A new nucleoporin-like protein interacts with both HIV-1 Rev nuclear export signal and CRM-1.

The HIV-1 Rev is a shuttling protein required for the nuclear export of unspliced and partially spliced viral mRNA. In this study, we have identified a new Rev-interacting protein, that specifically interacts with the Rev nuclear export signal both in yeast and mammalian cells. This protein has features found in nucleoporins including many phenylalanine-glycine repeats, a very high serine content, a putative zinc finger, and a coiled-coil domain; we thus called it NLP-1 (nucleoporin-like protein 1). In addition, gene expression analysis and wheat germ agglutinin chromatography experiments suggested that NLP-1 is an ubiquitous O-glycosylated nuclear protein. Recently, a cellular factor called CRM-1 has been shown to be an essential nuclear export factor interacting directly with nuclear export signals including the Rev nuclear export signal in a RanGTP-dependent manner. We show here that NLP-1, like the previously described Rev-interacting protein hRIP/Rab and several nucleoporins, also interacts with CRM-1 both in yeast and mammalian cells.     

VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway

Interference with nucleocytoplasmic transport is a strategy employed by certain viruses to compromise host cellular function. While it has been shown that the matrix (M) protein of the vesicular stomatitis virus (VSV) inhibits nuclear export of host cell mRNAs, the underlying mechanism has not been fully established. Here we show that VSV M protein binds the mRNA export factor Rae1/mrnp41. A mutant of M protein defective in Rae1 binding is unable to inhibit mRNA nuclear export. We further show that increased expression of Rae1 fully reverts the inhibition of mRNA export induced by M protein or following virus infection. We found that Rae1 is induced by interferon-gamma, a cytokine that plays a critical role in the immune response to viruses, such as VSV. Thus, these results demonstrate that VSV M protein blocks mRNA export by disrupting Rae1 function, which can be reverted by induction of Rae1 expression.     

Definition of a consensus transportin-specific nucleocytoplasmic transport signal

The low cytoplasmic and high nuclear concentration of the GTP-bound form of Ran provides directionality for both nuclear protein import and export. Both import and export factors bind RanGTP directly, yet this interaction produces opposite effects; in the former case, RanGTP binding induces nuclear cargo release, whereas in the latter, RanGTP binding induces nuclear cargo assembly. Therefore, nuclear import and export receptors and their protein recognition sites are predicted to be distinct. Nevertheless, the approximately 38-amino acid M9 sequence present in heterogeneous nuclear ribonucleoprotein A1 has been reported to serve as both a nuclear localization signal and a nuclear export signal, even though only one protein, the nuclear import factor transportin, has been shown to bind M9 directly. We have used a combination of mutational randomization followed by selection for transportin binding to exhaustively define amino acids in M9 that are critical for transportin binding in vivo. As expected, the resultant approximately 12-amino acid transportin-binding consensus sequence is also predictive of nuclear localization signal activity. Surprisingly, however, this extensive mutational analysis failed to dissect M9 nuclear localization signal and nuclear export signal function. Nevertheless, transportin appears unlikely to be the M9 export receptor, as RanGTP can be shown to block M9 binding by transportin not only in vitro, but also in the nucleus in vivo. This analysis therefore predicts the existence of a nuclear export receptor distinct from transportin that nevertheless shares a common protein-binding site on heterogeneous nuclear ribonucleoprotein A1.     

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.     

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.     

Nuclear export of the E1B 55-kDa and E4 34-kDa adenoviral oncoproteins mediated by a rev-like signal sequence.

The E1B 55-kDa and E4 34-kDa oncoproteins of adenovirus type 5 (abbreviated here as E1B-55kD and E4-34kD) promote the export of viral mRNA and inhibit the export of most cellular mRNA species. We show that the intracellular complex containing E1B-55kD and E4-34kD continuously shuttles between the nucleus and the cytoplasm, and may thus serve as a nucleocytoplasmic transporter for viral mRNA. We present evidence that within this complex, it is the E4-34kD protein that directs both nuclear import and nuclear export. E4-34kD contains a functional nuclear export signal similar to corresponding sequences found in the retroviral proteins rev and rex. This sequence element is required for nuclear export of the complex, and it can function autonomously when fused to a carrier protein and microinjected in HeLa cell nuclei. When E4-34kD is expressed alone, a portion of the protein that contains a predicted arginine-rich amphipathic alpha-helical structure mediates nuclear retention of the protein. This retention, however, can be abolished by the association with E1B-55kD or by a specific point mutation within the arginine-rich motif. The export of E4-34kD can be blocked by an HTLV-rex derived competitive inhibitor and overexpressed E4-34kD inhibits rev-mediated transport, suggesting that the export pathways accessed by the adenoviral and retroviral proteins share components. The interplay between two polypeptides as well as the involvement of a dominant nuclear retention domain are novel features that might contribute to the efficiency and regulation of the adenovirus export system.     

