Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters

The Ets factor Friend leukemia integration 1 (Fli-1) is an important regulator of megakaryocytic (Mk) differentiation. Here, we demonstrate two novel nuclear localization signals (NLSs) within Fli-1: one (NLS1) is located at the N terminus, and another (NLS2) is within the Ets domain. Nuclear accumulation of Fli-1 reflected the combined functional effects of the two discrete NLSs. Each NLS can independently direct nuclear transport of a carrier protein, with mutations within the NLSs affecting nuclear accumulation. NLS1 has a bipartite motif, whereas the NLS2 region contains a nonclassical NLS. Both NLSs bind importin alpha (IMPalpha) and IMPbeta, with NLS1 and NLS2 being predominantly recognized by IMPalpha and IMPbeta, respectively. Fli-1 also contains one nuclear export signal. Leptomycin B abolished its cytoplasmic accumulation, showing CRM1 dependency. We demonstrate that Ets domain binding to specific target DNA effectively blocks IMP binding, indicating that the targeted DNA binding plays a role in localizing Fli-1 to its destination and releasing IMPs for recycling back to the cytoplasm. Finally, by analyzing full-length Fli-1 carrying NLS1, NLS2, and combined NLS1-NLS2 mutations, we conclude that two functional NLSs exist in Fli-1 and that each NLS is sufficient to target Fli-1 to the nucleus for activation of Mk-specific genes.     

Efficiency of importin alpha/beta-mediated nuclear localization sequence recognition and nuclear import. Differential role of NTF2.

Little quantitative, kinetic information is available with respect to the process of nuclear import of conventional nuclear localization sequence (NLS)-containing proteins, which initially involves recognition and docking at the nuclear pore by importin alpha/beta. This study compares the binding and nuclear import properties of mouse (m) and yeast (y) importin (IMP) subunits with respect to the NLSs from the SV40 large tumor antigen (T-ag), and the Xenopus laevis phosphoprotein N1N2. m- and y-IMPalpha recognized both NLSs, with y-IMPalpha exhibiting higher affinity. m-IMPbeta greatly enhanced the binding of m-IMPalpha to the T-ag and N1N2 NLSs, but y-IMPbeta did not significantly affect the affinity of y-IMPalpha for the T-ag NLS. In contrast, y-IMPbeta enhanced y-IMPalpha binding to the NLS of N1N2, but to a lesser extent than the enhancement of m-IMPalpha binding by m-IMPbeta. NLS-dependent nuclear import was reconstituted in vitro using the different importin subunits together with the transport factors Ran and NTF2. Whereas T-ag NLS-mediated nuclear import did not exhibit an absolute requirement for NTF2, N1N2 NLS-mediated transport strictly required NTF2. High levels of NTF2 inhibited nuclear accumulation conferred by both NLSs. We conclude that different NLSs possess distinct nuclear import properties due to differences in recognition by importin and requirements for NTF2.     

A simple, solid-phase binding assay for the nuclear import receptor karyopherin alpha. Part 1: direct binding

The nuclear import receptor karyopherin alpha recognizes nuclear localization signals (NLSs), peptides that direct the transport of proteins into the nucleus. A simple, colorimetric assay has been developed to facilitate the identification and comparison of karyopherin ligands by direct and competitive binding using NLSs immobilized on the solid phase (TentaGel resin).     

A role for nucleosome assembly protein 1 in the nuclear transport of histones H2A and H2B

Import of core histones into the nucleus is a prerequisite for their deposition onto DNA and the assembly of chromatin. Here we demonstrate that nucleosome assembly protein 1 (Nap1p), a protein previously implicated in the deposition of histones H2A and H2B, is also involved in the transport of these two histones. We demonstrate that Nap1p can bind directly to Kap114p, the primary karyopherin/importin responsible for the nuclear import of H2A and H2B. Nap1p also serves as a bridge between Kap114p and the histone nuclear localization sequence (NLS). Nap1p acts cooperatively to increase the affinity of Kap114p for these NLSs. Nuclear accumulation of histone NLS-green fluorescent protein (GFP) reporters was decreased in deltanap1 cells. Furthermore, we demonstrate that Nap1p promotes the association of the H2A and H2B NLSs specifically with the karyopherin Kap114p. Localization studies demonstrate that Nap1p is a nucleocytoplasmic shuttling protein, and genetic experiments suggest that its shuttling is important for maintaining chromatin structure in vivo. We propose a model in which Nap1p links the nuclear transport of H2A and H2B to chromatin assembly.     

