Nuclear Localization Signal Receptor Research Articles

HIV-1 nuclear import: in search of a leader.

The ability of HIV-1 to use host cell nuclear import machinery to translocate the viral preintegration complex into the cell nucleus is the critical determinant in the replication of the virus in non-dividing cells, such as macrophages. In this review, we describe the viral and cellular factors involved in this process. The available data suggest that the process of HIV-1 nuclear import is driven by interaction between nuclear localization signals (NLSs) present on viral proteins matrix and integrase and the cellular NLS receptor, karyopherin alpha. However, this interaction by itself is weak and insufficient to insure effective import of the preintegration complex. Viral protein R (Vpr) functions to increase the affinity of interaction between viral NLSs and karyopherin alpha, thus substantially enhancing the karyophilic potential of the preintegration complex. Interestingly, some cells, in particular HeLa, seem to contain a factor which can substitute for the Vpr's activity, making HIV-1 replication in such cells Vpr-independent. We also describe a class of novel anti-HIV compounds which target the NLSs of HIV-1 and effectively block viral replication in T cells and macrophages.

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

The Nuclear Localization Sequences of the BRCA1 Protein Interact with the Importin-α Subunit of the Nuclear Transport Signal Receptor

The BRCA1 gene product is a nuclear phosphoprotein that is aberrantly localized in the cytoplasm of most breast cancer cells. In an attempt to elucidate the potential mechanism for the nuclear transport of BRCA1 protein, three regions of highly charged, basic residues, 503KRKRRP508, 606PKKNRLRRKS615, and 651KKKKYN656, were identified as potential nuclear localization signals (NLSs). These three regions were subsequently mutated to 503KLP508, 607KLS615, and 651KLN656, respectively. Wild-type and mutated proteins were tagged with the flag epitope, expressed in human DU145 cells, and detected with the M2 monoclonal antibody. In DU145 cells, the KLP mutant completely fails to localize in nuclei, whereas the KLS mutant is mostly cytoplasmic with occasional nuclear localization. The KLN protein is always located in nuclei. Consistently, hSRP1alpha (importin-alpha), a component of the NLS receptor complex, was identified in a yeast two-hybrid screen using BRCA1 as the bait. The specificity of the interaction between BRCA1 and importin-alpha was further demonstrated by showing that the 503KRKRRP508 and 606PKKNRLRRKS615 regions, but not 651KKKKYN656, are critical for this interaction. To determine if the cytoplasmic mislocation of endogenous BRCA1 in breast cancer cells is due to a deficiency of the cells, wild-type BRCA1 protein tagged with the flag epitope was ectopically expressed in six breast cancer cell lines. The analysis demonstrated that, in all six, this protein localized in the cytoplasm of these cells. In contrast, expression of the construct in four non-breast cancer cell lines resulted in nuclear localization. These data support the possibility that the mislocation of the BRCA1 protein in breast cancer cells may be due to a defect in the cellular machinery involved in the NLS receptor-mediated pathway of nuclear import.

A Novel Receptor-Mediated Nuclear Protein Import Pathway

Targeting of most nuclear proteins to the cell nucleus is initiated by interaction between the classical nuclear localization signals (NLSs) contained within them and the importin NLS receptor complex. We have recently delineated a novel 38 amino acid transport signal in the hnRNP A1 protein, termed M9, which confers bidirectional transport across the nuclear envelope. We show here that M9-mediated nuclear import occurs by a novel pathway that is independent of the well-characterized, importin-mediated classical NLS pathway. Additionally, we have identified a specific M9-interacting protein, termed transportin, which binds to wild-type M9 but not to transport-defective M9 mutants. Transportin is a 90 kDa protein, distantly related to importin β, and we show that it mediates the nuclear import of M9-containing proteins. These findings demonstrate that there are at least two receptor-mediated nuclear protein import pathways. Furthermore, as hnRNP A1 likely participates in mRNA export, it raises the possibility that transportin is a mediator of this process as well.

Nuclear import in permeabilized protoplasts from higher plants has unique features.

The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.

Dynamic localization of the nuclear import receptor and its interactions with transport factors.

Characterization of the interactions between soluble factors required for nuclear transport is key to understanding the process of nuclear trafficking. Using a synthetic lethal screen with the rna1-1 strain, we have identified a genetic interaction between Rna1p, a GTPase activating protein required for nuclear transport, and yeast importin-beta, a component of the nuclear localization signal receptor. By the use of fusion proteins, we demonstrate that Rna1p physically interacts with importin-beta. Mutants in importin-beta exhibit in vivo nuclear protein import defects, and importin-beta localizes to the nuclear envelope along with other proteins associated with the nuclear pore complex. In addition, we present evidence that importin-alpha, but not importin-beta, mislocalizes to the nucleus in cells where the GTPase Ran is likely to be in the GDP-bound state. We suggest a model of nuclear transport in which Ran-mediated hydrolysis of GTP is necessary for the import of importin-alpha and the nuclear localization signal-bearing substrate into the nucleus, while exchange of GDP for GTP on Ran is required for the export of both mRNA and importin-alpha from the nucleus.

