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Results for “Nuclear Localization Signals”

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PubMed2000

Two nuclear localization signals in the HIV-1 matrix protein regulate nuclear import of the HIV-1 pre-integration complex.

Replication of HIV-1 in non-dividing and slowly proliferating cell populations depends on active import of the viral pre-integration complex (PIC) into the cell nucleus. While it is commonly accepted that this process is mediated by an interaction between the HIV-1 PIC and the cellular nuclear import machinery, controversial results have been reported concerning the mechanisms of this interaction. Here, we demonstrate that a recently identified nuclear localization signal within the HIV-1 matrix protein (MA), MA NLS-2, together with previously described MA NLS-1, mediates nuclear import of the HIV-1 PIC. Inactivation of both MA NLSs precluded nuclear translocation of MA and rendered the virus defective in nuclear import and replication in non-dividing macrophage cultures, even when functional Vpr and integrase (IN), two more viral proteins implicated in HIV-1 nuclear import, were present. Taken together, these results indicate that Vpr does not function as an independent nuclear import factor and demonstrate that HIV-1 MA, by virtue of its two nuclear localization signals, regulates HIV-1 nuclear import.

Amino Acid Sequence↗

PubMed2004

Phosphorylation of nuclear localization signal inhibits the ligand-dependent nuclear import of aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor which plays a role as an intracellular mediator of the xenobiotic signaling pathway. We previously identified the minimum nuclear localization signal (NLS) of AhR(13-39): it is composed of two basic amino acid segments, AhR(13-16:RKRR) and AhR(37-39:KRH). In this study, we showed that the two protein kinase C (PKC) sites of Ser-12 and Ser-36 are located one amino acid upstream from each of the two segments, and that a ligand-dependent nuclear import of AhR is inhibited by substitution of aspartic acid for Ser-12 (S12D) or Ser-36 (S36D), which mimics the negative charge of phosphorylation. This observation was supported by microinjection analysis, an in vitro nuclear transport assay, and a luciferase reporter assay, suggesting a two-step mechanism in the ligand-dependent nuclear translocation of AhR.

Active Transport, Cell Nucleus↗

PubMed1995

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.

BACKGROUND: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS: We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS: Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Amino Acid Sequence↗

PubMed2003

[Influence of HBV gene immunization by introduction of nuclear localization signal into eukaryonized T7 expression system].

To investigate the effects of nuclear localization signal on immune response induced by eukaryonized T7 expression system. Nuclear localization signal (NLS) coding sequence was introduced into eukaryonized T7 expression system containing HBV M protein coding gene. Naked gene immunization was performed. The results showed that the expression efficiency of T7 system which containing nuclear localization signal in vitro was lower than that without NLS. In vivo study found that HBV specific immune response induced by NLS lacking eukaryonized T7 system was significantly higher than that in eukaryonized T7 expression system containing NLS. HBV specific Th1 and Th2 type immune responses were also observed in the mice immunized with T7-HBsAg plasmid lacking NLS, indicating that the nuclear localization signal might not be essential in T7 expression system.

Animals↗

PubMed1997

Identification of a novel nuclear localization signal in Sam68.

Sam68, a nuclear RNA binding protein, binds to Src and is phosphorylated at tyrosine residues in an M-phase specific manner. Here we identified a stretch of 24 amino acid residues in the COOH-terminal portion of Sam68 which function as a nuclear localization signal. This signal sequence bears no apparent homology to any other known nuclear localization sequence. However, this sequence was found to contain a motif, PPXXR (P, Pro; R, Arg), which is conserved in various RNA binding proteins including hnRNP proteins. Replacement of Arg in this motif with Ala abolished the nuclear accumulation of a GFP fusion protein, suggesting that this residue is important in translocating the protein to the nucleus.

3T3 Cells↗

PubMed2006

Staufen1 is imported into the nucleolus via a bipartite nuclear localization signal and several modulatory determinants.

Mammalian Stau1 (Staufen1), a modular protein composed of several dsRBDs (double-stranded RNA-binding domains), is probably involved in mRNA localization. Although Stau1 is mostly described in association with the rough endoplasmic reticulum and ribosomes in the cytoplasm, recent studies suggest that it may transit through the nucleus/nucleolus. Using a sensitive yeast import assay, we show that Stau1 is actively imported into the nucleus through a newly identified bipartite nuclear localization signal. As in yeast, the bipartite nuclear localization signal is necessary for Stau1 nuclear import in mammalian cells. It is also required for Stau1 nucleolar trafficking. However, Stau1 nuclear transit seems to be regulated by mechanisms that involve cytoplasmic retention and/or facilitated nuclear export. Cytoplasmic retention is mainly achieved through the action of dsRBD3, with dsRBD2 playing a supporting role in this function. Similarly, dsRBD3, but not its RNA-binding activity, is critical for Stau1 nucleolar trafficking. The function of dsRBD3 is strengthened or stabilized by the presence of dsRBD4 but prevented by the interdomain between dsRBD2 and dsRBD3. Altogether, these results suggest that Stau1 nuclear trafficking is a highly regulated process involving several determinants. The presence of Stau1 in the nucleus/nucleolus suggests that it may be involved in ribonucleoprotein formation in the nucleus and/or in other nuclear functions not necessarily related to mRNA transport.