Transportins 1 and 2 are redundant nuclear import factors for hnRNP A1 and HuR

Several mRNA-binding proteins, including hnRNP A1 and HuR, contain bidirectional transport signals that mediate both their nuclear import and export. Previously, Transportin 1 (Trn1) was identified as a mediator of hnRNP A1 import, whereas the closely related protein Transportin 2 (Trn2) was shown to interact with HuR. Here we have investigated the subfamily of transportins that consists of Trn1 (or Kap beta2A) and two alternatively spliced Trn2 isoforms (Trn2a and Trn2b), also called Trn2 and Kap beta2B. The sequence differences among these proteins could alter either their cargo specificity or their response to RanGTP and thus their function as import or export receptors. Using in vitro binding assays, we show that hnRNP A1 preferentially binds Trn1 and Trn2b versus Trn2a. HuR interacts with all three transportins, as well as weakly with Imp beta. The hnRNP A1 and HuR shuttling domains, called M9 and HNS, respectively, are sufficient for these interactions. Despite small differences in the binding of HuR and hnRNP A1 to the three transportins, in vitro interaction studies performed in the presence and absence of RanQ69LGTP indicate that all three transportins most likely act as import factors for HuR and hnRNP A1. In digitonin-permeabilized HeLa cells, both M9 and HNS peptides compete for the import of recombinant hnRNP A1 and HuR, indicating that HuR and hnRNP A1 import pathways are at least partially overlapping. Possible nucleocytoplasmic shuttling mechanisms for hnRNP A1 and HuR are discussed.     

Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion

The high mobility group box 1 (HMGB1) protein can be secreted by activated monocytes and macrophages and functions as a late mediator of sepsis. HMGB1 contains two nuclear localization signals (NLSs) for controlled nuclear transport, and acetylation of both NLSs of HMGB1 is involved in nuclear transport toward secretion. However, phosphorylation of HMGB1 and its relation to nuclear transport have not been shown. We show here that HMGB1 is phosphorylated and dynamically shuttled between cytoplasmic and nuclear compartments according to its phosphorylation state. Phosphorylation of HMGB1 was detected by metabolic labeling and Western blot analysis after treatments with TNF-alpha and okadaic acid, a phosphatase inhibitor. Hyperphosphorylated HMGB1 in RAW 264.7 and human monocytes was relocated to the cytoplasm. In a nuclear import assay, phosphorylated HMGB1 in the cytoplasm did not enter the nucleus. We mutated serine residues of either or both NLSs of HMGB1 to glutamic acid to simulate a phosphorylated state and examined the binding of HMGB1 to karyopherin-alpha1, which was identified as the nuclear import protein for HMGB1 in this study. Substitution to glutamic acid in either NLSs decreased the binding with karyopherin-alpha1 by approximately 50%; however, substitution of both NLSs showed no binding, and HMGB1 was relocated to the cytoplasm and subsequently secreted. These data support the hypothesis that HMGB1 could be phosphorylated and that the direction of transport is regulated by phosphorylation of both NLS regions.     

An unconventional NLS is critical for the nuclear import of the influenza A virus nucleoprotein and ribonucleoprotein

Replication of the RNAs of influenza virus occurs in the nucleus of infected cells. The nucleoprotein (NP) has been shown to be important for the import of the viral RNA into the nucleus and has been proposed to contain at least three different nuclear localization signals (NLSs). Here, an import assay in digitonin-permeabilized cells was used to further define the contribution of these NLSs. Mutation of the unconventional NLS impaired the nuclear import of the NP. A peptide bearing the unconventional NLS could inhibit the nuclear import of the NP in this import assay and prevent the NP-karyopherin alpha interaction in a binding assay confirming the crucial role of this signal. Interestingly, a peptide containing the SV40 T antigen NLS was unable to inhibit the nuclear import of NP or the NP-karyopherin alpha interaction, suggesting that the NP and the SV40 T antigen do not share a common binding site on karyopherin alpha. We also investigated the question of which NLS(s) is/are necessary for the viral ribonucleoprotein complex to enter the nucleus. We found that the peptide containing the unconventional NLS efficiently inhibited the nuclear import of the ribonucleoprotein complexes. This finding suggests that the unconventional NLS is the major signal necessary not only for the nuclear transport of free NP but also for the import of the ribonucleoprotein complexes. Finally, viral replication could be specifically inhibited by a membrane-permeable peptide containing the unconventional NLS, confirming the crucial role of this signal during the replicative cycle of the virus.     

