The nuclear export receptor Xpo1p forms distinct complexes with NES transport substrates and the yeast Ran binding protein 1 (Yrb1p)

Xpo1p (Crm1p) is the nuclear export receptor for proteins containing a leucine-rich nuclear export signal (NES). Xpo1p, the NES-containing protein, and GTP-bound Ran form a complex in the nucleus that translocates across the nuclear pore. We have identified Yrb1p as the major Xpo1p-binding protein in Saccharomyces cerevisiae extracts in the presence of GTP-bound Gsp1p (yeast Ran). Yrb1p is cytoplasmic at steady-state but shuttles continuously between the cytoplasm and the nucleus. Nuclear import of Yrb1p is mediated by two separate nuclear targeting signals. Export from the nucleus requires Xpo1p, but Yrb1p does not contain a leucine-rich NES. Instead, the interaction of Yrb1p with Xpo1p is mediated by Gsp1p-GTP. This novel type of export complex requires the acidic C-terminus of Gsp1p, which is dispensable for the binding to importin beta-like transport receptors. A similar complex with Xpo1p and Gsp1p-GTP can be formed by Yrb2p, a relative of Yrb1p predominantly located in the nucleus. Yrb1p also functions as a disassembly factor for NES/Xpo1p/Gsp1p-GTP complexes by displacing the NES protein from Xpo1p/Gsp1p. This Yrb1p/Xpo1p/Gsp1p complex is then completely dissociated after GTP hydrolysis catalyzed by the cytoplasmic GTPase activating protein Rna1p.     

Mutants in a yeast Ran binding protein are defective in nuclear transport.

Ran, a Ras-like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione-S-transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP-bound state. A major protein with the apparent size of 34 kDa co-purifies with the GTP-bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature-sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.     

Yrb2p, a Nup2p-related yeast protein, has a functional overlap with Rna1p, a yeast Ran-GTPase-activating protein.

The Ran-GTPase cycle is important for nucleus-cytosol exchange of macromolecules and other nuclear processes. We employed the two-hybrid method to identify proteins interacting with Ran and the Ran GTP/GDP exchange factor. Using PRP20, encoding the Ran GTP/GDP exchange factor, we identified YRB1, previously identified as a protein able to interact with human Ran GTP/GDP exchange factor RCC1 in the two-hybrid system. Using GSP1, encoding the yeast Ran, as bait, we isolated YRB2. YRB2 encodes a protein containing a Ran-binding motif similar to that found in Yrb1p and Nup2p. Yrb1p is located in the cytosol whereas Nup2p is nuclear. Similar to Yrb1p, Yrb2p bound to GTP-Gsp1p but not to GDP-Gsp1p and enhanced the GTPase-activating activity of Rna1p. However, unlike Yrb1p, Yrb2p did not inhibit the nucleotide-releasing activity of Prp20p. While overproduction of Yrb1p inhibited the growth of a mutant possessing a PRP20 mutation (srm1-1) and suppressed the rna1-1 mutation, overproduction of Yrb2p showed no effect on the growth of these mutants. Disruption of YRB2 made yeast cold sensitive and was synthetically lethal with rna1-1 but not with nup2delta. Nuclear protein import and the mRNA export were normal in strains possessing mutations of YRB2. We propose that Yrb2p is involved in the nuclear processes of the Ran-GTPase cycle which are not related to nucleus-cytosol exchange of macromolecules.     

Ran-binding protein 3 is a cofactor for Crm1-mediated nuclear protein export.

Crm1 is a member of the karyopherin family of nucleocytoplasmic transport receptors and mediates the export of proteins from the nucleus by forming a ternary complex with cargo and Ran:GTP. This complex translocates through the nuclear pores and dissociates in the cytosol. The yeast protein Yrb2p participates in this pathway and binds Crm1, but its mechanism of action has not been established. We show that the human orthologue of Yrb2p, Ran-binding protein 3 (RanBP3), acts as a cofactor for Crm1-mediated export in a permeabilized cell assay. RanBP3 binds directly to Crm1, and the complex possesses an enhanced affinity for both Ran:GTP and cargo. RanBP3 shuttles between the nucleus and the cytoplasm by a Crm1-dependent mechanism, and the Crm1--RanBP3-NES-Ran:GTP quarternary complex can associate with nucleoporins. We infer that this complex translocates through the nuclear pore to the cytoplasm where it is disassembled by RanBP1 and Ran GTPase--activating protein.     

