A comparison of the activity, sequence specificity, and CRM1-dependence of different nuclear export signals

Nuclear export sequences (NESs) have been identified in many cellular proteins, but it remains unclear how different NESs compare in activity. We describe a sensitive new in vivo export assay which we have used to assess the relative export activity of different types of NES. The most common type of export sequence resembles that first identified in the HIV-1 Rev protein and typically comprises a core of large hydrophobic amino acids that specify recognition by the CRM1 export receptor. We compared 10 previously identified Rev-type NESs in our assay, and whereas all were functional, the relative export activities of these signals varied considerably. We further identified 3 new Rev-type NESs from a computer database search, and each export signal was assigned a score of 1 to 9 and ranked in order of activity (e.g., PKI > c-ABL > Ran-BP1 > FMRP > PML > IkappaB-alpha > hdm2). The weakest NESs were found in the p53 tumor suppressor and the p53-regulated proteins p21 and hdm2, which are all normally localized to the nucleus. All of the Rev-type NESs were inactivated by mutation of key hydrophobic residues and by treatment with the CRM1-specific export inhibitor, leptomycin B. In contrast, a different type of export signal, the KNS shuttling element derived from hnRNP K, exhibited a modest export activity that was insensitive to leptomycin B treatment. KNS thus appears to mediate export via a CRM1-independent pathway. Mutagenesis of the KNS sequence identified, for the first time, specific serines and acidic residues necessary for its export activity, thereby distinguishing KNS from other types of nuclear transport signal. We have shown that different nuclear export signals can vary profoundly in activity and therefore conclude that the nuclear export rate of a specific shuttling protein largely depends on both the strength and the accessibility of its NES.     

NESdb: a database of NES-containing CRM1 cargoes

The leucine-rich nuclear export signal (NES) is the only known class of targeting signal that directs macromolecules out of the cell nucleus. NESs are short stretches of 8-15 amino acids with regularly spaced hydrophobic residues that bind the export karyopherin CRM1. NES-containing proteins are involved in numerous cellular and disease processes. We compiled a database named NESdb that contains 221 NES-containing CRM1 cargoes that were manually curated from the published literature. Each NESdb entry is annotated with information about sequence and structure of both the NES and the cargo protein, as well as information about experimental evidence of NES-mapping and CRM1-mediated nuclear export. NESdb will be updated regularly and will serve as an important resource for nuclear export signals. NESdb is freely available to nonprofit organizations at http://prodata.swmed.edu/LRNes.     

A supraphysiological nuclear export signal is required for parvovirus nuclear export

CRM1 exports proteins that carry a short leucine-rich peptide signal, the nuclear export signal (NES), from the nucleus. Regular NESs must have low affinity for CRM1 to function optimally. We previously generated artificial NESs with higher affinities for CRM1, termed supraphysiological NESs. Here we identify a supraphysiological NES in an endogenous protein, the NS2 protein of parvovirus Minute Virus of Mice (MVM). NS2 interacts with CRM1 without the requirement of RanGTP, whereas addition of RanGTP renders the complex highly stable. Mutation of a single hydrophobic residue that inactivates regular NESs lowers the affinity of the NS2 NES for CRM1 from supraphysiological to regular. Mutant MVM harboring this regular NES is compromised in viral nuclear export and productivity. In virus-infected mouse fibroblasts we observe colocalization of NS2, CRM1 and mature virions, which is dependent on the supraphysiological NS2 NES. We conclude that supraphysiological NESs exist in nature and that the supraphysiological NS2 NES has a critical role in active nuclear export of mature MVM particles before cell lysis.     

Nuclear export signal consensus sequences defined using a localization-based yeast selection system

Proteins bearing nuclear export signals (NESs) are translocated to the cytoplasm from the nucleus mainly through the CRM1-dependent pathway. However, the NES consensus sequence remains poorly defined, and there are currently no high-throughput methods for identifying NESs. In this study, we report the development of an efficient yeast selection system for detecting nuclear export activity as well as several reliable NES consensus sequences identified using this method. Our selection system is based on the nuclear export-dependent rescue of Tys1p, an essential cytoplasmic protein that has been artificially localized to the nucleus in a haploid Delta tys1 knockout strain. A screen of a random peptide library revealed 101 distinct CRM1-dependent NESs, which were classified into six patterns according to the conserved hydrophobic spacing. By combining mutational analyses, we have defined new NES consensus sequences with more specific and redundant residues than the traditional consensus sequence, which are consistent with most experimentally confirmed NESs. These NES consensus sequences should help identify functional NESs, and our selection system can be used to identify other targeting signals or proteins imported to specific subcellular compartments.     

