Nucleocytoplasmic transport: taking an inventory.

In eukaryotic cells, the enclosure of the genetic information in the nucleus allows the spatial and temporal separation of DNA replication and transcription from cytoplasmic protein synthesis. This compartmentalization not only permits a high level of regulation of these processes but at the same time necessitates a system of selective macromolecular transport between the nucleus and the cytoplasm. Transfer of macromolecules between both compartments is mediated by soluble receptors that interact with components of nuclear pore complexes (NPCs) to move their specific cargos. Transport occurs by way of a great variety of different pathways defined by individual receptors and accessory factors. Often, processes in substrate biogenesis that precede transport concurrently recruit transport factors to substrates, thus making transport responsive to correct and orderly synthesis of substrates. Some current challenges are to understand how transport factor-substrate interactions are controlled and integrated with sequential steps in substrate biogenesis, how large macromolecular complexes are restructured to fit through the NPC channel and to understand how transport factor-NPC interactions lead to actual translocation through the NPC.

RanBP3 influences interactions between CRM1 and its nuclear protein export substrates.

We investigated the role of RanBP3, a nuclear member of the Ran-binding protein 1 family, in CRM1-mediated protein export in higher eukaryotes. RanBP3 interacts directly with CRM1 and also forms a trimeric complex with CRM1 and RanGTP. However, RanBP3 does not bind to CRM1 like an export substrate. Instead, it can stabilize CRM1-export substrate interaction. Nuclear RanBP3 stimulates CRM1-dependent protein export in permeabilized cells. These data indicate that RanBP3 functions by a novel mechanism as a cofactor in recognition and export of certain CRM1 substrates. In vitro, RanBP3 binding to CRM1 affects the relative affinity of CRM1 for different substrates.

Multiple pathways contribute to nuclear import of core histones.

Nuclear import of the four core histones H2A, H2B, H3 and H4 is one of the main nuclear import activities during S-phase of the cell cycle. However, the molecular machinery facilitating nuclear import of core histones has not been elucidated. Here, we investigated the pathways by which histone import can occur. First, we show that core histone import can be competed by the BIB (beta-like import receptor binding) domain of ribosomal protein L23a suggesting that histone import is an importin mediated process. Secondly, affinity chromatography on immobilized core histones revealed that several members of the importin beta family of transport receptors are able to interact with core histones. Finally, we demonstrate that at least four known and one novel importin, importin 9, can mediate nuclear import of core histones into the nuclei of permeabilized cells. Our results suggest that multiple pathways of import exist to provide efficient nuclear uptake of these abundant, essential proteins.

TAP (NXF1) belongs to a multigene family of putative RNA export factors with a conserved modular architecture.

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity.

Identification of two novel RanGTP-binding proteins belonging to the importin beta superfamily.

Nucleo-cytoplasmic transport comprises a large number of distinct pathways, many of which are defined by members of the importin beta superfamily of nuclear transport receptors. These transport receptors all directly interact with RanGTP to modulate the compartment-specific binding of their transport substrates. To identify new members of the importin beta family, we used affinity chromatography on immobilized RanGTP and isolated Ran-binding protein (RanBP) 16 from HeLa cell extracts. RanBP16 and its close human homologue, RanBP17, are distant members of the importin beta family. Like the other members of the transport receptor superfamily, RanBP16 interacts with the nuclear pore complex and is able to enter the nucleus independent of energy and additional nuclear transport receptors.

Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes.

Transport receptors of the importin beta superfamily account for many of the nuclear import and export events in eukaryotic cells. They mediate translocation through nuclear pore complexes, shuttle between nucleus and cytoplasm and co-operate with the RanGTPase system to regulate their interactions with cargo molecules in a compartment-specific manner. We used affinity chromatography on immobilized RanGTP to isolate further candidate nuclear transport receptors and thereby identified exportin 4 as the most distant member of the importin beta family so far. Exportin 4 appears to be conserved amongst higher eukaryotes, but lacks obvious orthologues in yeast. It mediates nuclear export of eIF-5A (eukaryotic translation initiation factor 5A) and possibly that of other cargoes. The export signal in eIF-5A appears to be complex and to involve the hypusine modification that is unique to eIF-5A. We discuss possible cellular roles for nuclear export of eIF-5A.

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro.

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.

