Putative reaction intermediates in Crm1-mediated nuclear protein export

We discovered several novel interactions between proteins involved in Crm1-mediated nuclear export of the nuclear export signal containing human immunodeficiency virus type 1 protein Rev. First, a Rev/Crm1/RanGTP complex (where Ran is Ras-related nuclear protein) reacts with some nucleoporins (Nup42 and Nup159) but not others (NSP1, Nup116, and Nup1), forming a Nup/Crm1/RanGTP complex and concomitantly releasing Rev. Second, RanBP1 (or homologous proteins) can displace Nup and form a ternary RanBP1/RanGTP/Crm1 complex that can be disassembled by RanGAP via GTP hydrolysis. Third, and most surprisingly, RanBP1/RanGTP/Crm1 can be disassembled without GTP hydrolysis by the nucleotide exchange factor RanGEF. Recycling of a Ran/RanGEF complex by GTP and Mg2+ is stimulated by both Crm1 and Rev, allowing reformation of a Rev/Crm1/RanGTP complex. Based on these reactions we propose a model for Crm1-mediated export.     

RanGTP-Regulated Interactions of CRM1 with Nucleoporins and a Shuttling DEAD-Box Helicase

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.     

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins.

We have identified a novel human karyopherin (Kap) beta family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs.     

An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1

The karyopherin CRM1 mediates nuclear export of proteins and ribonucleoproteins bearing a leucine‐rich nuclear export signal (NES). To elucidate the precise mechanism by which NES‐cargos are dissociated from CRM1 in the cytoplasm, which is important for transport directionality, we determined a 2.0‐Å resolution crystal structure of yeast CRM1:RanBP1:RanGTP complex, an intermediate in the disassembly of the CRM1 nuclear export complex. The structure shows that on association of Ran‐binding domain (RanBD) of RanBP1 with CRM1:NES‐cargo:RanGTP complex, RanBD and the C‐terminal acidic tail of Ran induce a large movement of the intra‐HEAT9 loop of CRM1. The loop moves to the CRM1 inner surface immediately behind the NES‐binding site and causes conformational rearrangements in HEAT repeats 11 and 12 so that the hydrophobic NES‐binding cleft on the CRM1 outer surface closes, squeezing out the NES‐cargo. This allosteric mechanism accelerates dissociation of NES by over two orders of magnitude. Structure‐based mutagenesis indicated that the HEAT9 loop also functions as an allosteric autoinhibitor to stabilize CRM1 in a conformation that is unable to bind NES‐cargo in the absence of RanGTP.     

Novel interaction of the 60S ribosomal subunit export adapter Nmd3 at the nuclear pore complex

Nuclear export of the large (60S) ribosomal subunit depends on the adapter protein Nmd3 to provide a nuclear export signal (NES). The leucine-rich NES is recognized by the export receptor Crm1 to mediate export via interaction with the nuclear pore complex (NPC). Here, we show that certain mutant Nmd3 proteins that are impaired for binding to the 60S subunit accumulate at the nuclear envelope. These mutant proteins also show enhanced binding to Crm1, both in vivo and in vitro. Although their interaction with the NPC is dependent on recognition of the NES by Crm1, their interaction with Crm1 is not strictly dependent on RanGTP. Using a collection of GFP-tagged nucleoporin mutants, we identified several nucleoporins, including components of the Nup82 complex that copurified with the mutant Nmd3. The Nup82 complex is on the cytoplasmic face of the NPC and has previously been shown to be important as a terminal binding site for Crm1-mediated export. Mutations in the Nup82 complex led to accumulation of wild-type Nmd3 in the nucleoplasm, suggesting that the interaction of mutant Nmd3 with the Nup82 complex reflects a defect in the bona fide export pathway for the 60S subunit. These results suggest that in the absence of the ribosome, Nmd3 is not efficiently released from Crm1 at the NPC.     

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.     

Apoptotic regulation by the Crk adapter protein mediated by interactions with Wee1 and Crm1/exportin

The adapter protein Crk contains an SH2 domain and two SH3 domains. Through binding of particular ligands to the SH2 domain and the N-terminal SH3 domain, Crk has been implicated in a number of signaling processes, including regulation of cell growth, cell motility, and apoptosis. We report here that the C-terminal SH3 domain, never shown to bind any specific signaling molecules, contains a binding site for the nuclear export factor Crm1. We find that a mutant Crk protein, deficient in Crm1 binding, promotes apoptosis. Moreover, this nuclear export sequence mutant [NES(-) Crk] interacts strongly, through its SH2 domain, with the nuclear tyrosine kinase, Wee1. Collectively, these data suggest that a nuclear population of Crk bound to Wee1 promotes apoptotic death of mammalian cells.     