Nuclear export of proteins in plants: AtXPO1 is the export receptor for leucine-rich nuclear export signals in Arabidopsis thaliana

Transport across the nuclear envelope is mediated by transport receptors from the Importin beta family. We identified Exportin 1 from Arabidopsis (AtXPO1/AtCRM1) as the nuclear export receptor for proteins carrying leucine-rich nuclear export signals (NESs). AtXPO1 shares 42-50% identity with its functional homologues from humans and yeasts. We functionally characterised AtXPO1 by its interaction with NESs of animal and plant proteins, which is inhibited by the cytotoxin leptomycin B (LMB), and also by its interaction with the small GTPase Ran1 in the yeast two-hybrid system. Furthermore, we demonstrated the existence of a nuclear export pathway for proteins in plants. For the characterisation of nuclear export activities, we established an in vivo assay based on the localisation equilibrium of a GFP reporter protein fused to both a nuclear localisation signal (NLS) and an NES motif. Using this in vivo assay we demonstrated that the NES of the heterologous protein Rev is also functional in plants and that its export is inhibited by LMB. In addition, we identified a leucine-rich NES in the Arabidopsis protein AtRanBP1a. The NES, which is located at the carboxy terminus of the protein, is disrupted by mutating three long chain hydrophobic amino acid residues to alanine (L176A, L179A, V181A). In BY-2 protoplasts the NES of AtRanBP1a is functionally indistinguishable from the Rev NES. Our results demonstrate that the machinery for the nuclear export of proteins is functionally conserved in plants.     

Differential nucleocytoplasmic trafficking between the related endocytic proteins Eps15 and Eps15R.

Eps15 and Eps15R are constitutive components of clathrin-coated pits that are required for clathrin-dependent endocytosis. The most striking difference between these two related proteins is that Eps15R is also found in the nucleus, whereas Eps15 is excluded from this compartment at steady state. To better understand the individual functions of these two proteins, the mechanisms responsible for their different localization were investigated. Interestingly, some mutants of Eps15 were found in the nucleus. This nuclear localization was correlated with the loss of the last approximately 100 amino acids of Eps15, suggesting the presence of a nuclear export signal (NES) within this region. As expected, the last 25 amino acids contain a leucine-rich sequence matching with classical NESs, show a leptomycin B-sensitive nuclear export activity, and bind to the exportin CRM1 in a leucine residue-dependent manner. In contrast, no NES could be found in Eps15R, a result in keeping with its constitutive nuclear localization that appears to be regulated by alternative splicing. Altogether, these results are the first characterization of nucleocytoplasmic shuttling signals for endocytic proteins. They also provide an explanation for the different nuclear localization of Eps15 and Eps15R and further evidence for a possible nuclear function for Eps15 protein family members.     

Difference in nucleocytoplasmic shuttling sequences of rat and human constitutive active/androstane receptor

Fluorescence recovery after photobleaching (FRAP) in spontaneous multinuclear cells shows that both rat and human constitutive active/androstane receptors (CARs) are shuttling proteins with both nuclear localization signals (NLSs) and nuclear export signals (NESs). We previously identified two NLSs in rat CAR: NLS1 in the hinge region (residues 100-108) and NLS2 in the ligand-binding domain (residues 111-320). In the present study, we compared the intracellular localization signals between rat and human CARs. There was a marked difference in their intracellular localization in COS-7 cells because, unlike rat CAR, human CAR does not contain NLS1 due to an amino acid change at position 106. A CRM1-dependent leucine-rich NES, which is sensitive to an inhibitory effect of leptomycin B, was found in the cytoplasmic retention region previously identified within the ligand-binding domain of rat CAR (residues 220-258). We found that human CAR instead has a NES in the ligand-binding domain between residues 170 and 220. Also, we detected CRM1-independent C-terminal NESs between residues 317-358 of rat and human CARs. Removal of NLS1 by N-terminal truncation and mutation of xenochemical response signal caused rat CAR to localize in the cytoplasm of COS-7 cells, which we suspect is due to the masking of NLS2.     

