SEARCH · Search PubMed

Results for “nuclear localization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 127 records · Page 7Linked to original sources

PubMed2000

Functional characterization of nuclear localization signals in yeast Sm proteins.

In mammals, nuclear localization of U-snRNP particles requires the snRNA hypermethylated cap structure and the Sm core complex. The nature of the signal located within the Sm core proteins is still unknown, both in humans and yeast. Close examination of the sequences of the yeast SmB, SmD1, and SmD3 carboxyl-terminal domains reveals the presence of basic regions that are reminiscent of nuclear localization signals (NLSs). Fluorescence microscopy studies using green fluorescent protein (GFP)-fusion proteins indicate that both yeast SmB and SmD1 basic amino acid stretches exhibit nuclear localization properties. Accordingly, deletions or mutations in the NLS-like motifs of SmB and SmD1 dramatically reduce nuclear fluorescence of the GFP-Sm mutant fusion alleles. Phenotypic analyses indicate that the NLS-like motifs of SmB and SmD1 are functionally redundant: each NLS-like motif can be deleted without affecting yeast viability whereas a simultaneous deletion of both NLS-like motifs is lethal. Taken together, these findings suggest that, in the doughnut-like structure formed by the Sm core complex, the carboxyl-terminal extensions of Sm proteins may form an evolutionarily conserved basic amino acid-rich protuberance that functions as a nuclear localization determinant.

Alleles↗

PubMed2004

Multiple nuclear localization sequences allow modulation of 5-lipoxygenase nuclear import.

The nuclear import of proteins typically requires the presence of a nuclear localization sequence (NLS). Some proteins have more than one NLS, but the significance of having multiple NLSs is unclear. The enzyme 5-lipoxygenase (5-LO) has three NLSs that, unlike the tight cluster of basic residues of the classical SV40 large T antigen NLS, contain dispersed basic residues. When attached to green fluorescent protein (GFP), individual 5-LO NLSs caused quantitatively and statistically less import than the SV40 NLS. Combined 5-LO NLSs produced nuclear import that was comparable to that of the SV40 NLS. As expected, GFP/NLS proteins displayed relatively uniform import in all cells. However, a fusion protein of GFP plus the 5-LO protein, modified to contain only one functional NLS, produced some cells with import and some cells without import. A GFP/5-LO fusion protein containing two functional NLSs produced four identifiable levels of nuclear import. Quantitative and visual analysis of a population of cells expressing the intact GFP/5-LO protein, with three intact NLSs, indicated five levels of nuclear import. This suggested that the subcellular distribution of 5-LO may vary widely in normal cells of the body. Consistent with this, immunohistochemical staining of lung sections found that individual macrophages, in situ, displayed cell-specific levels of import of 5-LO. Since nuclear accumulation is known to affect 5-LO activity, multiple NLSs may allow graded regulation of activity via controlled import. Multiple NLSs on other proteins may likewise allow fine control of protein action through modulation of the level of import.

Active Transport, Cell Nucleus↗

PubMed2005

Characterization of nuclear localization signals and cytoplasmic retention region in the nuclear receptor CAR.

The constitutive androstane receptor (CAR) is a ligand/activator-dependent transactivation factor that resides in the cytoplasm and forms part of an as yet unidentified protein complex. Upon stimulation, CAR translocates into the nucleus where it modulates the transactivation of target genes. However, CAR exogenously expressed in rat liver RL-34 cells is located in the nucleus even in the absence of activators. By transiently transfecting RL-34 cells with various mutated rat CAR segments, we identified two nuclear localization signals: a basic amino acid-rich sequence (RRARQARRR) between amino acids 100 and 108; and an assembly of noncontiguous residues widely spread over amino acid residues 111 to 320 within the ligand binding domain. A C-terminal leucine-rich segment corresponding to a previously reported murine xenochemical response signal was not found to exhibit nuclear import activity in cultured cells. Using rat primary hepatocytes transfected with various CAR segments, we identified the region required for the cytoplasmic retention of CAR. Based on these results, the intracellular localization of CAR would be determined by the combined effects of nuclear localization signals, the xenochemical response signal, and the cytoplasmic retention region.