Random mutagenesis and functional analysis of the Ran-binding protein, RanBP1

Ran GTPase is required for nucleocytoplasmic transport of many types of cargo. Several proteins that recognize Ran in its GTP-bound state (Ran x GTP) possess a conserved Ran-binding domain (RanBD). Ran-binding protein-1 (RanBP1) has a single RanBD and is required for RanGAP-mediated GTP hydrolysis and release of Ran from nuclear transport receptors (karyopherins). In budding yeast (Saccharomyces cerevisiae), RanBP1 is encoded by the essential YRB1 gene; expression of mouse RanBP1 cDNA rescues the lethality of Yrb1-deficient cells. We generated libraries of mouse RanBP1 mutants and examined 11 mutants in vitro and for their ability to complement a temperature-sensitive yrb1 mutant (yrb1-51(ts)) in vivo. In 9 of the mutants, the alteration was a change in a residue (or 2 residues) that is conserved in all known RanBDs. However, 4 of these 9 mutants displayed biochemical properties indistinguishable from that of wild-type RanBP1. These mutants bound to Ran x GTP, stimulated RanGAP, inhibited the exchange activity of RCC1, and rescued growth of the yrb1-51(ts) yeast cells. Two of the 9 mutants altered in residues thought to be essential for interaction with Ran were unable to rescue growth of the yrb1(ts) mutant and did not bind detectably to Ran in vitro. However, one of these 2 mutants (and 2 others that were crippled in other RanBP1 functions) retained some ability to co-activate RanGAP. A truncated form of RanBP1 (lacking its nuclear export signal) was able to complement the yrb1(ts) mutation. When driven from the YRB1 promoter, 4 of the 5 mutants most impaired for Ran binding were unable to rescue growth of the yrb1(ts) cells; remarkably, these mutants could nevertheless form ternary complexes with importin-5 or importin-beta and Ran-GTP. The same mutants stimulated only inefficiently RanGAP-mediated GTP hydrolysis of the Ran x GTP x importin-5 complex. Thus, the essential biological activity of RanBP1 in budding yeast correlates not with Ran x GTP binding per se or with the ability to form ternary complexes with karyopherins, but with the capacity to potentiate RanGAP activity toward GTP-bound Ran in these complexes.     

Both ran and importins have the ability to function as nuclear mRNA export factors

The Ran protein regulates nucleocytoplasmic transport mediated by the karyopherin family of nuclear transport factors. Ran is converted to the active, GTP bound form in the nucleus and then binds to a conserved domain found in all karyopherins. This interaction induces cargo binding for exportins and cargo release for importins. In either case, the Ran.GTP is then transported to the cytoplasm by the karyopherin, where it is hydrolyzed to Ran.GDP. To ask whether Ran could function as a nuclear mRNA export factor, we fused Ran to the MS2 coat protein and inserted MS2 RNA-binding sites into an unspliced cat mRNA that is normally sequestered in the nucleus. Coexpression of MS2-Ran induced cat mRNA export and CAT enzyme expression as effectively as, for example, an MS2-Rev fusion protein. MS2-Ran dependent nuclear mRNA export was reduced by inhibitors specific for Crm1, but not blocked as was seen with MS2-Rev. Consistent with the hypothesis that Crm1 is not the only karyopherin cofactor for MS2-Ran mediated mRNA export, we show that not only Crm1 but also CAS, transportin, importin beta and exportin t can all export mRNA from the nucleus when tethered via the MS2 RNA-binding domain. In contrast, two shuttling hnRNPs, hnRNP A1 and hnRNP K, proved unable to function as nuclear RNA export factors when expressed as MS2 fusions. Together, these data argue that karyopherins that normally function to transport proteins into or out of the nucleus are also capable of exporting tethered mRNA molecules.     

