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At least 19 recordsLinked to original sources
PubMed1998

Differential importin-alpha recognition and nuclear transport by nuclear localization signals within the high-mobility-group DNA binding domains of lymphoid enhancer factor 1 and T-cell factor 1.

The transcription factor lymphoid enhancer factor 1 (LEF-1) is directed to the nucleus by a nine-amino-acid nuclear localization signal (NLS; KKKKRKREK) located in the high-mobility-group DNA binding domain. This NLS is recognized by two armadillo repeat proteins (pendulin/Rch1/alpha-P1/hSrp1alpha and Srp1/karyopherin-alpha/alpha-S1/NPI-1) which function in nuclear transport as the importin-alpha subunit of NLS receptors. T-cell factor 1 (TCF-1), a related transcription factor, contains a similar sequence (KKKRRSREK) in the identical position within its HMG DNA binding domain. We show that this sequence functions as an NLS in vivo but is not recognized by these two importin-alpha subtypes in a yeast two-hybrid assay and only weakly recognized in an in vitro binding assay. Transfer of the LEF-1 NLS to TCF-1 can confer pendulin/Rch1 binding, demonstrating that the NLS is the primary determinant for recognition. We have constructed a set of deletion mutations in pendulin/Rch1 to examine the differential NLS recognition more closely. We find that the entire armadillo repeat array of pendulin/Rch1 is necessary to maintain high affinity and specificity for the LEF-1 NLS versus the TCF-1 NLS. Importin-beta, the second subunit of the NLS receptor complex, does not influence in vitro NLS binding affinity or specificity. To test whether this differential recognition is indicative of distinct mechanisms of nuclear transport, the subcellular localization of LEF-1 and TCF-1 fused to green fluorescent protein (GFP)) was examined in an in vitro nuclear transport assay. GFP-LEF-1 readily localizes to the nucleus, whereas GFP-TCF-1 remains in the cytoplasm. Thus, LEF-1 and TCF-1 differ in several aspects of nuclear localization.

Animals↗
PubMed2005

Quantitative models of nuclear transport.

Nuclear pore complexes mediate the rapid trafficking of target macromolecules between the nucleus and the cytoplasm but exclude non-targets. Mathematical modeling helps to define the physical properties of a transport medium that can selectively enhance the permeation of some molecules but block others. Recent pioneering work has established a basis for quantitative modeling of nuclear translocation, and we expect this field to expand rapidly. A second area where modeling of nucleocytoplasmic transport has been prominently employed is in efforts to understand the regulatory networks by which signals pass between the nuclear and cytoplasmic compartments. Recent evidence suggests that the distinctive kinetics and spatial organization of nuclear transport processes can be used to efficiently propagate signals by new and unexpected pathways.

Active Transport, Cell Nucleus↗
PubMed1992

Nuclear transport and nuclear pores in yeast.

The central features of nuclear import have been conserved during evolution. In yeast the nuclear accumulation of proteins follows the same selective and active transport mechanisms known from higher eukaryotes. Yeast nuclear proteins contain nuclear localization sequences (NLS) which are presumably recognized by receptors in the cytoplasm and the nuclear envelope. Subsequent to this recognition step, nuclear proteins are translocated into the nucleus via the nuclear pore complexes. The structure of the yeast nuclear pore complex resembles that of higher eukaryotes. Recently, the first putative components of the yeast nuclear import machinery have been cloned and sequenced. The genetically amenable yeast system allows for an efficient structural and functional analysis of these components. Due to the evolutionary conservation potential insights into the nuclear import mechanisms in yeast can be transferred to higher eukaryotes. Thus, yeast can be considered as a eukaryotic model system to study nuclear transport.

Biological Evolution↗
PubMed1996

Evidence for an inhibitory feedback loop regulating simian virus 40 large T-antigen fusion protein nuclear transport.

Nuclear protein import is central to eukaryotic cell function. It is dependent on ATP, temperature and cytosolic factors, and requires specific targeting sequences called nuclear localization signals (NLSs). Nuclear import kinetics was studied in vitro using digitonin-permeabilized cells of the HTC rat hepatoma cell line and a fluorescently labelled beta-galactosidase fusion protein carrying amino acids 111-135 of the simian virus 40 large T-antigen (T-ag), including the NLS. Nuclear accumulation was rapid, reaching steady-state after about 80 min at 37 degrees C (t1/2 at about 17 min). Surprisingly, maximal nuclear concentration was found to be directly proportional to the concentration of the cytosolic extract and of cytoplasmic T-ag protein. Neither preincubation of cells for 1 h at 37 degrees C before the addition of T-ag protein nor the addition of fresh transport medium after 1 h and continuation of the incubation for another hour affected the maximal nuclear concentration. If cells were allowed to accumulate T-ag protein for 1 h before the addition of fresh transport medium containing different concentrations of T-ag protein and incubated for a further hour, the maximal nuclear concentration did not change unless the concentration of T-ag protein in the second transport mixture exceeded that in the first, in which case the nuclear concentration increased. Nuclear import of T-ag thus appeared (i) to be strictly unidirectional over 2 h at 37 degrees C and (ii) to be regulated by an inhibitory feedback loop, whereby the cytosolic concentration of protein appears to determine directly the precise end point of nuclear accumulation. This study represents the first characterization of this previously undescribed mechanism of regulation of nuclear protein import.