Deciphering the cellular pathway for transport of poly(A)-binding protein II.

Poly(A)-binding protein II (PABP2) is an abundant nuclear protein that binds with high affinity to nascent poly(A) tails, stimulating their extension and controlling their length. In the cytoplasm, a distinct protein (PABP1) binds to poly(A) tails and participates in mRNA translation and stability. How cytoplasmic PABP1 substitutes for nuclear PABP2 is still unknown. Here we report that PABP2 shuttles back and forth between nucleus and cytoplasm by a carrier-mediated mechanism. A potential novel type of nuclear localization signal exists at the C-terminus of the protein, a domain that is highly enriched in methylated arginines. PABP2 binds directly to transportin in a RanGTP-sensitive manner, suggesting an involvement of this transport receptor in mediating import of the protein into the nucleus. Although PABP2 is small enough to diffuse passively through the nuclear pores, protein fusion experiments reveal the existence of a facilitated export pathway. Accordingly, no transport of PABP2 to the cytoplasm occurs at 4 degrees C. In contrast, export of PABP2 continues in the absence of transcription, indicating that transport to the cytoplasm is independent of mRNA traffic. Thus, rather than leaving the nucleus as a passive passenger of mRNAs, the data suggest that PABP2 interacts with the nuclear export machinery and may therefore contribute to mRNA transport.

The C-terminal domain of TAP interacts with the nuclear pore complex and promotes export of specific CTE-bearing RNA substrates.

Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process include TAP/Mex67p and RAE1/Gle2p and are distinct from the nuclear transport receptors of the beta-related, Ran-binding protein family. Mex67p is essential for mRNA export in yeast. Its vertebrate homolog TAP has been implicated in the export of cellular mRNAs and of simian type D viral RNAs bearing the constitutive transport element (CTE). Here we show that TAP is predominantly localized in the nucleoplasm and at both the nucleoplasmic and cytoplasmic faces of the nuclear pore complex (NPC). TAP interacts with multiple components of the NPC including the nucleoporins CAN, Nup98, Nup153, p62, and with three major NPC subcomplexes. The nucleoporin-binding domain of TAP comprises residues 508-619. In HeLa cells, this domain is necessary and sufficient to target GFP-TAP fusions to the nuclear rim. Moreover, the isolated domain strongly competes multiple export pathways in vivo, probably by blocking binding sites on the NPC that are shared with other transport receptors. Microinjection experiments implicate this domain in the export of specific CTE-containing RNAs. Finally, we show that TAP interacts with transportin and with two proteins implicated in the export of cellular mRNAs: RAE1/hGle2 and E1B-AP5. The interaction of TAP with nucleoporins, its direct binding to the CTE RNA, and its association with two mRNP binding proteins suggest that TAP is an RNA export mediator that may bridge the interaction between specific RNP export substrates and the NPC.

Transport between the cell nucleus and the cytoplasm.

The compartmentation of eukaryotic cells requires all nuclear proteins to be imported from the cytoplasm, whereas, for example, transfer RNAs, messenger RNAs, and ribosomes are made in the nucleus and need to be exported to the cytoplasm. Nuclear import and export proceed through nuclear pore complexes and can occur along a great number of distinct pathways, many of which are mediated by importin beta-related nuclear transport receptors. These receptors shuttle between nucleus and cytoplasm, and they bind transport substrates either directly or via adapter molecules. They all cooperate with the RanGTPase system to regulate the interactions with their cargoes. Another focus of our review is nuclear export of messenger RNA, which apparently largely relies on export mediators distinct from importin beta-related factors. We discuss mechanistic aspects and the energetics of transport receptor function and describe a number of pathways in detail.

Structural view of the Ran-Importin beta interaction at 2.3 A resolution.

Transport receptors of the Importin beta family shuttle between the nucleus and cytoplasm and mediate transport of macromolecules through nuclear pore complexes. They interact specifically with the GTP-binding protein Ran, which in turn regulates their interaction with cargo. Here, we report the three-dimensional structure of a complex between Ran bound to the nonhydrolyzable GTP analog GppNHp and a 462-residue fragment from Importin beta. The structure of Importin beta shows 10 tandem repeats resembling HEAT and Armadillo motifs. They form an irregular crescent, the concave site of which forms the interface with Ran-triphosphate. The importin-binding site of Ran does not overlap with that of the Ran-binding domain of RanBP2.

