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.     

Galectin-3 expression and subcellular localization in senescent human fibroblasts

Galectin-3 is a galactose/lactose-binding protein (M(r) approximately 30,000), identified as a required factor in the splicing of pre-mRNA. In the LG1 strain of human diploid fibroblasts, galectin-3 could be found in both the nucleus and the cytoplasm of young, proliferating cells. In contrast, the protein was predominantly cytoplasmic in senescent LG1 cells that have lost replicative competence through in vitro culture. Incubation of young cells with leptomycin B, a drug that disrupts the interaction between the leucine-rich nuclear export signal and its receptor, resulted in the accumulation of galectin-3 inside the nucleus. In senescent cells, galectin-3 staining remained cytoplasmic even in the presence of the drug, thus suggesting that the observed localization in the cytoplasm was due to a lack of nuclear import. In heterodikaryons derived from fusion of young and senescent LG1 cells, the predominant phenotype was galectin-3 in both nuclei. These results suggest that senescent LG1 cells might lack a factor(s) specifically required for galectin-3 nuclear import.     

Shuttling of galectin-3 between the nucleus and cytoplasm

In previous studies, we documented that galectin-3 (M(r) approximately 30,000) is a pre-mRNA splicing factor. Recently, galectin-3 was identified as a component of a nuclear and cytoplasmic complex, the survival of motor neuron complex, through its interaction with Gemin4. To test the possibility that galectin-3 may shuttle between the nucleus and the cytoplasm, human fibroblasts (LG-1) were fused with mouse fibroblasts (3T3). The monoclonal antibody NCL-GAL3, which recognizes human galectin-3 but not the mouse homolog, was used to monitor the localization of human galectin-3 in heterodikaryons. Human galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Addition of the antibiotic leptomycin B, which inhibits nuclear export of galectin-3, decreased the percentage of heterodikaryons showing human galectin-3 in both nuclei. In a parallel experiment, mouse 3T3 fibroblasts, which express galectin-3, were fused with fibroblasts derived from a mouse in which the galectin-3 gene was inactivated. Mouse galectin-3 localized to both nuclei of a large percentage of heterodikaryons. Again, addition of leptomycin B restricted the presence of galectin-3 to one nucleus of a heterodikaryon. The results from both heterodikaryon assays suggest that galectin-3 can exit one nucleus, travel through the cytoplasm, and enter the second nucleus, matching the definition of shuttling.     

A novel transferable nuclear export signal mediates CRM1-independent nucleocytoplasmic shuttling of the human cytomegalovirus transactivator protein pUL69.

The best studied nuclear export processes are mediated by classical leucine-rich nuclear export signals that specify recognition by the CRM1 export receptor. However, details concerning alternative nuclear export signals and pathways are beginning to emerge. Within the family of Herpesviridae, a set of homologous regulatory proteins that are exemplified by the ICP27 of herpes simplex virus were described recently as nucleocytoplasmic shuttling proteins. Here we report that pUL69 of the beta-herpesvirus human cytomegalovirus is a nuclear protein that is able to shuttle between the nucleus and the cytoplasm independently of virus-encoded cofactors. In contrast to proteins containing a leucine-rich export signal, the shuttling activity of pUL69 was not affected by leptomycin B, indicating that pUL69 trafficking is not mediated by the export receptor CRM1. Importantly, we identified and characterized a novel type of transferable, leptomycin B-insensitive export signal that is distinct from other export signals described previously and is required for pUL69-mediated activation of gene expression. These data suggest that pUL69 is exported via a novel nuclear export pathway, based on a so far unique nuclear export signal of 28 amino acids.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b’s efficient suppression of RNA silencing

The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm.     

Coordinated nuclear export of 60S ribosomal subunits and NMD3 in vertebrates

60S and 40S ribosomal subunits are assembled in the nucleolus and exported from the nucleus to the cytoplasm independently of each other. We show that in vertebrate cells, transport of both subunits requires the export receptor CRM1 and Ran.GTP. Export of 60S subunits is coupled with that of the nucleo- cytoplasmic shuttling protein NMD3. Human NMD3 (hNMD3) contains a CRM-1-dependent leucine-rich nuclear export signal (NES) and a complex, dispersed nuclear localization signal (NLS), the basic region of which is also required for nucleolar accumulation. When present in Xenopus oocytes, both wild-type and export-defective mutant hNMD3 proteins bind to newly made nuclear 60S pre-export particles at a late step of subunit maturation. The export-defective hNMD3, but not the wild-type protein, inhibits export of 60S subunits from oocyte nuclei. These results indicate that the NES mutant protein competes with endogenous wild-type frog NMD3 for binding to nascent 60S subunits, thereby preventing their export. We propose that NMD3 acts as an adaptor for CRM1-Ran.GTP-mediated 60S subunit export, by a mechanism that is conserved from vertebrates to yeast.     