Fluorescence resonance energy transfer biosensors that detect Ran conformational changes and a Ran x GDP-importin-beta -RanBP1 complex in vitro and in intact cells

The Ran GTPase plays a central role in nucleocytoplasmic transport. Association of Ran x GTP with transport carriers (karyopherins) triggers the loading/unloading of export or import cargo, respectively. The C-terminal tail of Ran x GTP is deployed in an extended conformation when associated with a Ran binding domain or importins. To monitor tail orientation, a Ran-GFP fusion was labeled with the fluorophore Alexa546. Fluorescence resonance energy transfer (FRET) occurs efficiently between the green fluorescent protein (GFP) and Alexa546 for Ran x GDP and Ran x GTP, suggesting that the tail is tethered in both states. However, Ran x GTP complexes with importin-beta, RanBP1, and Crm1 all show reduced FRET consistent with tail extension. Displacement of the C-terminal tail of Ran by karyopherins may be a general mechanism to facilitate RanBP1 binding. A Ran x GDP-RanBP1-importin-beta complex also displayed a low FRET signal. To detect this complex in vivo, a bipartite biosensor consisting of Ran-Alexa546 plus GST-GFP-RanBP1, was co-injected into the cytoplasm of cells. The Ran redistributed predominantly to the nucleus, and RanBP1 remained cytoplasmic. Nonetheless, a robust cytoplasmic FRET signal was detectable, which suggests that a significant fraction of cytoplasmic Ran.GDP may exist in a ternary complex with RanBP1 and importins.     

Importin-11, a nuclear import receptor for the ubiquitin-conjugating enzyme, UbcM2

Importins are members of a family of transport receptors (karyopherins) that mediate the nucleocytoplasmic transport of protein and RNA cargoes. We identified importin-11 as a potential new human member of this family, on the basis of limited similarity to the Saccharomyces cerevisiae protein, Lph2p, and cloned the complete open reading frame. Importin-11 interacts with the Ran GTPase, and constitutively shuttles between the nuclear and cytoplasmic compartments. A yeast dihybrid screen identified UbcM2, an E2-type ubiquitin-conjugating enzyme, as a binding partner and potential transport cargo for importin-11. Importin-11 and UbcM2 interact directly, and the complex is disassembled by Ran:GTP but not by Ran:GDP. UbcM2 is constitutively nuclear and shuttles between the nuclear and cytoplasmic compartments. Nuclear import of UbcM2 requires Ran and importin-11, and is inhibited by wheatgerm agglutinin, energy depletion or dominant interfering mutants of Ran and importin-beta. These data establish importin-11 as a new member of the karyopherin family of transport receptors, and identify UbcM2 as a nuclear member of the E2 ubiquitin-conjugating enzyme family.     

Nucleocytoplasmic transport of proteins

In eukaryotic cells, the movement of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC)--a large protein complex spanning the nuclear envelope. The nuclear transport of proteins is usually mediated by a family of transport receptors known as karyopherins. Karyopherins bind to their cargoes via recognition of nuclear localization signal (NLS) for nuclear import or nuclear export signal (NES) for export to form a transport complex. Its transport through NPC is facilitated by transient interactions between the karyopherins and NPC components. The interactions of karyopherins with their cargoes are regulated by GTPase Ran. In the current review, we describe the NPC structure, NLS, and NES, as well as the model of classic Ran-dependent transport, with special emphasis on existing alternative mechanisms; we also propose a classification of the basic mechanisms of protein transport regulation.     

The histones H2A/H2B and H3/H4 are imported into the yeast nucleus by different mechanisms

Proteins are imported from the cytoplasm into the nucleus by importin beta-related transport receptors. The yeast Saccharomyces cerevisiae contains ten of these importins, but only two of them are essential. After transfer through the nuclear pore, importins release their cargo upon binding to the Ran GTPase, the key regulator of nuclear transport. We investigated the import of the core histones in yeast and found that four importins are involved. The essential Pse1p and the nonessential importins Kap114p, Kap104p, and Yrb4p/Kap123p specifically bind to histones H2A and H2B. Release of H2 histones from importins requires Ran-GTP and DNA simultaneously suggesting a function of the importins in intranuclear targeting. H3 and H4 associate mainly with Pse1p and the dissociation requires Ran but not DNA, which points to a different import mechanism. Import of green fluorescent protein fusions to H2A and H2B requires primarily Pse1p and Kap114p, whereas Yrb4p plays an auxiliary role. Pse1p is predominantly necessary for nuclear uptake of H3 and H4, while Kap104p and Yrb4p also support import. We conclude from our in vivo and in vitro experiments that import of the essential histones is mediated mainly by the essential importin Pse1p, while the non-essential Kap114p functions in a parallel import pathway for H2A and H2B.     