Nucleocytoplasmic traffic of CPEB1 and accumulation in Crm1 nucleolar bodies

The translational regulator CPEB1 plays a major role in the control of maternal mRNA in oocytes, as well as of subsynaptic mRNAs in neurons. Although mainly cytoplasmic, we found that CPEB1 protein is continuously shuttling between nucleus and cytoplasm. Its export is controlled by two redundant NES motifs dependent on the nuclear export receptor Crm1. In the nucleus, CPEB1 accumulates in a few foci most often associated with nucleoli. These foci are different from previously identified nuclear bodies. They contain Crm1 and were called Crm1 nucleolar bodies (CNoBs). CNoBs depend on RNA polymerase I activity, indicating a role in ribosome biogenesis. However, although they form in the nucleolus, they never migrate to the nuclear envelope, precluding a role as a mediator for ribosome export. They could rather constitute a platform providing factors for ribosome assembly or export. The behavior of CPEB1 in CNoBs raises the possibility that it is involved in ribosome biogenesis.     

Interaction of the hepatitis B virus X protein with the Crm1-dependent nuclear export pathway

The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFkappaB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFkappaB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

Characterization of the nuclear export signal of polypyrimidine tract-binding protein.

The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, we have shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for its activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor.     

The NES-Crm1p export pathway is not a major mRNA export route in Saccharomyces cerevisiae.

Nuclear export signal (NES)-containing proteins are recognized by the NES receptor CRM1/Crm1p (also called exportin 1/Xpo1p). In vertebrates and Schizosaccharomyces pombe, the toxin leptomycin B (LMB) inhibits CRM1-mediated export by interacting directly with CRM1 and disrupting the trimeric Ran-GTP-CRM1-NES export complex. In Saccharomyces cerevisiae, LMB is not toxic and is apparently unable to interact with Crm1p. A second difference between the systems is that LMB has no effect on mRNA export in vertebrate systems, whereas there is evidence that S.cerevisiae Crm1p plays a role in mRNA export. Here we show that a single amino acid change converts S. cerevisiae Crm1p from being LMB insensitive to fully LMB sensitive, indicating that Crm1p is the only relevant LMB target. This new strain has no phenotype, but LMB has a rapid and potent inhibitory effect on NES-mediated export. In situ hybridization assays show that LMB also causes nuclear accumulation of poly(A)+ RNA but with a significant delay compared with the effect on NES-mediated export. Biochemical assays indicate little or no LMB effect on cytoplasmic protein synthesis, indicating that the NES-Crm1p pathway is not a major mRNA export route in S.cerevisiae. We conclude that Crm1p structure and function is conserved from S.cerevisiae to man.     

The Mobile FG Nucleoporin Nup98 Is a Cofactor for Crm1-dependent Protein Export

Nup98 is a mobile nucleoporin that forms distinct dots in the nucleus, and, although a role for Nup98 in nuclear transport has been suggested, its precise function remains unclear. Here, we show that Nup98 plays an important role in Crm1-mediated nuclear protein export. Nuclear, but not cytoplasmic, dots of EGFP-tagged Nup98 disappeared rapidly after cell treatment with leptomycin B, a specific inhibitor of the nuclear export receptor, Crm1. Mutational analysis demonstrated that Nup98 physically and functionally interacts with Crm1 in a RanGTP-dependent manner through its N-terminal phenylalanine-glycine (FG) repeat region. Moreover, the activity of the Nup98-Crm1 complex was modulated by RanBP3, a known cofactor for Crm1-mediated nuclear export. Finally, cytoplasmic microinjection of anti-Nup98 inhibited the Crm1-dependent nuclear export of proteins, concomitant with the accumulation of anti-Nup98 in the nucleus. These results clearly demonstrate that Nup98 functions as a novel shuttling cofactor for Crm1-mediated nuclear export in conjunction with RanBP3.     

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.     