Animals↗

PubMed2004

Probing essential residues for cellular uptake with a cationic nuclear localization signal sequence.

The nuclear localization signal sequence (NLS) of the transcription factor NF-kappaB is a cationic peptide with the ability to cross the cytoplasmic membrane and facilitate the delivery of attached cargo, such as DNA and proteins, to cells. Previous research had pointed to the essential role of cationic residues, therefore, the importance of residues within the NLS of NF-kappaB was evaluated for cellular uptake using an alanine replacement strategy. Although it was expected that removal of the cationic groups would have the greatest effect on membrane translocation, the most significant decreases in cellular uptake occurred with the replacement of the hydrophilic Q6 (80%) and the hydrophobic L8 (70%) residues. Replacement of the positively charged residues resulted in 30-40% decrease in cellular uptake, indicating that electrostatic interactions are not the primary driving force for membrane translocation.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Synthetic peptides as nuclear localization signals.

The nuclear envelope defines a compartment boundary which is penetrated by pores that mediate a remarkable transport process. Precursor RNAs are retained in the nucleus, while processed messenger RNA, transfer RNA and ribosomal subunits are transported to the cytoplasm. Proteins destined for the nucleus become localized soon after synthesis and again following mitosis, while cytoplasmic proteins are excluded. The process is highly specific: a single base change in vertebrate initiator tRNAMet (tRNAiMet) reduces the rate of export 20-fold; a point mutation within the simian virus 40 (SV40) large-T antigen, converting Lys 128 to Thr or Asn, prevents import. Lys 128 lies within a short 'signal' sequence which, when fused to large non-nuclear proteins, causes their accumulation in nuclei. Regions of other eukaryotic proteins also seem to contain nuclear localization signals, although a single consensus sequence has not emerged. We report here that a synthetic peptide containing 10 residues of large-T antigen sequence serves as a nuclear localization signal when cross-linked to bovine serum albumin (BSA) or immunoglobulin G (IgG) and microinjected in Xenopus oocytes. Substitution of Thr at the position of Lys 128 in this peptide renders it six- to sevenfold less effective. The uptake of peptide-linked BSA is saturable, and the rate is diminished by co-injection of free peptide. These findings are indicative of a receptor-mediated uptake process. With the use of anti-peptide antibodies, a family of proteins is revealed in nuclear but not cytoplasmic extracts of human lymphocytes which contain large-T antigen-like sequences.

Animals↗

PubMed2005

Stabilization of PML nuclear localization by conjugation and oligomerization of SUMO-3.

The PML gene of acute promyelocytic leukemia (APL) encodes a cell-growth and tumor suppressor. PML localizes to discrete nuclear bodies (NBs) that are disrupted in APL cells, resulting from a reciprocal chromosome translocation t (15;17). Here we show that the nuclear localization of PML is also regulated by SUMO-3, one of the three recently identified SUMO isoforms in human cells. SUMO-3 bears similar subcellular distribution to those of SUMO-1 and -2 in the interphase nuclear body, which is colocalized with PML protein. However, both SUMO-2 and -3 are also localized to nucleoli, a region lacking SUMO-1. Immunoprecipitated PML protein bears SUMO-3 moiety in a covalently modified form, supporting the notion that PML is conjugated by SUMO-3. To determine the functional relevance of SUMO-3 conjugation on PML molecular dynamics, we suppressed SUMO-3 protein expression using a siRNA-mediated approach. Depletion of SUMO-3 markedly reduced the number of PML-containing NBa and their integrity, which is rescued by exogenous expression of SUMO-3 but not SUMO-1 or SUMO-2. The specific requirement of SUMO-3 for PML nuclear localization is validated by expression of SUMO-3 conjugation defective mutant. Moreover, we demonstrate that oligomerization of SUMO-3 is required for PML retention in the nucleus. Taken together, our studies provide first line of evidence showing that SUMO-3 is essential for PML localization and offer novel insight into the pathobiochemistry of APL.