Dynamic localization of the nuclear import receptor and its interactions with transport factors

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin- beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal- bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.     

The mammalian Mog1 protein is a guanine nucleotide release factor for Ran

Ran is a Ras-related GTPase that is essential for the transport of protein and RNA between the nucleus and the cytoplasm. Proteins that regulate the GTPase cycle and subcellular distribution of Ran include the cytoplasmic GTPase-activating protein (RanGAP) and its co-factors (RanBP1, RanBP2), the nuclear guanine nucleotide exchange factor (RanGEF), and the Ran import receptor (NTF2). The recent identification of the Saccharomyces cerevisiae protein Mog1p as a suppressor of temperature-sensitive Ran mutations suggests that additional regulatory proteins remain to be characterized. Here, we describe the identification and biochemical characterization of murine Mog1, which, like its yeast orthologue, is a nuclear protein that binds specifically to RanGTP. We show that Mog1 stimulates the release of GTP from Ran, indicating that Mog1 functions as a guanine nucleotide release factor in vitro. Following GTP release, Mog1 remains bound to nucleotide-free Ran in a conformation that prevents rebinding of the guanine nucleotide. These properties distinguish Mog1 from the well characterized RanGEF and suggest an unanticipated mechanism for modulating nuclear levels of RanGTP.     

Yeast Ran-binding protein 1 (Yrb1) shuttles between the nucleus and cytoplasm and is exported from the nucleus via a CRM1 (XPO1)-dependent pathway

The RanGTP-binding protein RanBP1, which is located in the cytoplasm, has been implicated in release of nuclear export complexes from the cytoplasmic side of the nuclear pore complex. Here we show that Yrb1 (the yeast homolog of RanBP1) shuttles between the nucleus and the cytoplasm. Nuclear import of Yrb1 is a facilitated process that requires a short basic sequence within the Ran-binding domain (RBD). By contrast, nuclear export of Yrb1 requires an intact RBD, which forms a ternary complex with the Xpo1 (Crm1) NES receptor in the presence of RanGTP. Nuclear export of Yrb1, however, is insensitive towards leptomycin B, suggesting a novel type of substrate recognition between Yrb1 and Xpo1. Taken together, these data suggest that ongoing nuclear import and export is an important feature of Yrb1 function in vivo.     

Nuclear export of tRNA

Summary Exit of tRNA from the nucleus was shown, long time ago, to be a saturable and carrier-mediated process. Nevertheless, only recently, progress in the field of nucleocytoplasmic transport gave first insight into the mechanism of tRNA nuclear export. A nuclear export receptor for tRNA (Los1p/Xpo-t), belonging to the importin (karyopherin) family, has been characterized in yeast and mammalian cells. Mature tRNA molecules can associate with Los1p/ Xpo-t and the GTP-bound form of the small GTPase Ran to form an export complex in the nucleus. This complex translocates through the nuclear-pore complexes and dissociates upon GTP hydrolysis in the cytoplasm. Genetic studies in yeast have, however, shown thatLOS1 is not essential, unless additional steps in the tRNA biogenesis pathway are impaired, suggesting the existence of additional tRNA nuclear export routes. Furthermore, modification and aminoacylation of tRNA may also be important for efficient transport of tRNA into the cytoplasm.     

