei competitively inhibited nuclear export of 5S ribosomal RNA and U1 snRNA, but had no effect on the export of mRNA, tRNA or ribosomes (43). These and other data (113,114) indicate that Rev functions as an adaptor for linking export of pre-viral RNA to a cellular export pathway, and that cellular cargo for this pathway may include 5S RNA-TFIIIA complexes, some U snRNPs, as well as several NES proteins (such as PKI, I kappa B) with no obvious role in RNA metabolism. So far, no carrier of the importin-beta/transportin family has been implicated in exporting proteins with Rev/PKI-type NESs. However, cellular proteins able to interact with such NESs, and thus potentially involved in the corresponding export pathway, have been identified. These include human hRIP/RAB1 (115,116) and yeast Rip1p (117). Both proteins contain FG repeats reminiscent of nucleoporins, but display little sequence similarity otherwise. Yeast Rip1p appears to be concentrated at the nuclear envelope (117), but the bulk of hRip/RAB1 is found in the nucleoplasm (115,116). Recent data suggest that NESs may interact with FG-repeats in several nucleoporins indicating that translocation of NES proteins through the NPC involves sequential interactions with particular nucleoporins (27).

Another protein with a Rev/PKI type NES has been identified in yeast (118). This protein, termed Gle1p, was isolated in a screen for synthetic lethality with the GLFG-repeat NPC component Nup100. Mutational inactivation of the Gle1p NES impaired poly(A) sup + RNA export, leading the authors to propose that Gle1p may be an essential mRNA export factor, and that Rev might mediate viral RNA export by mimicking Gle1p function (118). However, this proposal appears to conflict with the finding that the NES of Rev did not compete with mRNA export in Xenopus oocytes (43), and further studies will be required to clarify the role of Gle1p.

A role for hnRNP proteins in mRNA export. Shuttling hnRNP proteins, exemplified by protein A1 of vertebrates (46,106) and the structurally related Npl3p of yeast (103-119), have been proposed to play prominent roles in mRNA export (13). In support of this view, the M9 signal confers export upon reporter proteins in the absence of any RNA binding (46), and export of hnRNP protein A1 does not depend on ongoing RNA transcription, although, curiously, re-import does (102). As described above, nuclear import of protein A1 is mediated by the carrier transportin (81). Because the M9 signal specifies export as well as import (46), it will be interesting to explore the possibility that transportin might also mediate the export of RNP complexes carrying A1 proteins.

Cap-binding proteins in U snRNA export. The 5' ends of all RNA polymerase II transcripts, including pre-mRNAs and most U snRNAs, acquire a monomethylated guanosine (m sup 7 G) cap structure. This m sup 7 G cap is implicated in pre-mRNA processing and mRNA translation; in addition, it constitutes one of probably several signals important for export of U snRNAs (108,120,121). The m sup 7 G cap is recognized in the nucleus by a protein complex, termed CBC (cap-binding complex), which consists of two cap-binding proteins, CBP80 and CBP20 (122). So far, no export signals have been defined on CBPs, and the precise role of these proteins remains to be clarified. In particular, they are not required for viability in yeast. Furthermore, the m sup 7 G cap is not essential for export of mRNA, although CBPs do accompany hnRNPs through the NPC, perhaps contributing to ensure that the 5' cap takes the lead during hnRNP translocation (105).

Coupling of import and export To Top

Import and export reactions are likely to be coupled at multiple levels. A first link stems from the fact that several proteins implicated in nucleocytoplasmic transport shuttle rapidly between the nucleus and the cytoplasm. Hence, sustained transport of cargo in one direction requires continuous recyling of carriers and factors in the other direction. A second link may be provided by Ran. Definitely required for protein import, the Ran-GTP/GDP cycle may be important also for the export of at least some classes of RNA (16), albeit apparently not all (69,123). A recent provocative study suggests an unexpected, additional link between import and export (124). Specifically, both in yeast and in Xenopus oocytes, a nuclear complex between importin-alpha, CBC and m sup 7 G-capped RNA was detected, suggesting that CBC might shuttle while bound to importin-alpha. Most interestingly, cytoplasmic importin-beta was shown to displace the importin-alpha-CBC complex from capped RNA, suggesting an attractive mechanism for releasing RNA cargo specifically in the cytoplasm (124).