Nuclear export of yeast signal recognition particle lacking Srp54p by the Xpo1p/Crm1p NES-dependent pathway

BACKGROUND: The movement of macromolecules through the nuclear pores requires energy and transport receptors that bind both cargo and nuclear pores. Different molecules/complexes often require different transport receptors. The signal recognition particle (SRP) is a conserved cytosolic ribonucleoprotein that targets proteins to the endoplasmic reticulum. Previous studies have shown that the export of SRP RNA from the nucleus requires trans-acting factors and that SRP may be at least partly assembled in the nucleus, but little else is known about how it is assembled and exported into the cytoplasm. RESULTS: Of the six proteins that constitute the yeast SRP, we found that all except Srp54p were imported into the nucleus. Four of these had nucleolar pools. The same four proteins are required for stability of the yeast SRP RNA scR1, suggesting that they assemble with the RNA in the nucleus to form a central core SRP. This core SRP was a competent export substrate. Of the remaining components, Sec65p entered the nucleus and was assembled onto the core particle there, whereas Srp54p was solely cytoplasmic. The export of SRP from the nucleus required the transport receptor Xpo1p/Crm1p and Yrb2p, both components of the pathway that exports leucine-rich nuclear export signal (NES)-containing proteins from the nucleus. CONCLUSIONS: The SRP is assembled in the nucleus into a complex lacking only Srp54p. It is then exported through the NES pathway into the cytoplasm where Srp54p binds to it. This transport route for a ribonucleoprotein complex is so far unique in yeast.     

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 import and export signals in control of Nrf2

Nrf2 binds to the antioxidant response element and regulates expression and antioxidant induction of a battery of chemopreventive genes. In this study, we have identified nuclear import and export signals of Nrf2 and show that the nuclear import and export of Nrf2 is regulated by antioxidants. We demonstrate that Nrf2 contains a bipartite nuclear localization signal (NLS) and a leucine-rich nuclear export signal, which regulate Nrf2 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nrf2 accumulates in the nucleus within 15 min of antioxidant treatment and is exported out of nucleus by 8 h after treatment. Nrf2 mutant lacking the NLS failed to enter the nucleus and displayed diminished expression and induction of the downstream NAD(P)H:quinone oxidoreductase 1 gene. The Nrf2 NLS sequence, when fused to green fluorescence protein, resulted in the nuclear accumulation of green fluorescence protein, indicating that this signal sequence was sufficient to direct nuclear localization of Nrf2. A nuclear export signal (NES) was characterized in the C terminus of Nrf2, the deletion of which caused Nrf2 to accumulate predominantly in the nucleus. The Nrf2 NES was sensitive to leptomycin B and could function as an independent export signal when fused to a heterologous protein. Further studies demonstrate that NES-mediated nuclear export of Nrf2 is required for degradation of Nrf2 in the cytosol. These results led to the conclusion that Nrf2 localization between cytosol and nucleus is controlled by both nuclear import and export of Nrf2, and the overall distribution of Nrf2 is probably the result from a balance between these two processes. Antioxidants change this balance in favor of nuclear accumulation of Nrf2, leading to activation of chemopreventive proteins. Once this is achieved, Nrf2 exits the nucleus for binding to INrf2 and degradation.     

CRM1 mediates nuclear export of nonstructural protein 2 from parvovirus minute virus of mice.

The nonstructural protein 2 (NS2) from parvovirus minute virus of mice (MVMp) is a 25-kDa polypeptide which localizes preferentially to the cytoplasm and associates with cellular proteins in cytoplasm. These lines of evidence suggest that NS2 is positively exported from the nucleus to cytoplasm and functions in cytoplasm. We report here that nuclear export of NS2 is inhibited by leptomycin B (LMB), a drug that specifically blocks nuclear export signal (NES)-chromosomal region maintenance 1 (CRM1) interactions. CRM1 binds specifically to the 81- to 106-amino-acid (aa) region of NS2, and the region of NS2 actually functions as a NES. Interestingly, this region appears to be distinct from a typical NES sequence, which consists of leucine-rich sequences. These results indicate that NS2 protein is continuously exported from the nucleus by a CRM1-dependent mechanism and suggest that CRM1 also exports to distinct type of NESs.     