Structural basis for the specificity of bipartite nuclear localization sequence binding by importin-alpha

Importin-alpha is the nuclear import receptor that recognizes cargo proteins carrying conventional basic monopartite and bipartite nuclear localization sequences (NLSs) and facilitates their transport into the nucleus. Bipartite NLSs contain two clusters of basic residues, connected by linkers of variable lengths. To determine the structural basis of the recognition of diverse bipartite NLSs by mammalian importin-alpha, we co-crystallized a non-autoinhibited mouse receptor protein with peptides corresponding to the NLSs from human retinoblastoma protein and Xenopus laevis phosphoprotein N1N2, containing diverse sequences and lengths of the linker. We show that the basic clusters interact analogously in both NLSs, but the linker sequences adopt different conformations, whereas both make specific contacts with the receptor. The available data allow us to draw general conclusions about the specificity of NLS binding by importin-alpha and facilitate an improved definition of the consensus sequence of a conventional basic/bipartite NLS (KRX10-12KRRK) that can be used to identify novel nuclear proteins.     

Nuclear localization signal and protein context both mediate importin alpha specificity of nuclear import substrates

The "classical" nuclear protein import pathway depends on importin alpha and importin beta. Importin alpha binds nuclear localization signal (NLS)-bearing proteins and functions as an adapter to access the importin beta-dependent import pathway. In humans, only one importin beta is known to interact with importin alpha, while six alpha importins have been described. Various experimental approaches provided evidence that several substrates are transported specifically by particular alpha importins. Whether the NLS is sufficient to mediate importin alpha specificity is unclear. To address this question, we exchanged the NLSs of two well-characterized import substrates, the seven-bladed propeller protein RCC1, preferentially transported into the nucleus by importin alpha3, and the less specifically imported substrate nucleoplasmin. In vitro binding studies and nuclear import assays revealed that both NLS and protein context contribute to the specificity of importin alpha binding and transport.     

An ATP-dependent, Ran-independent mechanism for nuclear import of the U1A and U2B" spliceosome proteins

Nuclear import of the two uracil-rich small nuclear ribonucleoprotein (U snRNP) components U1A and U2B" is mediated by unusually long and complex nuclear localization signals (NLSs). Here we investigate nuclear import of U1A and U2B" in vitro and demonstrate that it occurs by an active, saturable process. Several lines of evidence suggest that import of the two proteins occurs by an import mechanism different to those characterized previously. No cross competition is seen with a variety of previously studied NLSs. In contrast to import mediated by members of the importin-beta family of nucleocytoplasmic transport receptors, U1A/U2B" import is not inhibited by either nonhydrolyzable guanosine triphosphate (GTP) analogues or by a mutant of the GTPase Ran that is incapable of GTP hydrolysis. Adenosine triphosphate is capable of supporting U1A and U2B" import, whereas neither nonhydrolyzable adenosine triphosphate analogues nor GTP can do so. U1A and U2B" import in vitro does not require the addition of soluble cytosolic proteins, but a factor or factors required for U1A and U2B" import remains tightly associated with the nuclear fraction of conventionally permeabilized cells. This activity can be solubilized in the presence of elevated MgCl(2). These data suggest that U1A and U2B" import into the nucleus occurs by a hitherto uncharacterized mechanism.     

FUS-NLS/Transportin 1 Complex Structure Provides Insights into the Nuclear Targeting Mechanism of FUS and the Implications in ALS

The C-terminal nuclear localization sequence of FUsed in Sarcoma (FUS-NLS) is critical for its nuclear import mediated by transportin (Trn1). Familial amyotrophic lateral sclerosis (ALS) related mutations are clustered in FUS-NLS. We report here the structural, biochemical and cell biological characterization of the FUS-NLS and its clinical implications. The crystal structure of the FUS-NLS/Trn1 complex shows extensive contacts between the two proteins and a unique α-helical structure in the FUS-NLS. The binding affinity between Trn1 and FUS-NLS (wide-type and 12 ALS-associated mutants) was determined. As compared to the wide-type FUS-NLS (K_D = 1.7 nM), each ALS-associated mutation caused a decreased affinity and the range of this reduction varied widely from 1.4-fold over 700-fold. The affinity of the mutants correlated with the extent of impaired nuclear localization, and more importantly, with the duration of disease progression in ALS patients. This study provides a comprehensive understanding of the nuclear targeting mechanism of FUS and illustrates the significance of FUS-NLS in ALS.     