Regulation of nucleocytoplasmic transport To Top

Both nuclear transport rates and the functional sizes of NPCs are significantly greater in proliferating cells than in quiescent cells, and they are increased further in neoplastically transformed cells (125). This indicates that the NPC and/or the transport machinery are subject to controls that affect entire classes of cargo. These generalized controls are likely to relate to overall cell metabolism (for example, to events such as ribosome production); how they operate is not well understood. More specific types of regulation concern individual types of cargo. The best evidence for regulated RNA export stems from studies on virally infected cells. During adenovirus or influenza virus infection, for instance, export of viral RNA is favoured over export of cellular mRNA, and the cytoplasmic accumulation of early and late viral transcripts is also regulated at the level of export (13). In the case of cellular RNAs, regulated export may contribute to determine not only the abundance, but also the spatial distribution of transcripts, particularly in response to stress (such as heat shock) (123) or during early development (13). The mechanisms underlying these regulations are unknown. Export of proteins with NESs may often depend on protein-protein or protein-RNA interactions favouring the exposure of such signals (44). On the other hand, export of slowly shuttling proteins appears to be limited primarily by intranuclear retention (9).

In the case of import, much attention has been focused on regulated nuclear entry of signalling proteins. For many prominent signal transduction pathways, specific proteins have been identified that translocate from the cytoplasm to the nucleus in response to appropriate agonists (for example, hormones or growth factors), and often these proteins are kinases (or phosphatases) or transcription factors (6,7,125). It is beyond the scope of this review to survey signal transduction to the nucleus. Instead, the following brief discussion is meant to highlight a few general principles of regulated nuclear transport, and to illustrate its importance for cell physiology. At first glance, the nucleocytoplasmic distribution of signalling proteins appears to be controlled by many different mechanisms. Closer inspection shows, however, that most of these represent variations on two major themes: one is based on anchoring and release, the other on masking and unmasking of NLSs (6). Both the release from anchoring sites and the functional activation of NLSs is brought about by similar mechanisms, particularly ligand binding, phosphorylation or proteolysis (or a combination of these).

One extreme example of anchoring is provided by transcription factors involved in cholesterol homeostasis. These factors, termed sterol-regulatory element binding proteins (SREBPs) are inserted into the endoplasmic reticulum membrane; thus, their liberation and nuclear translocation, in response to sterol depletion, requires proteolytic cleavage of the transmembrane domain (126). Membranes are involved also in the cytoplasmic anchoring of other transcription factors and of certain signalling protein kinases. The prototypic example for a protein kinase translocating to the cell nucleus is PKA (6). Inactive PKA holoenzyme is anchored to cytoplasmic membraneous organelles. In response to appropriate hormonal stimulation, binding of cyclic AMP to regulatory subunits then releases active catalytic (C) subunits. These enter the nucleus, apparently by diffusion, where they phosphorylate CREB and other transcription factors. Binding of C subunits to PKI, the small polypeptide inhibitor of PKA described above, causes exposure of the NES of PKI and rapid export of the C subunit-PKI complex (44). Other protein kinases whose nucleocytoplasmic distribution changes in response to appropriate signals include MAP kinases and certain isoforms of protein kinase C; similarly, the cell-cycle regulatory protein kinase p34 sup cdc2/cyclin B undergoes a striking relocalization to the nucleus shortly before the onset of mitosis. However, the mechanisms controlling the redistributions of these latter kinases are not well understood.

Examples for masking and unmasking of NLSs are plenty, and in many cases NLS function is regulated by phosphorylation (6,7). Inactivation of a NLS may result from direct phosphorylation of residues within the NLS or in its immediate vicinity, as observed with the yeast transcription factor Swi5p whose nucleocytoplasmic distribution changes during the cell cycle (127). Alternatively, phosphorylation (or dephosphorylation) may cause conformational changes exposing a NLS, or may alter protein-protein interactions. Prominent examples for the latter mechanism are the NF kappa B/Rel transcription factors (135). In this case, NLSs are masked by interactions with inhibitors of the I kappa B family, and nuclear translocation of NF kappa B/Rel requires inactivation of I kappa B, usually by phosphorylation and proteolysis. Interestingly, I kappa B appears to contain a NES, and may thus play a role also in terminating NF kappa B signalling through a negative-feedback loop (113,128): once in the nucleus, NF kappa B will in fact induce the synthesis of I kappa B; newly synthesized inhibitor may then enter the nucleus, displace NF kappa B from DNA and cause its export from the nucleus.