Animals↗
PubMed2006

Oncogenic conversion of the thyroid hormone receptor by altered nuclear transport.

Nuclear receptors (NRs) are transcription factors whose activity is modulated by ligand binding. These receptors are at the core of complex signaling pathways and act as integrators of many cellular signals. In the last decade our understanding of NRs has greatly evolved. In particular, regulation of NR subcellular dynamics has emerged as central to their activity. Research on the subcellular distribution of the thyroid hormone receptor (TR) has revealed new dimensions in the complexity of NR regulation, and points to the possibility that NR mislocalization plays a key role in oncogenesis. For many years, TR was thought to reside exclusively in the nucleus. It is now known that TR is a dynamic protein that shuttles between the nucleus and cytoplasm. TR is localized to the nucleus in a phosphorylated form, suggesting that compartment-specific phosphorylation mediates cross-talk between multiple cell signaling pathways. The oncoprotein v-ErbA, a viral-derived dominant negative variant of TR is actively exported to the cytoplasm by the CRM1 export receptor. Strikingly, the oncoprotein causes mislocalization of cellular TR and some of its coactivators by direct interaction. Here, we offer some perspectives on the role of subcellular trafficking in the oncogenic conversion of TR, and propose a new model for oncoprotein dominant negative activity.

Journal Article↗
PubMed1990

A protein recognized by antibodies to Asp-Asp-Asp-Glu-Asp shows specific binding activity to heterogeneous nuclear transport signals.

Nuclear proteins contain a signal, termed the nuclear transport signal, that specifies their selective transport into the nucleus. Previously we reported that antibodies to Asp-Asp-Asp-Glu-Asp (DDDED) inhibited nuclear transport of nuclear proteins in vivo. We therefore tried to detect a cellular receptor of nuclear transport signals as a protein that reacted with both anti-DDDED antibody and nuclear transport signal sequences. Using two steps of affinity chromatography, anti-DDDED-Sepharose and nucleoplasmin-Sepharose, we obtained a protein of 69 kDa (p69) from the nuclear pore fraction that showed these characters. This p69 recognized by anti-DDDED antibody interacted specifically with SV40 large T antigen and nucleoplasmin transport signals.

Amino Acid Sequence↗
PubMed2007

Stem cells transform into a cardiac phenotype with remodeling of the nuclear transport machinery.

Nuclear transport of transcription factors is a critical step in stem cell commitment to a tissue-specific lineage. While it is recognized that nuclear pores are gatekeepers of nucleocytoplasmic exchange, it is unknown how the nuclear transport machinery becomes competent to support genetic reprogramming and cell differentiation. Here, we report the dynamics of nuclear transport factor expression and nuclear pore microanatomy during cardiac differentiation of embryonic stem cells. Cardiac progeny derived from pluripotent stem cells displayed a distinct proteomic profile characterized by the emergence of cardiac-specific proteins. This profile correlated with the nuclear translocation of cardiac transcription factors. The nuclear transport genes, including nucleoporins, importins, exportins, transportins, and Ran-related factors, were globally downregulated at the genomic level, streamlining the differentiation program underlying stem cell-derived cardiogenesis. Establishment of the cardiac molecular phenotype was associated with an increased density of nuclear pores spanning the nuclear envelope. At nanoscale resolution, individual nuclear pores exhibited conformational changes resulting in the expansion of the pore diameter and an augmented probability of conduit occupancy. Thus, embryonic stem cells undergo adaptive remodeling of the nuclear transport infrastructure associated with nuclear translocation of cardiac transcription factors and execution of the cardiogenic program, underscoring the plasticity of the nucleocytoplasmic trafficking machinery in accommodating differentiation requirements.

Active Transport, Cell Nucleus↗
PubMed1996

Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

Amino Acid Sequence↗
PubMedDate not supplied

The nuclear pore complex, nuclear transport, and apoptosis.

The nuclear pore complex (NPC) is the sole gateway between the nucleus and the cytoplasm of interphase eukaryotic cells, and it mediates all trafficking between these 2 cellular compartments. As such, the NPC and nuclear transport play central roles in translocating death signals from the cell membrane to the nucleus where they initiate biochemical and morphological changes occurring during apoptosis. Recent findings suggest that the correlation between the NPC, nuclear transport, and apoptosis goes beyond the simple fact that NPCs mediate nuclear transport of key players involved in the cell death program. In this context, the accessibility of key regulators of apoptosis appears to be highly modulated by nuclear transport (e.g., impaired nuclear import might be an apoptotic trigger). In this review, recent findings concerning the unexpected tight link between NPCs, nuclear transport, and apoptosis will be presented and critically discussed.