Three leucine-rich sequences and the N-terminal region of double-stranded RNA-activated protein kinase (PKR) are responsible for its cytoplasmic localization

The double-stranded RNA-activated-protein kinase PKR was originally identified as a ribosomal protein that regulates protein synthesis at the translational level. While PKR locates predominantly to the cytoplasm, nuclear or nucleolar species of PKR have been detected. Here, we demonstrate that PKR possesses three leucine-rich sequences resembling nuclear export signals (NESs). Enhanced green fluorescent protein (EGFP) fused to one of these sequences and transfected in COS-1 cells exhibited predominant cytoplasmic staining, which was abrogated by a leucine to alanine substitution. In addition, Leptomycin B (LMB), an inhibitor of NES-mediated nuclear export, inhibited the cytoplasmic localization of EGFP-NES, indicating the potential activity of these stretches as NESs. Although EGFP fused to a PKR with three NES mutations still located to the cytoplasm, an additional N-terminal deletion impaired the cytoplasmic predominance, suggesting that the N-terminal region is also required for localization. These results suggest that the cytoplasmic localization of PKR is regulated by NESs as well as the N-terminal sequence.     

Influenza A virus NS2 protein mediates vRNP nuclear export through NES-independent interaction with hCRM1

For nuclear export of proteins, the formation of a ternary export complex composed of the export substrate, a cellular export factor and Ran-GTP is crucial. CRM1 is a cellular export factor for proteins containing leucine-rich nuclear export signals (NESs). Although the NES sequence is crucial for nuclear export, its exact role in the formation of the ternary export complex is controversial. Here we demonstrate an interaction between human CRM1 (hCRM1) and influenza A virus NS2 protein, which contains an NES motif in its N-terminal region. Replacement of the hydrophobic amino acids in the NES motif did not abolish NS2's interaction with hCRM1. Using our recently established systems for the generation of influenza virus or virus-like particles from cloned cDNAs, we found that NS2 is essential for nuclear export of influenza virus ribonucleoprotein (RNP) complexes, and that alteration of the NS2-NES abrogated this event and influenza virus generation. These findings suggest that the NS2-NES is not crucial for the interaction of this protein with hCRM1, but is for the formation of the ternary export complex with Ran-GTP.     

NESmapper: Accurate Prediction of Leucine-Rich Nuclear Export Signals Using Activity-Based Profiles

The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear–cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at http://sourceforge.net/projects/nesmapper.

This is a PLOS Computational Biology Software Article

     

Leucine-rich nuclear-export signals: born to be weak

CRM1 mediates the nuclear export of proteins exposing leucine-rich nuclear-export signals (NESs). Most NESs bind to CRM1 with relatively low affinity. Recently, higher-affinity NESs were selected from a 15-mer random peptide library. Unexpectedly, complexes between high-affinity NESs and CRM1 accumulate at the cytoplasmic filaments of the nuclear pore complex (NPC). This finding suggests that high-affinity NES binding to CRM1 impairs the efficient release of export complexes from the NPC, explaining why leucine-rich NESs have evolved to be weak.     

The yeast splicing factor Prp40p contains functional leucine-rich nuclear export signals that are essential for splicing

To investigate the function of the essential U1 snRNP protein Prp40p, we performed a synthetic lethal screen in Saccharomyces cerevisiae. Using an allele of PRP40 that deletes 47 internal residues and causes only a slight growth defect, we identified aphenotypic mutations in three distinct complementation groups that conferred synthetic lethality. The synthetic phenotypes caused by these mutations were suppressed by wild-type copies of CRM1 (XPO1), YNL187w, and SME1, respectively. The strains whose synthetic phenotypes were suppressed by CRM1 contained no mutations in the CRM1 coding sequence or promoter. This indicates that overexpression of CRM1 confers dosage suppression of the synthetic lethality. Interestingly, PRP40 and YNL187w encode proteins with putative leucine-rich nuclear export signal (NES) sequences that fit the consensus sequence recognized by Crm1p. One of Prp40p's two NESs lies within the internal deletion. We demonstrate here that the NES sequences of Prp40p are functional for nuclear export in a leptomycin B-sensitive manner. Furthermore, mutation of these NES sequences confers temperature-sensitive growth and a pre-mRNA splicing defect. Although we do not expect that yeast snRNPs undergo compartmentalized biogenesis like their metazoan counterparts, our results suggest that Prp40p and Ynl187wp contain redundant NESs that aid in an important, Crm1p-mediated nuclear export event.     

Deciphering the Nuclear Import Pathway for the Cytoskeletal Red Cell Protein 4.1R

The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.     