An N-terminal Nuclear Export Signal Regulates Trafficking and Aggregation of Huntingtin (Htt) Protein Exon 1

Huntington disease is a dominantly inherited neurodegenerative condition caused by polyglutamine expansion in the N terminus of the huntingtin protein (Htt). The first 17 amino acids (N17) of Htt play a key role in regulating its toxicity and aggregation. Both nuclear export and cytoplasm retention functions have been ascribed to N17. We have determined that N17 acts as a nuclear export sequence (NES) within Htt exon and when fused to yellow fluorescent protein. We have defined amino acids within N17 that constitute the nuclear export sequence (NES). Mutation of any of the conserved residues increases nuclear accumulation of Htt exon 1. Nuclear export of Htt is sensitive to leptomycin B and is reduced by knockdown of exportin 1. In HEK293 cells, NES mutations decrease overall Htt aggregation but increase the fraction of cells with nuclear inclusions. In primary cultured neurons, NES mutations increase nuclear accumulation and increase overall aggregation. This work defines a bona fide nuclear export sequence within N17 and links it to effects on protein aggregation. This may help explain the important role of N17 in controlling Htt toxicity.     

Transport of galectin-3 between the nucleus and cytoplasm. II. Identification of the signal for nuclear export

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. Previous studies have shown that incubation of fibroblasts with leptomycin B resulted in the accumulation of galectin-3 in the nucleus, suggesting that the export of galectin-3 from the nucleus may be mediated by the CRM1 receptor. A candidate nuclear export signal fitting the consensus sequence recognized by CRM1 can be found between residues 240 and 255 of the murine galectin-3 sequence. This sequence was engineered into the pRev(1.4) reporter system, in which candidate sequences can be tested for nuclear export activity in terms of counteracting the nuclear localization signal present in the Rev(1.4) protein. Rev(1.4)-galectin-3(240-255) exhibited nuclear export activity that was sensitive to inhibition by leptomycin B. Site-directed mutagenesis of Leu247 and Ile249 in the galectin-3 nuclear export signal decreased nuclear export activity, consistent with the notion that these two positions correspond to the critical residues identified in the nuclear export signal of the cAMP-dependent protein kinase inhibitor. The nuclear export signal activity was also analyzed in the context of a full-length galectin-3 fusion protein; galectin-3(1-263; L247A) showed more nuclear localization than wild-type, implicating Leu247 as critical to the function of the nuclear export signal. These results indicate that residues 240-255 of the galectin-3 polypeptide contain a leucine-rich nuclear export signal that overlaps with the region (residues 252-258) identified as important for nuclear localization.     

The influenza virus NEP (NS2 protein) mediates the nuclear export of viral ribonucleoproteins.

Nuclear import and export of viral nucleic acids is crucial for the replication cycle of many viruses, and elucidation of the mechanism of these steps may provide a paradigm for understanding general biological processes. Influenza virus replicates its RNA genome in the nucleus of infected cells. The influenza virus NS2 protein, which had no previously assigned function, was shown to mediate the nuclear export of virion RNAs by acting as an adaptor between viral ribonucleoprotein complexes and the nuclear export machinery of the cell. A functional domain on the NS2 with characteristics of a nuclear export signal was mapped: it interacts with cellular nucleoporins, can functionally replace the effector domain of the human immunodeficiency virus type 1 (HIV-1) Rev protein and mediates rapid nuclear export when cross-linked to a reporter protein. Microinjection of anti-NS2 antibodies into infected cells inhibited nuclear export of viral ribonucleoproteins, suggesting that the Rev-like NS2 mediates this process. Therefore, we have renamed this Rev-like factor the influenza virus nuclear export protein or NEP. We propose a model by which NEP acts as a protein adaptor molecule bridging viral ribonucleoproteins and the nuclear pore complex.     