Amino Acid Sequence↗

PubMed1990

Homeodomain of yeast repressor alpha 2 contains a nuclear localization signal.

The yeast repressor alpha 2 is shown, by analysis of deletion-bearing alpha 2-beta-galactosidase hybrid proteins, to have two structurally distinct nuclear localization signals. The cellular location of hybrid proteins was determined by indirect immunofluorescence and optical sectioning of whole fixed yeast cells. The two nuclear localization signals are far apart in the alpha 2 primary structure and do not have any sequence homology. One signal is, as reported previously, within the aminoterminal 13 amino acids of alpha 2. Deletion of only this aminoterminal signal has no evident effect on nuclear localization. The second signal is in a central portion of alpha 2, within the alpha 2 homeodomain. Since this signal is within the amino terminus of the alpha 2 homeodomain, the homeodomain mediates nuclear localization in addition to, and independently of, DNA binding. Deletion of only this second signal results in inefficient localization and accumulation of mutant protein at discrete sites on the nuclear envelope assumed to be nuclear pores. We propose that the two signals in alpha 2 are functionally distinct and act at different steps in a localization pathway.

Base Sequence↗

PubMed2003

Antibody fragments selected by phage display against the nuclear localization signal of the HIV-1 Vpr protein inhibit nuclear import in permeabilized and intact cultured cells.

The HIV-1 Vpr protein harbors a nuclear localization signal in its N-terminal domain. A peptide bearing this domain and which is designated VprN has been used as a target to screen a phage display single chain Fv (scFv) library. Here we report the isolation of anti-VprN scFv fragments from this library. The purified scFv fragments were able to bind the VprN peptide in an ELISA-based system and to inhibit VprN-mediated nuclear import in permeabilized as well as in intact microinjected cells. Furthermore, the anti-VprN scFv fragments recognized the full-length recombinant Vpr protein and inhibited its nuclear import. The same scFv fragments did not inhibit nuclear import mediated by the nuclear localization signal of the SV40 large T-antigen demonstrating a specific effect. The use of the described inhibitory anti-VprN scFv fragments to study nuclear import of viral karyophilic proteins and their therapeutic potential is discussed.

Active Transport, Cell Nucleus↗

PubMed2002

Molecular basis for the recognition of a nonclassical nuclear localization signal by importin beta.

Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.

Active Transport, Cell Nucleus↗

PubMed1995

Two conserved serines in the nuclear localization signal flanking region are involved in the nuclear targeting of human lamin A.

The nuclear lamins are karyophilic proteins located at the nucleoplasmic surface of the inner nuclear membrane. We have constructed mutants immediately N-terminal to the nuclear localization signal of human lamin A to identify sites regulating the nuclear transport of the protein. Using an in vitro transport assay, we determined the short-term kinetics of nucleocytoplasmic transport of wild type and mutant proteins. The double mutation of two putative protein kinase C sites (serine 403/404-->alanine) reduced the rate of nuclear import for the mutant protein. Inhibition of phosphorylation in wild type lamin A by the specific protein kinase C inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) and staurosporine or treatment with acid or alkaline phosphatase decreased the nuclear import of the protein. We suggest that transport of human lamin A into the nucleus is regulated by phosphorylations of protein kinase C sites in the sequence N-terminal to the nuclear localization signal.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1998

Functional characterization of a plant importin alpha homologue. Nuclear localization signal (NLS)-selective binding and mediation of nuclear import of nls proteins in vitro.

Nuclear import of most nuclear proteins is initiated by recognition of the nuclear localization signal (NLS) by importin alpha. We recently isolated an importin alpha homologue from rice (rice importin alpha1) and demonstrated that transcription of the gene is down-regulated by light in rice leaves. To address the function of rice importin alpha1 in the process of nuclear import of proteins, we performed in vitro binding and nuclear import assays. The rice importin alpha1 showed specific binding to fusion proteins containing either monopartite or bipartite NLSs, but not to a fusion protein containing a Matalpha-2-type NLS, suggesting that there exists selective binding of rice importin alpha1 to different plant NLSs. The rice importin alpha1 is also capable of forming a complex with mouse importin beta and NLS protein in vitro. An in vitro nuclear import assay using permeabilized HeLa cells revealed that rice importin alpha1, in conjunction with other vertebrate transport factors, mediates the nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data provide strong, direct evidence suggesting that rice importin alpha1 functions as a component of the NLS receptor in plant cells.