Identification of a Conserved Loop in Mog1 that Releases GTP from Ran

Ran regulates nuclear import and export pathways by coordinating the assembly and disassembly of transport complexes. These transport reactions are linked to the GTPase cycle and subcellular distribution of Ran. Mog1 is an evolutionarily conserved nuclear protein that binds RanGTP and stimulates guanine nucleotide release, suggesting Mog1 regulates the nuclear transport functions of Ran. In the present study, we have characterized the nuclear import pathway of Mog1, and we have defined the domain in Mog1 that stimulates GTP release from Ran. In permeabilized cells, nuclear import of Mog1 is independent of exogenously added factors, and is inhibited by wheat germ agglutinin, indicating that translocation of Mog1 involves physical interactions with the nuclear pore complex. In contrast to RanGEF, which is restricted to the nucleus, Mog1 shuttles between the nucleus and the cytoplasm. Single-point mutations in acidic residues of Mog1 (Asp25, Asp34, Glu37) dramatically reduce GTP release and Ran binding activity, whereas mutation of a single basic residue (Arg30) renders Mog1 hyperactive for GTP release. These mutations map within a conserved, solvent-exposed loop in Mog1 that is functionally similar to the β-wedge used by RanGEF to promote nucleotide release from Ran. These data suggest that Mog1 and RanGEF use similar mechanisms to facilitate guanine nucleotide release from Ran.     

Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan;124(1-2):217]

We have investigated a possible involvement of GTPases in nuclear protein import using an in vitro transport system involving digitonin- permeabilized cells supplemented with exogenous cytosol. Transport in this system was measured with a novel ELISA-based assay that allows rapid quantitative analysis. GTP gamma S and other nonhydrolyzable analogues of GTP were found to rapidly inhibit the rate of in vitro nuclear import. Transport inhibition by GTP gamma S was dependent on the concentrations of permeabilized cells and cytosol, and was strongly enhanced by a cytosolic factor(s). The predominant cytosolic component responsible for this inhibition was found in a 20-30-kD fraction in molecular sieving chromatography. Furthermore, a component(s) of this 20-30-kD fraction was itself required for efficient nuclear import. Biochemical complementation with bacterially expressed protein demonstrated that this essential GTP gamma S-sensitive transport factor was Ran/TC4, a previously described GTPase of the Ras superfamily found in both nucleus and cytoplasm. Ran/TC4 and its guanine nucleotide release protein RCC1 have previously been implicated in DNA replication, cell cycle checkpoint control, and RNA synthesis, processing and export. Our results suggest that Ran/TC4 serves to integrate nuclear protein import with these other nuclear activities.     

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.     

Review: Transport of tRNA out of the Nucleus—Direct Channeling to the Ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin β superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.     

Review: transport of tRNA out of the nucleus-direct channeling to the ribosome?

Although tRNA was the first substrate whose export from the nuclei of eukaryotic cells had been shown to be carrier-mediated and active, it has only been in the last 2 years that the first mechanistic details of this nucleocytoplasmic transport pathway have begun to emerge. A member of the importin/karyopherin beta superfamily, Los1p in yeast and Xpo-t in vertebrates, has been shown to export tRNA in cooperation with the small GTPase Ran (Gsp1p) from the nucleus into the cytoplasm, where tRNA becomes available for translation. However, Los1p is not essential for viability in yeast cells, suggesting that alternative tRNA export pathways exist. Recent results show that aminoacylation and a translation factor are also required for efficient nuclear tRNA export. Thus, protein translation and nuclear export of tRNA appear to be coupled processes.     

Formation of a Trimeric Xpo1-Ran[GTP]-Ded1 Exportin Complex Modulates ATPase and Helicase Activities of Ded1

The DEAD-box RNA helicase Ded1, which is essential in yeast and known as DDX3 in humans, shuttles between the nucleus and cytoplasm and takes part in several basic processes including RNA processing and translation. A key interacting partner of Ded1 is the exportin Xpo1, which together with the GTP-bound state of the small GTPase Ran, facilitates unidirectional transport of Ded1 out of the nucleus. Here we demonstrate that Xpo1 and Ran[GTP] together reduce the RNA-stimulated ATPase and helicase activities of Ded1. Binding and inhibition of Ded1 by Xpo1 depend on the affinity of the Ded1 nuclear export sequence (NES) for Xpo1 and the presence of Ran[GTP]. Association with Xpo1/Ran[GTP] reduces RNA-stimulated ATPase activity of Ded1 by increasing the apparent KM for the RNA substrate. Despite the increased KM, the Ded1:Xpo1:Ran[GTP] ternary complex retains the ability to bind single stranded RNA, suggesting that Xpo1/Ran[GTP] may modulate the substrate specificity of Ded1. These results demonstrate that, in addition to transport, exportins such as Xpo1 also have the capability to alter enzymatic activities of their cargo.