The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin beta 1

The sex-determining factor SRY is a DNA-binding protein that diverts primordial gonads from the ovarian pathway toward male differentiation to form testes. It gains access to the nucleus through two distinct nuclear localization signals (NLSs) that flank the high mobility group (HMG) DNA-binding domain, but the mechanisms through which these NLSs operate have not been studied. In this study, we reconstitute the nuclear import of SRY in vitro, demonstrating a lack of requirement for exogenous factors for nuclear accumulation and a significant reduction in nuclear transport in the presence of antibodies to importin beta but not importin alpha. Using a range of quantitative binding assays including enzyme-linked immunosorbent assay, fluorescence polarization, and native gel mobility electrophoresis, we assess the binding of importins to SRY, demonstrating a high affinity recognition (in the low nm range) by Imp beta independent of Imp alpha. In assessing the contribution of each NLS, we found that the N-terminal NLS was recognized poorly by importins, whereas the C-terminal NLS was bound by importin beta with similar affinity to SRY. We also found that RanGTP, but not RanGDP, could dissociate the SRY-importin beta complex in solution using FP. We describe a novel double-fluorescent label DNA binding assay to demonstrate mutual exclusivity between importin beta recognition and DNA binding on the part of SRY, which may represent an alternative release mechanism upon nuclear entry. This study represents the first characterization of the nuclear import pathway for a HMG domain-containing protein. Importantly, it demonstrates for the first time that recognition of SRY by Imp beta is of comparable affinity to that with which Imp alpha/beta recognizes conventional NLS-containing substrates.     

Importin beta recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin alpha

Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin beta and not by importin alpha. The sequence of PTHrP responsible for binding was mapped to amino acids 66-94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of approximately 2 nM for importin beta). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin beta, together with the GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin alpha, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin alpha and perhaps also of NTF2.     

Structural Characterisation of the Nuclear Import Receptor Importin Alpha in Complex with the Bipartite NLS of Prp20

The translocation of macromolecules into the nucleus is a fundamental eukaryotic process, regulating gene expression, cell division and differentiation, but which is impaired in a range of significant diseases including cancer and viral infection. The import of proteins into the nucleus is generally initiated by a specific, high affinity interaction between nuclear localisation signals (NLSs) and nuclear import receptors in the cytoplasm, and terminated through the disassembly of these complexes in the nucleus. For classical NLSs (cNLSs), this import is mediated by the importin-α (IMPα) adaptor protein, which in turn binds to IMPβ to mediate translocation of nuclear cargo across the nuclear envelope. The interaction and disassembly of import receptor:cargo complexes is reliant on the differential localisation of nucleotide bound Ran across the envelope, maintained in its low affinity, GDP-bound form in the cytoplasm, and its high affinity, GTP-bound form in the nucleus. This in turn is maintained by the differential localisation of Ran regulating proteins, with RanGAP in the cytoplasm maintaining Ran in its GDP-bound form, and RanGEF (Prp20 in yeast) in the nucleus maintaining Ran in its GTP-bound form. Here, we describe the 2.1 Å resolution x-ray crystal structure of IMPα in complex with the NLS of Prp20. We observe 1,091 Ų of buried surface area mediated by an extensive array of contacts involving residues on armadillo repeats 2-7, utilising both the major and minor NLS binding sites of IMPα to contact bipartite NLS clusters ¹⁷RAKKMSK²³ and ³KR⁴, respectively. One notable feature of the major site is the insertion of Prp20NLS Ala¹⁸ between the P0 and P1 NLS sites, noted in only a few classical bipartite NLSs. This study provides a detailed account of the binding mechanism enabling Prp20 interaction with the nuclear import receptor, and additional new information for the interaction between IMPα and cargo.     

NLSdb: database of nuclear localization signals

NLSdb is a database of nuclear localization signals (NLSs) and of nuclear proteins. NLSs are short stretches of residues mediating transport of nuclear proteins into the nucleus. The database contains 114 experimentally determined NLSs that were obtained through an extensive literature search. Using 'in silico mutagenesis' this set was extended to 308 experimental and potential NLSs. This final set matched over 43% of all known nuclear proteins and matches no currently known non-nuclear protein. NLSdb contains over 6000 predicted nuclear proteins and their targeting signals from the PDB and SWISS-PROT/TrEMBL databases. The database also contains over 12 500 predicted nuclear proteins from six entirely sequenced eukaryotic proteomes (Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cerevisiae). NLS motifs often co-localize with DNA-binding regions. This observation was used to also annotate over 1500 DNA-binding proteins. NLSdb can be accessed via the web site: http://cubic.bioc.columbia.edu/db/NLSdb/.     

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.