Blotting, Western↗

PubMed2002

Nuclear localization signal of murine CMP-Neu5Ac synthetase includes residues required for both nuclear targeting and enzymatic activity.

5-N-Acetylneuraminic acid (Neu5Ac) is the major sialic acid derivative found in animal cells. As a component of cell surface glycoconjugates, Neu5Ac is pivotal to numerous cellular recognition and communication processes including host-parasite interactions. A prerequisite for the synthesis of sialylated glycoconjugates is the activation of Neu5Ac to cytidine-monophosphate N-acetylneuraminic acid (CMP-Neu5Ac). The reaction is catalyzed by CMP-Neu5Ac-synthetase (syn), which, for unknown reasons, resides in the nucleus. Sequence analysis of the cloned murine CMP-Neu5Ac synthetase identified three clusters of basic amino acids (BC1-BC3) that might function as nuclear localization signals (NLS). In the present study chimeric protein and mutagenesis strategies were used to show that BC1 and BC2 are active NLS sequences when attached to the green fluorescent protein (enhanced GFP), but only BC2 is necessary and sufficient to mediate the nuclear import of CMP-Neu5Ac synthetase. Site-directed mutations identified the residues K(198)RXR to be essential for nuclear transport and Arg(202) to be necessary to complete the transport process. Cytoplasmic forms of CMP-Neu5Ac synthetase generated by single site mutations in BC2 demonstrated that (i) enzyme activity is independent of nuclear localization, and (ii) Arg(199) and Arg(202) are involved in both nuclear transport and synthetase activity. Comparison of all known and predicted CMP-sialic acid synthetases reveals Arg(202) and Gln(203) as highly conserved in evolution and critically important for optimal synthetase activity but not for nuclear localization. Combined, the data demonstrate that nuclear transport and enzyme activity are independent functions that share some common amino acid requirements in CMP-Neu5Ac synthetase.

3T3 Cells↗

PubMed2003

Nuclear localization of 5-lipoxygenase as a determinant of leukotriene B4 synthetic capacity.

The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation.

Active Transport, Cell Nucleus↗

PubMed2007

Nuclear localization of liver X receptor alpha and beta is differentially regulated.

Activity of nuclear receptors is regulated by their nuclear localization. Liver X receptors (LXR) alpha and beta are nuclear receptors that regulate transcription of genes for cholesterol metabolism, cholesterol transport, and lipogenesis. While LXR alpha and beta are very similar in structure and exhibit similar ligand binding properties, their physiological roles are quite different. Since the LXRs fall into a class of receptors that move between the nucleus and cytoplasm, experiments were conducted to determine whether LXR alpha and LXR beta show differences in their nuclear localization pattern. To determine the location of each receptor, cell lines stably expressing yellow fluorescent protein (YFP) chimeras with either LXR alpha or LXR beta were examined. Retention in the nucleus of the chimeric proteins in the presence or absence of ligands was assessed using fluorescence microscopy coupled with digitonin permeabilization assays. Surprisingly, differences were found between LXR alpha and LXR beta. Whereas unliganded LXR alpha was retained in the nucleus, unliganded LXR beta was partially exported. Mutations were then introduced into putative nuclear localization sequences (NLS) to determine which sequences are important for nuclear localization and function. Mutation in one such sequence abolished nuclear localization of LXR alpha, whereas the analogous change in LXR beta had a much less dramatic effect. Mutations in analogous putative NLS also differentially affected transcriptional activation by LXR alpha and LXR beta. These data demonstrate for the first time that nuclear retention and localization as well as function of LXR alpha and LXR beta are differentially regulated.

Active Transport, Cell Nucleus↗

PubMedDate not supplied

Synthesis and binding properties of oligonucleotides carrying nuclear localization sequences.