An amino-terminal motif functions as a second nuclear export sequence in BRCA1

Mutations in the breast cancer susceptibility gene 1 (BRCA1) account for a substantial percentage of familial breast and ovarian cancers. Although BRCA1 is thought to function within the nucleus, it has also been located in the cytoplasm. In addition, BRCA1 accumulates in the nucleus of cells treated with leptomycin B, an inhibitor of chromosome region maintenance 1-mediated nuclear export, indicative of its active nuclear export via this pathway. The nuclear export signal in BRCA1 has been described as consisting of amino acid residues 81-99. However, a number of other tumor suppressors have multiple nuclear export sequences, and we sought to determine whether BRCA1 did also. Here, we report that BRCA1 contains a second nuclear export sequence that comprises amino acid residues 22-30. By use of the human immunodeficiency virus-1 Rev complementation assay, this sequence was shown to confer export capability to an export-defective Rev fusion protein. The level of export activity was comparable with that of residues 81-99 comprising the previously reported nuclear export sequence in BRCA1. Mutation of leucine 28 to an alanine reduced nuclear export by approximately 75%. In MCF-7 cells stably transfected with a BRCA1 cDNA containing mutations in this novel sequence or the previously reported export sequence, BRCA1 accumulated in the nucleus. These data imply that BRCA1 contains at least two leucine-dependent nuclear export sequences.     

Nuclear export in plants. Use of geminivirus movement proteins for a cell-based export assay.

The nuclear export of proteins and RNAs has been studied in heterokaryons or by microinjecting test substrates into nuclei of HeLa cells or Xenopus oocytes. We have previously shown that the two movement proteins BR1 and BL1 encoded by the plant pathogenic squash leaf curl virus act in a coordinated manner to facilitate virus cell-to-cell movement and that one of these (BR1) is a nuclear shuttle protein. By using a novel in vivo cell-based assay for nuclear export in which nuclear-localized BR1 is trapped by BL1 and redirected to the cortical cytoplasm, we demonstrate that residues 177 to 198 of BR1 contain a leucine-rich nuclear export signal (NES) of the type found in the Rev protein encoded by the human immunodeficiency virus and in Xenopus TFIIIA. We further show that the TFIIIA NES can functionally replace the NES of BR1 in both nuclear export and viral infectivity. These findings suggest that this basic pathway for nuclear export is highly conserved among plant and animal cells and in yeast.     

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.     

Nuclear export of African swine fever virus p37 protein occurs through two distinct pathways and is mediated by three independent signals

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle.     

Characterization of a novel transferable CRM-1-independent nuclear export signal in a herpesvirus tegument protein that shuttles between the nucleus and cytoplasm

A new group of nucleocytoplasmic shuttling proteins has recently been identified in the structural proteins encoded by several alphaherpesvirus UL47 genes. Nuclear import and export signals for the bovine herpesvirus type 1 UL47 protein (VP8 or bUL47) have been described previously. Here, we study the trafficking of bUL47 in detail and identify an import signal different from that shown before. It comprises a 20-residue N-terminal peptide that is fully transferable and targets a large, normally cytosolic protein to the nucleus. A conserved RRPRRS motif within this peptide was shown to be essential but not sufficient for nuclear targeting. Using interspecies heterokaryon assays, we further demonstrate that the export activity of the published leucine-rich nuclear export signal (NES) is also transferable to a large protein but is functionally weak compared to the activity of the HIV-1 Rev NES. We show that nuclear export dictated by this bUL47 NES is sensitive to leptomycin B (LMB) and therefore dependent on the export receptor CRM-1. However, nuclear export of full-length bUL47 is fully resistant to LMB, suggesting the presence of an additional NES. We go on to identify a second NES in bUL47 within a 28-residue peptide that is in close proximity to but entirely separable from the N-terminal import signal, and we use fluorescence loss in photobleaching to confirm its activity. This NES is resistant to leptomycin B, and therefore utilizes an export receptor other than CRM-1. As this new sequence bears little similarity to other export signals so far defined, we suggest it may be involved in bUL47 export from the nucleus via a novel cellular receptor.     

Nucleocytoplasmic shuttling of human inositol phosphate multikinase is influenced by CK2 phosphorylation

Human inositol phosphate multikinase (IPMK) is a multifunctional protein in cellular signal transduction, namely, a multispecific inositol phosphate kinase, phosphatidylinositol 3-kinase, and a scaffold within the mTOR-raptor complex. To fulfill these nuclear and cytoplasmic functions, intracellular targeting of IPMK needs to be regulated. We show here that IPMK, which has been considered to be a preferentially nuclear protein, is a nucleocytoplasmic shuttling protein, whose nuclear export is mediated by classical nuclear export receptor CRM1. We identified a functional nuclear export signal (NES) additionally to its previously described nuclear import signal (NLS). Furthermore, we describe a mechanism by which the activity of the IPMK-NLS is controlled. Protein kinase CK2 binds endogenous IPMK and phosphorylates it at serine 284. Interestingly, this phosphorylation can decrease nuclear localization of IPMK cell type specifically. A controlled nuclear import of IPMK may direct its actions either toward nuclear inositol phosphate (InsPx) metabolism or cytoplasmic actions on InsPx, phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P?], as well as mTOR-raptor.     

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.