Animals↗

PubMed1997

Characterization of a Drosophila phosphorylation-dependent nuclear-localization-signal-binding protein.

A 94 kDa nuclear-localization-signal (NLS)-binding protein was purified from Drosophila embryos. The NLS of the simian-virus-40 T-antigen is specifically bound by the dephosphorylated form of the protein. After phosphorylation, the affinity of the protein for the NLS is sharply decreased. In the dephosphorylated form, p94 (protein of 94 kDa) is the major NLS-binding protein in Drosophila embryos. Immunoprecipitation confirmed the ATP-dependent phosphorylation of p94, and co-precipitation of two additional phosphorylated proteins, indicated that the NLS-binding protein is part of a larger complex in Drosophila embryos. In agreement with the immunoprecipitation results, cross-linking experiments demonstrated the interaction of p94 with three additional proteins. These protein-protein interactions were also phosphorylation-dependent.

Adenosine Triphosphate↗

PubMed2005

A serine/threonine-rich motif is one of three nuclear localization signals that determine unidirectional transport of the mineralocorticoid receptor to the nucleus.

The mineralocorticoid receptor (MR) is a tightly regulated nuclear hormone receptor that selectively transmits corticosteroid signals. Steroid treatment transforms MR from a transcriptionally inert state, in which it is distributed equally between the nucleus and cytoplasm, to an active completely nuclear transcription factor. We report here that MR is an atypical nuclear hormone receptor that moves unidirectionally from the cytoplasm to the nucleus. We show that nuclear import of MR is controlled through three nuclear localization signals (NLSs) of distinct types. Nuclear localization of naive MR was mediated primarily through a novel serine/threonine-rich NLS (NL0) in the receptor N terminus. Specific amino acid substitutions that mimicked phosphorylation selectively enhanced or repressed NL0 activity, highlighting the potential for active regulation of this new type of NLS. The second NLS (NL2) within the ligand-binding domain also lacks a recognizable basic motif. Nuclear transfer through this signal was strictly dependent on steroid agonist, but was independent of the interaction of MR with coactivator proteins. The third MR NLS (NL1) is a bipartite basic motif localized to the C terminus of the MR DNA-binding domain with properties distinct from those of NL1 of the closely related glucocorticoid receptor. NL1 acted in concert with NL0 and NL2 to stimulate nuclear uptake of the agonist-treated receptor, but also directed the complete nuclear localization of MR in response to treatment with steroid antagonist. These results present MR as a nuclear hormone receptor whose unidirectional transfer to the nucleus may be regulated through multiple pathways.

Active Transport, Cell Nucleus↗

PubMed2005

Nucleocytoplasmic shuttling of the rabies virus P protein requires a nuclear localization signal and a CRM1-dependent nuclear export signal.

Rabies virus P protein is a co-factor of the viral RNA polymerase. It has been shown previously that P mRNA directs the synthesis of four N-terminally truncated P products P2, P3, P4, and P5 due to translational initiation by a leaky scanning mechanism at internal Met codons. Whereas P and P2 are located in the cytoplasm, P3, P4, and P5 are found in the nucleus. Here, we have analyzed the molecular basis of the subcellular localization of these proteins. Using deletion mutants fused to GFP protein, we show the presence of a nuclear localization signal (NLS) in the C-terminal part of P (172-297). This domain contains a short lysine-rich stretch ((211)KKYK(214)) located in close proximity with arginine 260 as revealed by the crystal structure of P. We demonstrate the critical role of lysine 214 and arginine 260 in NLS activity. In the presence of Leptomycin B, P is retained in the nucleus indicating that it contains a CRM1-dependent nuclear export signal (NES). The subcellular distribution of P deletion mutants indicates that the domain responsible for export is the amino-terminal part of the protein. The use of fusion proteins that have amino terminal fragments of P fused to beta-galactosidase containing the NLS of SV40 T antigen allows us to identify a NES between residues 49 and 58. The localization of NLS and NES determines the cellular distribution of the P gene products.

Active Transport, Cell Nucleus↗

PubMed1997

Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1.