The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1.

Import of proteins into the nucleus proceeds through nuclear pore complexes and is largely mediated by nuclear transport receptors of the importin beta family that use direct RanGTP-binding to regulate the interaction with their cargoes. We investigated nuclear import of the linker histone H1 and found that two receptors, importin beta (Impbeta) and importin 7 (Imp7, RanBP7), play a critical role in this process. Individually, the two import receptors bind H1 weakly, but binding is strong for the Impbeta/Imp7 heterodimer. Consistent with this, import of H1 into nuclei of permeabilized mammalian cells requires exogenous Impbeta together with Imp7. Import by the Imp7/Impbeta heterodimer is strictly Ran dependent, the Ran-requiring step most likely being the disassembly of the cargo-receptor complex following translocation into the nucleus. Disassembly is brought about by direct binding of RanGTP to Impbeta and Imp7, whereby the two Ran-binding sites act synergistically. However, whereas an Impbeta/RanGTP interaction appears essential for H1 import, Ran-binding to Imp7 is dispensable. Thus, Imp7 can function in two modes. Its Ran-binding site is essential when operating as an autonomous import receptor, i.e. independently of Impbeta. Within the Impbeta/Imp7 heterodimer, however, Imp7 plays a more passive role than Impbeta and resembles an import adapter.

Coordination of tRNA nuclear export with processing of tRNA.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

CRM1-mediated recycling of snurportin 1 to the cytoplasm.

Importin beta is a major mediator of import into the cell nucleus. Importin beta binds cargo molecules either directly or via two types of adapter molecules, importin alpha, for import of proteins with a classical nuclear localization signal (NLS), or snurportin 1, for import of m3G-capped U snRNPs. Both adapters have an NH2-terminal importin beta-binding domain for binding to, and import by, importin beta, and both need to be returned to the cytoplasm after having delivered their cargoes to the nucleus. We have shown previously that CAS mediates export of importin alpha. Here we show that snurportin 1 is exported by CRM1, the receptor for leucine-rich nuclear export signals (NESs). However, the interaction of CRM1 with snurportin 1 differs from that with previously characterized NESs. First, CRM1 binds snurportin 1 50-fold stronger than the Rev protein and 5,000-fold stronger than the minimum Rev activation domain. Second, snurportin 1 interacts with CRM1 not through a short peptide but rather via a large domain that allows regulation of affinity. Strikingly, snurportin 1 has a low affinity for CRM1 when bound to its m3G-capped import substrate, and a high affinity when substrate-free. This mechanism appears crucial for productive import cycles as it can ensure that CRM1 only exports snurportin 1 that has already released its import substrate in the nucleus.

The translocation of transportin-cargo complexes through nuclear pores is independent of both Ran and energy.

Active transport between nucleus and cytoplasm proceeds through nuclear pore complexes (NPCs) and is mediated largely by shuttling transport receptors that use direct RanGTP binding to coordinate loading and unloading of cargo [1] [2] [3] [4]. Import receptors such as importin beta or transportin bind their substrates at low RanGTP levels in the cytoplasm and release them upon encountering RanGTP in the nucleus, where a high RanGTP concentration is predicted. This substrate release is, in the case of import by the importin alpha/beta heterodimer, coupled directly to importin beta release from the NPCs. If the importin beta -RanGTP interaction is prevented, import intermediates arrest at the nuclear side of the NPCs [5] [6]. This arrest makes it difficult to probe directly the Ran and energy requirements of the actual translocation from the cytoplasmic to the nuclear side of the NPC, which immediately precedes substrate release. Here, we have shown that in the case of transportin, dissociation of transportin-substrate complexes is uncoupled from transportin release from NPCs. This allowed us to dissect the requirements of translocation through the NPC, substrate release and transportin recycling. Surprisingly, translocation of transportin-substrate complexes into the nucleus requires neither Ran nor nucleoside triphosphates (NTPs). It is only nuclear RanGTP, not GTP hydrolysis, that is needed for dissociation of transportin-substrate complexes and for re-export of transportin to the cytoplasm. GTP hydrolysis is apparently required only to restore the import competence of the re-exported transportin and, thus, for multiple rounds of transportin-dependent import. In addition, we provide evidence that at least one type of substrate can also complete NPC passage mediated by importin beta independently of Ran and energy.