Cofactor requirements for nuclear export of Rev response element (RRE)- and constitutive transport element (CTE)-containing retroviral RNAs. An unexpected role for actin

Nuclear export of proteins containing leucine-rich nuclear export signals (NESs) is mediated by the export receptor CRM1/exportin1. However, additional protein factors interacting with leucine-rich NESs have been described. Here, we investigate human immunodeficiency virus type 1 (HIV-1) Rev-mediated nuclear export and Mason-Pfizer monkey virus (MPMV) constitutive transport element (CTE)-mediated nuclear export in microinjected Xenopus laevis oocytes. We show that eukaryotic initiation factor 5A (eIF-5A) is essential for Rev and Rev-mediated viral RNA export, but not for nuclear export of CTE RNA. In vitro binding studies demonstrate that eIF-5A is required for efficient interaction of Rev-NES with CRM1/exportin1 and that eIF-5A interacts with the nucleoporins CAN/nup214, nup153, nup98, and nup62. Quite unexpectedly, nuclear actin was also identified as an eIF-5A binding protein. We show that actin is associated with the nucleoplasmic filaments of nuclear pore complexes and is critically involved in export processes. Finally, actin- and energy-dependent nuclear export of HIV-1 Rev is reconstituted by using a novel in vitro egg extract system. In summary, our data provide evidence that actin plays an important functional role in nuclear export not only of retroviral RNAs but also of host proteins such as protein kinase inhibitor (PKI).     

Analysis of the activity of nuclear export signals using fluorescent BSA conjugates

Here we demonstrate that fluorescein-labeled BSA conjugated with a mixture of nuclear import and export signals can be used to evaluate export activity. The method is based on the assumption that the intracellular distribution of the labeled conjugate [nuclear/cytoplasmic (N/C) fluorescent ratio] is dependent on the relative activity of the import versus the export signals. Using BALB/c cells as a model system, it was shown that this assumption is correct. Thus, the N/C fluorescent ratio increased significantly when an active leucine-rich nuclear export signal was replaced with its inactive mutant form in conjugates that also contained classical nuclear import signals. This approach was then used to demonstrate that the same leucine-rich nuclear export signal, which functions in vertebrates, is also active in amoebae.     

Nucleocytoplasmic shuttling of receptor-interacting protein 3 (RIP3): identification of novel nuclear export and import signals in RIP3

Receptor-interacting protein 3 (RIP3), a member of the RIP Ser/Thr kinase family, has been characterized as a pro-apoptotic protein involved in the tumor necrosis factor receptor-1 signaling pathway. In this study, we have mapped a minimal region of RIP3 sufficient for apoptosis induction to a fragment of 31 amino acids in length. This minimal region also functions as an unconventional nuclear localization signal sufficient to confer the import of full-length RIP3 to the nucleus to trigger apoptosis, suggesting that RIP3 is able to play an apoptosis-inducing role in the nucleus. In addition, we have characterized two novel leucine-rich nuclear export signals (NESs) that are responsible for the nuclear export of RIP3 to the cytoplasm via a chromosome region maintenance 1 (CRM1)-dependent pathway and an extra leucine-rich NES in the N terminus of RIP3 that contributes to the cytoplasmic distribution in a CRM1-independent manner. Thus, we provide the first evidence that RIP3 acts a nucleocytoplasmic shuttling protein, which presents a possible link between death receptor signaling and nuclear apoptosis.     

Nuclear accumulation of SHIP1 mutants derived from AML patients leads to increased proliferation of leukemic cells

The inositol 5-phosphatase SHIP1 acts as negative regulator of intracellular signaling in myeloid cells and is a tumor suppressor in myeloid leukemogenesis. After relocalization from the cytoplasm to the plasma membrane SHIP1 terminates PI3-kinase mediated signaling processes. Furthermore, SHIP1 is also found in distinct puncta in the cell nucleus and nuclear SHIP1 has a pro-proliferative function. Here we report the identification of five nuclear export signals (NESs) which regulate together with the two known nuclear localization signals (NLSs) the nucleocytoplasmic shuttling of SHIP1. Mutation of NLSs reduced the nuclear import and mutation of NESs decreased the nuclear export of SHIP1 in the acute myeloid leukemia (AML) cell line UKE-1. Interestingly, four SHIP1 mutants (K210R, N508D, V684E, Q1153L) derived from AML patients showed a nuclear accumulation after expression in UKE-1 cells. In addition, overexpression of the AML patient-derived mutation N508D caused an increased proliferation rate of UKE-1 cells in comparison to wild type SHIP1. Furthermore, we identified serine and tyrosine phosphorylation as a molecular mechanism for the regulation of nucleocytoplasmic shuttling of SHIP1 where tyrosine phosphorylation of distinct residues i.e. Y864, Y914, Y1021 reduces nuclear localization, whereas serine phosphorylation at S933 enhances nuclear localization of SHIP1.In summary, our data further implicate nuclear SHIP1 in cellular signaling and suggest that enhanced accumulation of SHIP1 mutants in the nucleus may be a contributory factor of abnormally high proliferation of AML cells.