Inhibition of Human Immunodeficiency Virus Rev and Human T-Cell Leukemia Virus Rex Function, but Not Mason-Pfizer Monkey Virus Constitutive Transport Element Activity, by a Mutant Human Nucleoporin Targeted to Crm1

The hypothesis that the cellular protein Crm1 mediates human immunodeficiency virus type 1 (HIV-1) Rev-dependent nuclear export posits that Crm1 can directly interact both with the Rev nuclear export signal (NES) and with cellular nucleoporins. Here, we demonstrate that Crm1 is indeed able to interact with active but not defective forms of the HIV-1 Rev NES and of NESs found in other retroviral nuclear export factors. In addition, we demonstrate that Crm1 can bind the Rev NES when Rev is assembled onto the Rev response element RNA target and that Crm1, like Rev, is a nucleocytoplasmic shuttle protein. Crm1 also specifically binds the Rev NES in vitro, although this latter interaction is detectable only in the presence of added Ran · GTP. Overexpression of a truncated, defective form of the nucleoporin Nup214/CAN, termed ΔCAN, that retains Crm1 binding ability resulted in the effective inhibition of HIV-1 Rev or human T-cell leukemia virus Rex-dependent gene expression. In contrast, ΔCAN had no significant affect on Mason-Pfizer monkey virus constitutive transport element (MPMV CTE)-dependent nuclear RNA export or on the expression of RNAs dependent on the cellular mRNA export pathway. As a result, ΔCAN specifically blocked late, but not early, HIV-1 gene expression in HIV-1-infected cells. These data strongly validate Crm1 as a cellular cofactor for HIV-1 Rev and demonstrate that the MPMV CTE nuclear RNA export pathway uses a distinct, Crm1-independent mechanism. In addition, these data identify a novel and highly potent inhibitor of leucine-rich NES-dependent nuclear export.     

Nuclear export receptor Xpo1/Crm1 is physically and functionally linked to the spindle pole body in budding yeast

The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the gamma-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.     

Distinct nuclear import and export pathways mediated by members of the karyopherin β family

Transport of proteins into and out of the nucleus occurs through nuclear pore complexes (NPCs) and is mediated by the interaction of transport factors with nucleoporins at the NPC. Nuclear import of proteins containing classical nuclear localization signals (NLSs) is mediated by a heterodimeric protein complex, composed of karyopherin α and β1, that docks via β1 the NLS-protein to the NPC. The GTPase Ran; the RanGDP binding protein, p10; and the RanGTP binding protein, RanBP1 are involved in translocation of the docked NLS-protein into the nucleus. Recently, new distinct nuclear import and export pathways that are mediated by members of the karyopherin β family have been discovered. Karyopherin β2 mediates import of mRNA binding proteins, whereas karyopherin β3 and β4 mediate import of a set of ribosomal proteins. Two other β karyopherin family members, CRM1 and CAS, mediate export of proteins containing leucine-rich nuclear export signals (NES) and reexport of karyopherin α, respectively. This growing family contains new members that constitute potential transport factors for cargoes yet to be identified in the future. The common features of the members of karyopherin β family are the ability to bind RanGTP and the ability to interact directly with nucleoporins at the NPC. The challenge for the future will be to identify the distinct or, perhaps, overlapping cargo(es) for each member of the karyopherin β superfamily and to characterize the molecular mechanisms of translocation of karyopherins together with their cargoes through the NPC. J. Cell. Biochem. 70:231–239, 1998.© 1998 Wiley-Liss, Inc.     

The Survivin-Crm1 interaction is essential for chromosomal passenger complex localization and function

The chromosomal passenger complex (CPC) of Aurora-B, Borealin, INCENP (inner centromere protein) and Survivin coordinates essential chromosomal and cytoskeletal events during mitosis. Here, we show that the nuclear export receptor Crm1 is crucially involved in tethering the CPC to the centromere by interacting with a leucine-rich nuclear export signal (NES), evolutionarily conserved in all mammalian Survivin proteins. We show that inhibition of the Survivin-Crm1 interaction by treatment with leptomycin B or by RNA-interference-mediated Crm1 depletion prevents centromeric targeting of Survivin. The genetic inactivation of the Survivin-Crm1 interaction by mutation of the NES affects the correct localization and function of Survivin and the CPC during mitosis. By contrast, CPC assembly does not seem to require the Survivin-Crm1 interaction. Our report shows the functional significance of the Survivin-Crm1 interface and provides a novel link between the mitotic effector Crm1 and the CPC.