Identification of a tRNA-specific nuclear export receptor.

In eukaryotes, tRNAs are synthesized in the nucleus and after several maturation steps exported to the cytoplasm. Here, we identify exportin-t as a specific mediator of tRNA export. It is a RanGTP-binding, importin beta-related factor with predominantly nuclear localization. It shuttles rapidly between nucleus and cytoplasm and interacts with nuclear pore complexes. Exportin-t binds tRNA directly and with high affinity. Its cellular concentration in Xenopus oocytes was found to be rate-limiting for export of all tRNAs tested, as judged by microinjection experiments. RanGTP regulates the substrate-exportin-t interaction such that tRNA can be preferentially bound in the nucleus and released in the cytoplasm.

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus.

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.

Yrb4p, a yeast ran-GTP-binding protein involved in import of ribosomal protein L25 into the nucleus.

Gsp1p, the essential yeast Ran homologue, is a key regulator of transport across the nuclear pore complex (NPC). We report the identification of Yrb4p, a novel Gsp1p binding protein. The 123 kDa protein was isolated from Saccharomyces cerevisiae cells and found to be related to importin-beta, the mediator of nuclear localization signal (NLS)-dependent import into the nucleus, and to Pse1p. Like importin-beta, Yrb4p and Pse1p specifically bind to Gsp1p-GTP, protecting it from GTP hydrolysis and nucleotide exchange. The GTPase block of Gsp1p complexed to Yrb4p or Pse1p is released by Yrb1p, which contains a Gsp1p binding domain distinct from that of Yrb4p. This might reflect an in vivo function for Yrb1p. Cells disrupted for YRB4 are defective in nuclear import of ribosomal protein L25, but show no defect in the import of proteins containing classical NLSs. Expression of a Yrb4p mutant deficient in Gsp1p-binding is dominant-lethal and blocks bidirectional traffic across the NPC in wild-type cells. L25 binds to Yrb4p and Pse1p and is released by Gsp1p-GTP. Consistent with its putative role as an import receptor for L25-like proteins, Yrb4p localizes to the cytoplasm, the nucleoplasm and the NPC.

Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.

NLS proteins are transported into the nucleus by the importin alpha/beta heterodimer. Importin alpha binds the NLS, while importin beta mediates translocation through the nuclear pore complex. After translocation, RanGTP, whose predicted concentration is high in the nucleus and low in the cytoplasm, binds importin beta and displaces importin alpha. Importin alpha must then be returned to the cytoplasm, leaving the NLS protein behind. Here, we report that the previously identified CAS protein mediates importin alpha re-export. CAS binds strongly to importin alpha only in the presence of RanGTP, forming an importin alpha/CAS/RanGTP complex. Importin alpha is released from this complex in the cytoplasm by the combined action of RanBP1 and RanGAP1. CAS binds preferentially to NLS-free importin alpha, explaining why import substrates stay in the nucleus.

A novel class of RanGTP binding proteins.

The importin-alpha/beta complex and the GTPase Ran mediate nuclear import of proteins with a classical nuclear localization signal. Although Ran has been implicated also in a variety of other processes, such as cell cycle progression, a direct function of Ran has so far only been demonstrated for importin-mediated nuclear import. We have now identified an entire class of approximately 20 potential Ran targets that share a sequence motif related to the Ran-binding site of importin-beta. We have confirmed specific RanGTP binding for some of them, namely for two novel factors, RanBP7 and RanBP8, for CAS, Pse1p, and Msn5p, and for the cell cycle regulator Cse1p from Saccharomyces cerevisiae. We have studied RanBP7 in more detail. Similar to importin-beta, it prevents the activation of Ran's GTPase by RanGAP1 and inhibits nucleotide exchange on RanGTP. RanBP7 binds directly to nuclear pore complexes where it competes for binding sites with importin-beta, transportin, and apparently also with the mediators of mRNA and U snRNA export. Furthermore, we provide evidence for a Ran-dependent transport cycle of RanBP7 and demonstrate that RanBP7 can cross the nuclear envelope rapidly and in both directions. On the basis of these results, we propose that RanBP7 might represent a nuclear transport factor that carries an as yet unknown cargo, which could apply as well for this entire class of related RanGTP-binding proteins.