Nuclear protein import requires a nuclear localization signal (NLS) receptor and at least three other cytoplasmic factors. The alpha subunit of the NLS receptor, Rag cohort 1 (Rch1), enters the nucleus, probably in a complex with the beta subunit of the receptor, as well as other import factors and the import substrate. To learn more about which factors and/or events end the import reaction and how the import factors return to the cytoplasm, we have studied nucleocytoplasmic shuttling of Rch1 in vivo. Recombinant Rch1 microinjected into Vero or tsBN2 cells was found primarily in the cytoplasm. Rch1 injected into the nucleus was rapidly exported in a temperature-dependent manner. In contrast, a mutant of Rch1 lacking the first 243 residues accumulated in the nuclei of Vero cells after cytoplasmic injection. After nuclear injection, the truncated Rch1 was retained in the nucleus, but either Rch1 residues 207-217 or a heterologous nuclear export signal, but not a mutant form of residues 207-217, restored nuclear export. Loss of the nuclear transport factor RCC1 (regulator of chromosome condensation) at the nonpermissive temperature in the thermosensitive mutant cell line tsBN2 caused nuclear accumulation of wild-type Rch1 injected into the cytoplasm. However, free Rch1 injected into nuclei of tsBN2 cells at the nonpermissive temperature was exported. These results suggested that RCC1 acts at an earlier step in Rch1 recycling, possibly the disassembly of an import complex that contains Rch1 and the import substrate. Consistent with this possibility, incubation of purified RanGTP and RCC1 with NLS receptor and import substrate prevented assembly of receptor/substrate complexes or stimulated their disassembly.

Animals↗

PubMed1998

A constitutive nuclear localization signal from the second zinc-finger of orphan nuclear receptor TR2.

The orphan nuclear receptor TR2 and its truncated isoform deleted in the ligand binding domain (LBD) were localized exclusively in the nuclei as revealed by two methods of detection. An anti-hemagglutinin (HA) antibody detected specific nuclear localization of HA-tagged receptors and the green fluorescent protein (GFP)-tagged receptors were found to be distributed in the nuclei of living cells. By deletion analyses, the sequence responsible for targeting this receptor into the nucleus was defined. A stretch of 20 amino acid residues (KDCVINKHHRNRCQYCRLQR) within the second zinc-finger of this receptor is required for its nuclear localization and this signal is constitutively active. No nuclear localization signal was found in the N-terminus or the LBD. The GFP-tagged receptor remained biologically active, as evidenced by its repressive activity on the reporter that carried a binding site for this receptor, a direct repeat-5 (DR5). An electrophoretic mobility shift assay was performed to characterize the binding property of TR2 and its truncated isoform. TR2 bound to the DR5 as dimers whereas its truncated isoform bound as monomers.

Animals↗

PubMed1992

Nuclear localization signals, but not putative leucine zipper motifs, are essential for nuclear transport of hepatitis delta antigen.

Hepatitis delta antigen (HDAg) is the only known protein of hepatitis delta virus and was previously shown to localize in the nucleoplasm of infected liver cells. In this study, nuclear localization signals of HDAg were defined by expressing various domains of the antigen in both hepatic and nonhepatic cells as beta-galactosidase fusion proteins. A cytochemical staining assay demonstrated that a domain from amino acid residues 35 to 88 of HDAg was able to facilitate transport to the nucleus of the originally cytoplasm-localized protein beta-galactosidase. Two nuclear localization signals, NLS1 and NLS2, which are similar to those of simian virus 40 T antigen and polyomavirus T antigen, respectively, were identified. Either NLS1 or NLS2 alone was sufficient for the nuclear transport of HDAg. However, a fusion protein (N65Z) containing beta-galactosidase and the N-terminal 65 amino acids of HDAg, containing NLS1, was localized exclusively in the cytoplasm and perinuclear region. A possible hydrophobic subdomain between amino acid residues 50 and 65 may block the function of NLS1. Nevertheless, N65Z could enter the nuclei of transfected cells when it was coexpressed with full-length HDAg. Entry into the nucleus may be mediated by the coiled-coil structure rather than the putative leucine zipper motif located between amino acid residues 35 and 65. The existence of two independent nuclear localization signals may ensure the proper functioning of HDAg in the multiplication of delta virus in the nucleus. In addition, two putative casein kinase II sites (SRSE-5 and SREE-126) that may be important in controlling the rate of nuclear transport were found in HDAg.

Amino Acid Sequence↗

PubMed1991

A yeast protein that binds nuclear localization signals: purification localization, and antibody inhibition of binding activity.

Short stretches of amino acids, termed nuclear localization sequences (NLS), can mediate assembly of proteins into the nucleus. Proteins from the yeast, Saccharomyces cerevisiae, have been identified that specifically recognize nuclear localization peptides (Silver, P., I. Sadler, and M. A. Osborne. 1989. J. Cell Biol. 109:983-989). We now further define the role of one of these NLS-binding proteins in nuclear protein localization. The NLS-binding protein of 70-kD molecular mass can be purified from salt extracts of nuclei. Antibodies raised against the NLS-binding protein localized the protein mainly to the nucleus with minor amounts in the cytoplasm. These antibodies also inhibited the association of NLS-protein conjugates with nuclei. Incubation of nuclei with proteases coupled to agarose removed NLS-binding protein activity. Extracts enriched for NLS-binding proteins can be added back to salt or protease-treated nuclei to restore NLS-binding activity. These results suggest that the first step of nuclear protein import can be reconstituted in vitro.

Amino Acid Sequence↗