Ran Binding Domains Promote the Interaction of Ran with p97/β-Karyopherin, Linking the Docking and Translocation Steps of Nuclear Import

Nuclear protein import is accomplished by two sequential events; docking at the nuclear pore complex followed by ATP-dependent translocation across the nuclear envelope. Docking of nuclear targeted proteins requires a 56-kDa nuclear localization signal receptor (alpha-karyopherin, importin-alpha, SRP1 alpha) and a 97-kDa protein (beta-karyopherin, importin-beta). Components necessary for translocation include the Ran/TC4 GTPase and NTF2/B-2. The functions of these factors at a molecular level remain unclear. We have now found that a complex of Ran, in the GTP-bound state, with either the Ran binding protein, RanBP1, or an isolated Ran binding domain binds with high affinity and specificity to beta-karyopherin to form a ternary complex. We find that a C-terminal truncation mutant of Ran, delta-DE Ran, also binds to beta-karyopherin and that delta-DE Ran can associate with a cytosolic, multiprotein complex that contains beta-karyopherin and another delta-DE Ran binding protein of 115/120 kDa. These data suggest a physical link between docking and translocation mediated by a Ran GTPase-Ran binding protein complex.

Most Nuclear Proteins Are Imported by a Single Pathway

Using kinetic competition analysis, we show that the import of most Xenopus oocyte nuclear proteins is directed by a single family of nuclear localization signals (NLS). The transport of microinjected [35S]methionine-labeled nuclear proteins into oocyte nuclei was challenged with saturating concentrations of SV40 large T-antigen NLS peptide-BSA (P(Lys)-BSA) and was assessed by SDS-acrylamide gel electrophoresis and fluorography. P(Lys)-BSA was effective at competing the import of virtually all larger polypeptides but had a less marked effect on the mediated import of many smaller polypeptides. Wheat germ agglutinin, an inhibitor of nuclear pore complex function, enhanced the competitive effects of P(Lys)-BSA on the import of both large and small polypeptides. These data indicate that most 35 S-labeled Xenopus oocyte nuclear proteins, including nucleoplasmin, contain NLSs that are functionally related to the T-antigen NLS and probably employ the same cytosolic NLS receptor(s).

Identification of binding proteins for nuclear localization signals of the glucocorticoid and thyroid hormone receptors.

Nuclear entry of proteins the size of the glucocorticoid and thyroid hormone receptors appears to be mediated by an interaction of nuclear localization signals (NLSs) within the proteins and specific NLS-binding proteins. NLSs have been identified in the hinge region of both receptors. We have identified the cellular binding proteins of the glucocorticoid receptor NLS and the thyroid hormone receptor NLS after cross-linking of radiolabeled signal peptides to subcellular fractions. Two S49 lymphoma cytosolic polypeptides of 60 and 76 kilodaltons (kDa) were specifically bound to either the glucocorticoid or thyroid hormone receptor NLS. The two binding sites demonstrated saturable binding. A competitive binding assay showed that the binding sites were specific for NLSs and that a mutated NLS was a poor competitor for the binding of labeled glucocorticoid receptor NLS. However, competition studies with peptides unrelated to NLSs, yet resembling NLSs in that they had a net positive charge, revealed that the 60-kDa entity demonstrated greater specificity for binding to NLSs than did the 76-kDa polypeptide. Glucocorticoid receptor NLS and thyroid hormone receptor NLS-binding proteins of 60 and 76 kDa were also identified in nuclear fractions. Although the unoccupied glucocorticoid receptor resides in the cytoplasm, while the unoccupied thyroid hormone receptor is always found in the nucleus, the hinge NLS interactions do not specify these different localizations of the unoccupied receptors. Rather, the data support roles for the hinge NLS in general steps of nuclear import and the 60-kDa cross-linked product as a chaperone of both receptors into the nucleus. Its cellular localization also suggests a role for the 76-kDa cross-linked product as a chaperone, but its relatively less stringent binding specificity may indicate that this polypeptide has a different physiological function.

Identification of a human protein that interacts with nuclear localization signals.

Through a series of label transfer experiments, we have identified a HeLa cell nuclear protein that interacts with nuclear localization signals (NLSs). The protein has a molecular weight of 66,000 and an isoelectric point of approximately 6. It associates with a synthetic peptide that contains the SV-40 T antigen NLS peptide but not with an analogous peptide in which an asparagine is substituted for an essential lysine (un-NLS peptide). In addition to these peptides, several proteins have been tested as label donors. With the proteins, there is a correlation between nuclear localization (assayed with lysolecithin-permeabilized cells) and label transfer to the 66-kD protein. The NLS peptide (but not the un-NLS peptide) competes with the proteins in label transfer experiments, but neither wheat germ agglutinin nor ATP has an effect. These results suggest that the 66-kD protein functions as an NLS receptor in the first step of nuclear localization. In the course of this work, we have observed that the Staphylococcus aureus protein A is a strongly karyophilic protein. Its dramatic nuclear localization properties suggest that it may have multiple copies of an NLS.