Nucleocytoplasmic Shuttling Activity of Ataxin-3

Spinocerebellar ataxia type-3, also known as Machado-Joseph Disease (MJD), is one of many inherited neurodegenerative disorders caused by polyglutamine-encoding CAG repeat expansions in otherwise unrelated genes. Disease protein misfolding and aggregation, often within the nucleus of affected neurons, characterize polyglutamine disorders. Several evidences have implicated the nucleus as the primary site of pathogenesis for MJD. However, the molecular determinants for the nucleocytoplasmic transport of human ataxin-3 (Atx3), the protein which is mutated in patients with MJD, are not characterized.In order to characterize the nuclear shuttling activity of Atx3, we performed yeast nuclear import assays and found that Atx3 is actively imported into the nucleus, by means of a classical nuclear localizing sequence formed by a cluster of lysine and arginine residues. On the other hand, when active nuclear export was inhibited using leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, both endogenous Atx3 and transfected GFP-Atx3 accumulated inside the nucleus of a subpopulation of COS-7 cells, whereas both proteins are normally predominant in the cytoplasm.Additionally, using a Rev(1.4)-GFP nuclear export assay, we performed an extensive analysis of six putative aliphatic nuclear export motifs identified in Atx3 amino acid sequence. Although none of the tested peptide sequences were found to drive nuclear export when isolated, we have successfully mapped the region of Atx3 responsible for its CRM1-independent nuclear export activity. Curiously, the N-terminal Josephin domain alone is exported into the cytoplasm, but the nuclear export activity of Atx3 is significantly enhanced in a longer construct that is truncated after the two ubiquitin interaction motifs, upstream from the polyQ tract.Our data show that Atx3 is actively imported to and exported from the cell nucleus, and that its nuclear export activity is dependent on a motif located at its N-terminal region. Since pathological Atx3 aggregates in the nucleus of affected neurons in MJD, and there is in vivo evidence that nuclear localization of Atx3 is required for the manifestation of symptoms in MJD, defects in the nucleocytoplasmic shuttling activity of the protein may be involved in the nuclear accumulation and aggregation of expanded Atx3.     

Insights into the function of the CRM1 cofactor RanBP3 from the structure of its Ran-binding domain

Proteins bearing a leucine-rich nuclear export signal (NES) are exported from the nucleus by the transport factor CRM1, which forms a cooperative ternary complex with the NES-bearing cargo and with the small GTPase Ran. CRM1-mediated export is regulated by RanBP3, a Ran-interacting nuclear protein. Unlike the related proteins RanBP1 and RanBP2, which promote disassembly of the export complex in the cytosol, RanBP3 acts as a CRM1 cofactor, enhancing NES export by stabilizing the export complex in the nucleus. RanBP3 also alters the cargo selectivity of CRM1, promoting recognition of the NES of HIV-1 Rev and of other cargos while deterring recognition of the import adaptor protein Snurportin1. Here we report the crystal structure of the Ran-binding domain (RBD) from RanBP3 and compare it to RBD structures from RanBP1 and RanBP2 in complex with Ran and CRM1. Differences among these structures suggest why RanBP3 binds Ran with unusually low affinity, how RanBP3 modulates the cargo selectivity of CRM1, and why RanBP3 promotes assembly rather than disassembly of the export complex. The comparison of RBD structures thus provides an insight into the functional diversity of Ran-binding proteins.     

Nuclear import of Upf3p is mediated by importin-alpha/-beta and export to the cytoplasm is required for a functional nonsense-mediated mRNA decay pathway in yeast

Upf3p, which is required for nonsense-mediated mRNA decay (NMD) in yeast, is primarily cytoplasmic but accumulates inside the nucleus when UPF3 is overexpressed or when upf3 mutations prevent nuclear export. Upf3p physically interacts with Srp1p (importin-alpha). Upf3p fails to be imported into the nucleus in a temperature-sensitive srp1-31 strain, indicating that nuclear import is mediated by the importin-alpha/beta heterodimer. Nuclear export of Upf3p is mediated by a leucine-rich nuclear export sequence (NES-A), but export is not dependent on the Crm1p exportin. Mutations identified in NES-A prevent nuclear export and confer an Nmd(-) phenotype. The addition of a functional NES element to an export-defective upf(-) allele restores export and partially restores an Nmd(+) phenotype. Our findings support a model in which the movement of Upf3p between the nucleus and the cytoplasm is required for a fully functional NMD pathway. We also found that overexpression of Upf2p suppresses the Nmd(-) phenotype in mutant strains carrying nes-A alleles but has no effect on the localization of Upf3p. To explain these results, we suggest that the mutations in NES-A that impair nuclear export cause additional defects in the function of Upf3p that are not rectified by restoration of export alone.     

CRM1-mediated nuclear export determines the cytoplasmic localization of the antiapoptotic protein Survivin

Survivin is a member of the inhibitor of apoptosis (IAP) family of negative regulators of programmed cell death that is frequently overexpressed in human tumors. Survivin is not only involved in the regulation of apoptosis, but is also known to play a role in the control of cell cycle progression at the G2/M phase. Survivin is a predominantly cytoplasmic protein expressed in a cell cycle-dependent manner, but the mechanism(s) that determine its nuclear-cytoplasmic localization have not been described. In this study, we report that Survivin is a nuclear shuttling protein that is actively exported from the nucleus via the CRM1-dependent pathway. Nuclear export of Survivin is independent of the export of other shuttling proteins that control the G2/M phase transition, such as cyclin B1 and cdc25. The carboxy-terminal domain of Survivin is both necessary and sufficient for its nuclear export, although this region does not contain a functional leucine-rich nuclear export signal. Differences in the amino acid sequence of this region determine the dramatically different localization of Survivin (in the cytoplasm) and its splicing variant Survivin-DeltaEx3 (in the nucleus). The carboxy-terminal end of Survivin-DeltaEx3 contains a bipartite nuclear localization signal, not present in Survivin, which mediates its strong nuclear accumulation. These data suggest that active transport between the nucleus and cytoplasm may constitute an important regulatory mechanism for Survivin function.