Finally, ligand-induced unmasking of NLSs has long been shown to control the nucleocytoplasmic distribution of nuclear receptors, such as those for glucocorticoid hormones. However, many nuclear receptors are likely to shuttle between the nucleus and the cytoplasm (129), and differences in steady-state distributions may reflect quantitative rather than qualitative differences in signalling mechanisms (6). This may serve as a reminder that regulatory mechanisms based on the compartmentalization of signalling proteins should not be expected to control cellular responses in an all-or-nothing manner. Instead, they are likely to modulate the kinetics and amplitudes of responses, and they are ideally suited for rapid signal transmission.

Conclusions and perspectives To Top

Since the early 1980s, when the first evidence was obtained for signal-mediated nucleocytoplasmic transport (107,130), and NPCs were firmly established as gateways for translocation (17), progress has been remarkable. The identification of shuttling proteins (8,10) has fostered the realization that transport mechanisms may involve components that move back and forth between the nucleus and the cytoplasm. The establishment of permeabilized cell systems for studying protein import in vitro (70,71) has allowed the functional characterization of soluble factors and shuttling carriers (72,131,132), and has brought to light the conspicuous role of the Ran-GTP/GDP cycle (52,53). In comparison, our understanding of RNA export remains rudimentary, and the development of reliable cell-free systems for studying export is eagerly awaited. Electron microscopy has shown that NPCs are beautiful structures (5,19,20), but the inner life of these structures remains a mystery, and detailed structural analyses of NPC components and transport factors by methods such as X-ray crystallography have barely begun (133,134). Also, answers to several fundamental questions are still lacking: for example, how many distinct transport pathways are there? Do they all use common factors, or do some operate on completely different principles? How many distinct carriers are there for protein import, and how many for export (if export uses carriers at all)? How is cargo translocated through the NPC, how is directionality achieved, and how are carriers recycled? Finally, how does cargo reach the NPC; does this occur by diffusion or does it involve structural tracks? Although these questions remain open, the newly acquired knowledge of transport mechanisms and their regulation already invites exploration of potential applications. In particular, it might be possible to target effector molecules to the nucleus, or conversely, block the nuclear translocation of particular signalling molecules, and thereby control the expression of specific genes, cell proliferation or differentiation. Furthermore, a better understanding of nucleoprotein transport during viral infection might prove useful for designing antiviral therapies and for designing delivery vectors for gene therapy.

Acknowledgements. My apologies go to those authors whose work could not be directly cited owing to space limitations. I thank N. Roggli for artwork, U. Aebi (Basel) and H. Mehlin (Stockholm) for generously providing *(Figure 1)* and *(Figure 5)*, respectively, and U. Aebi, A. Fry, P. Grandi, E. Izaurralde, A. Lamond, and R. Laskey for helpful comments on the manuscript.

Correspondence should be addressed to E.A.N. (e-mail: nigg@sc2a.unige.ch).

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132. Moor, M. S. & Blobel, G. The two steps of nuclear import, targeting to the nuclear envelope and translocation through the nuclear pore, require different cytosolic factors. Cell 69, 939-950 (1992). [Back to Conclusions and pers..]

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134. Bullock, T. L., Clarkson, W. D., Kent, H. M. & Stewart, M. The 1.6 angstroms resolution crystal structure of nuclear transport factor 2 (NTF2). J. Mol. Biol. 260, 422-431 (1996). [Back to Conclusions and pers..]

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136. Jarnik, M. & Aebi, U. Toward a more complete 3-D structure of the nuclear pore complex. J. Struct. Biol. 107, 291-308 (1991). [Back to GRAPHICS:Figure 1]

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