The synthesis of oligonucleotides carrying nuclear localization peptide sequences is described using two strategies: first, oligonucleotides carrying a thiol group at the 5' end were reacted with maleimido peptides; second, peptide and oligonucleotide were prepared stepwise on the same support, yielding oligonucleotide-3'-peptide conjugates. This second approach was thoroughly studied. Using amino acids and small peptides as model compounds, some side reactions were analyzed, detected, and minimized. Oligonucleotides complementary to Ha-ras gene and carrying nuclear localization peptides at the 3' and 5' ends were prepared. Melting temperature studies showed that duplexes containing nuclear localization peptides were more stable than duplexes with unmodified oligonucleotides. Moreover, oligonucleotide-peptide conjugates maintain a good mismatch discrimination when they bind to their target RNA.

Amino Acid Sequence↗

PubMed1989

Identification and characterization of a nuclear localization sequence-binding protein in yeast.

Nuclear proteins contain specific regions that are required for entry into the nucleus. Using ligand blotting, we have shown that a 67-kDa yeast nuclear envelope protein (p67) recognizes synthetic peptides containing the yeast histone H2B or simian virus 40 large tumor antigen nuclear localization sequence. Both free peptide and peptide conjugated to human serum albumin are recognized. The interaction between p67 and the nuclear localization sequences is specific; neither a mutant peptide that is incompetent for nuclear transport in vivo nor HSA can interact with p67 on blots. Moreover, although the wild-type peptide competes for binding to p67, the mutant peptides do not. p67 appears to be located at the nuclear envelope and is not present in other subcellular fractions. The nuclear localization sequence-binding protein is not extracted from the nuclear envelope with nonionic detergents and only partially extracted with high-salt buffer or 8 M urea, suggestive of a tight association with the nuclear envelope. Together our results are consistent with a role for p67 in nuclear transport.

Amino Acid Sequence↗

PubMed2005

RETRACTED: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) has nuclear localization signal-like sequences for nuclear import mediated by major vault protein.

Although phosphatase and tensin homologue deleted on chromosome 10 (PTEN) localization in the nucleus and cytoplasm is established, the mechanism is unknown. PTEN is a tumor suppressor phosphatase that causes cell cycle arrest and/or apoptosis. Nuclear-cytoplasmic compartmentalization may be a novel mechanism in regulating these events. PTEN does not contain a traditional nuclear localization sequence (NLS); however, we identified putative NLS-like sequences, which we analyzed by site-directed mutagenesis and localization studies in MCF-7 cells. Two double site mutations exhibited nuclear localization defects. Furthermore, unlike wild-type PTEN, double NLS mutant PTEN did not interact with major vault protein (MVP), a previously hypothesized nuclear-cytoplasmic transport protein. We conclude that these two NLS-like sequences are required for PTEN nuclear import that is mediated by MVP. Further, we show that this MVP-mediated nuclear import is independent of PTEN phosphorylation and of the lipid and protein phosphatase activities of PTEN.

Breast Neoplasms↗

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↗

PubMed2003

Structural requirements for nuclear localization of GCMa/Gcm-1.

GCM proteins constitute a small transcription factor family. Nuclear localization of Drosophila GCM is mediated by a typical bipartite nuclear localization sequence (NLS) close to the DNA-binding GCM domain. Here, we have analyzed nuclear localization of the mammalian GCM proteins. Whereas GCMb/Gcm-2 contained a classical bipartite NLS, nuclear localization of GCMa/Gcm-1 was mediated by two regions without resemblance to known NLS, one corresponding to the amino-terminal part of the GCM domain, the second defined as a tyrosine-and-proline-rich carboxy-terminal region. Nuclear import was counteracted by an amino-terminal nuclear export activity. This complex regulation of subcellular localization has important implications for GCMa/Gcm-1 function.

Active Transport, Cell Nucleus↗

PubMed1999

Analysis of nuclear localization signals using a green fluorescent protein-fusion protein library.

We describe here an efficient method for identifying intracellular localization signals in proteins with stereospecific intracellular localizations in culture cells. The method involves rapid fluorescence screening of cells transfected with a cDNA library in which cDNAs are fused to the gene encoding the Aequorea victoria green fluorescent protein (GFP). We analyzed nuclear localization and nuclear localization signals (NLSs) in a model application of this method. As a result, we identified classical NLSs in 75% of nuclear localized proteins. We identified some novel NLS candidates among the classical NLS-negative sequences whose nuclear localization was also identified in another cell line and with other molecular tag sequences. This method will be useful for identifying intracellular localization signals and for more detailed analysis of intracellular architecture.

Amino Acid Sequence↗

PubMed2006

Parafibromin is a nuclear protein with a functional monopartite nuclear localization signal.

Parafibromin is a nuclear protein with a tumour suppressor role in the development of non-hereditary and hereditary parathyroid carcinomas, and the hyperparathyroidism-jaw tumour (HPT-JT) syndrome, which is associated with renal and uterine tumours. Nuclear localization signal(s), (NLS(s)), of the 61 kDa parafibromin remain to be defined. Utilization of computer-prediction programmes, identified five NLSs (three bipartite (BP) and two monopartite (MP)). To investigate their functionality, wild-type (WT) and mutant parafibromin constructs tagged with enhanced green fluorescent protein or cMyc were transiently expressed in COS-7 cells, or human embryonic kidney 293 (HEK293) cells, and their subcellular locations determined by confocal fluorescence microscopy. Western blot analyses of nuclear and cytoplasmic fractions from the transfected cells were also performed. WT parafibromin localized to the nucleus and deletions or mutations of the three predicted BP and one of the predicted MP NLSs did not affect this localization. In contrast, deletions or mutations of a MP NLS, at residues 136-139, resulted in loss of nuclear localization. Furthermore, the critical basic residues, KKXR, of this MP NLS were found to be evolutionarily conserved, and over 60% of all parafibromin mutations lead to a loss of this NLS. Thus, an important functional domain of parafibromin, consisting of an evolutionarily conserved MP NLS, has been identified.

Amino Acid Sequence↗

PubMed2006

Epstein-Barr virus nuclear antigen 3A contains six nuclear-localization signals.

The Epstein-Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein-Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63-66 and 375-381) were computer-predicted, whilst the remaining three (aa 394-398, 573-578 and 598-603) were defined functionally in this study.

Epstein-Barr Virus Nuclear Antigens↗

PubMed2003

A nonconventional nuclear localization signal within the UL84 protein of human cytomegalovirus mediates nuclear import via the importin alpha/beta pathway.

The open reading frame UL84 of human cytomegalovirus encodes a multifunctional regulatory protein which is required for viral DNA replication and binds with high affinity to the immediate-early transactivator IE2-p86. Although the exact role of pUL84 in DNA replication is unknown, the nuclear localization of this protein is a prerequisite for this function. To investigate whether the activities of pUL84 are modulated by cellular proteins we used the Saccharomyces cerevisiae two-hybrid system to screen a cDNA-library for interacting proteins. Strong interactions were found between pUL84 and four members of the importin alpha protein family. These interactions could be confirmed in vitro by pull down experiments and in vivo by coimmunoprecipitation analysis from transfected cells. Using in vitro transport assays we showed that the pUL84 nuclear import required importin alpha, importin beta, and Ran, thus following the classical importin-mediated import pathway. Deletion mutagenesis of pUL84 revealed a domain of 282 amino acids which is required for binding to the importin alpha proteins. Its function as a nuclear localization signal (NLS) was confirmed by fusion to heterologous proteins. Although containing a cluster of basic amino acids similar to classical NLSs, this cluster did not contain the NLS activity. Thus, a complex structure appears to be essential for importin alpha binding and import activity.

Active Transport, Cell Nucleus↗