Active Transport, Cell Nucleus↗
PubMed2005

Interactions between Mad1p and the nuclear transport machinery in the yeast Saccharomyces cerevisiae.

In addition to its role in nucleocytoplasmic transport, the nuclear pore complex (NPC) acts as a docking site for proteins whose apparent primary cellular functions are unrelated to nuclear transport, including Mad1p and Mad2p, two proteins of the spindle assembly checkpoint (SAC) machinery. To understand this relationship, we have mapped domains of yeast Saccharomyces cerevisiae Mad1p that interact with the nuclear transport machinery, including further defining its interactions with the NPC. We showed that a Kap95p/Kap60p-dependent nuclear localization signal, positioned in the C-terminal third of Mad1p, is required for its efficient targeting to the NPC. At the NPC, Mad1p interacts with Nup53p and a presumed Nup60p/Mlp1p/Mlp2p complex through two coiled coil regions within its N terminus. When the SAC is activated, a portion of Mad1p is recruited to kinetochores through an interaction that is mediated by the C-terminal region of Mad1p and requires energy. We showed using photobleaching analysis that in nocodazole-arrested cells Mad1p rapidly cycles between the Mlp proteins and kinetochores. Our further analysis also showed that only the C terminus of Mad1p is required for SAC function and that the NPC, through Nup53p, may act to regulate the duration of the SAC response.

Active Transport, Cell Nucleus↗
PubMed2004

Computational and biochemical identification of a nuclear pore complex binding site on the nuclear transport carrier NTF2.

Nuclear transport carriers interact with proteins of the nuclear pore complex (NPC) to transport their cargo across the nuclear envelope. One such carrier is nuclear transport factor 2 (NTF2), whose import cargo is the small GTPase Ran. A domain highly homologous to the small NTF2 protein (14kDa) is also found in a number of additional proteins, which together make up the NTF2 domain containing superfamily of proteins. Using structural, computational and biochemical analysis we have identified a functional site that is present throughout this superfamily, and our results indicate that this site functions as an NPC binding site in NTF2. Previously we showed that a D23A mutant of NTF2 exhibits increased affinity for the NPC. The mechanism of this mutation, however, was unknown as this region of NTF2 had not been implicated in binding to NPC proteins. Here we show that the D23A mutation in NTF2 does not result in gross structural changes affecting other known NPC binding sites. Instead, the D23 residue is located in an evolutionarily important region in the NTF2 domain containing superfamily, that in NTF2, is involved in binding to the NPC.

Active Transport, Cell Nucleus↗
PubMed2004

Genome-wide localization of the nuclear transport machinery couples transcriptional status and nuclear organization.

The association of genes with the nuclear pore complex (NPC) and nuclear transport factors has been implicated in transcriptional regulation. We therefore examined the association of components of the nuclear transport machinery including karyopherins, nucleoporins, and the Ran guanine-nucleotide exchange factor (RanGEF) with the Saccharomyces cerevisiae genome. We find that most nucleoporins and karyopherins preferentially associate with a subset of highly transcribed genes and with genes that possess Rap1 binding sites whereas the RanGEF preferentially associates with transcriptionally inactive genes. Consistent with coupling of transcription to the nuclear pore, we show that transcriptional activation of the GAL genes results in their association with nuclear pore proteins, relocation to the nuclear periphery, and loss of RanGEF association. Taken together, these results indicate that the organization of the genome is coupled via transcriptional state to the nuclear transport machinery.

Active Transport, Cell Nucleus↗
PubMed2001

Mechanism of specific nuclear transport of adriamycin: the mode of nuclear translocation of adriamycin-proteasome complex.

Adriamycin (ADM), an anthracycline anticancer agent, is selectively stored in the nuclei of a variety of proliferating cells, but the precise mechanism of specific nuclear transport of ADM is not well known. Recently, we demonstrated that ADM shows high binding affinity to the cytoplasmic proteasomes of L1210 mouse leukemia cells and that taken up ADM by the cells selectively binds to proteasomes. Nuclear targeting of proteasome in proliferating cells may be mediated by the nuclear localization signals that are found in several of the alpha-type subunits of the 20S proteasome. To confirm nuclear transport of the ADM-proteasome complex, we synthesized a photoactive ADM analogue, N-(p-azidohenzoyl)-ADM, and generated a photoaffinity-labeled proteasome complex. The 26S proteasome purified from the cytosol of L1210 cells had a high affinity to N-(p-azidobenzoyl)-ADM. SDS-PAGE analysis of the photoaffinity-labeled proteasome showed that low molecular weight bands (approximately 21-31 kDa) of 20S proteasome had the highest photoaffinity. The photoaffinity-labeled proteasome was distributed in the cytoplasm and nuclei of digitonin-permeabilized L1210 and B-16 mouse melanoma cells in the presence of the cytosolic fraction and ATP. The rate of nuclear translocation of the proteasome was low in the absence of ATP. These results suggest that the proteasome is a specific translocator of ADM from the cytoplasm to the nucleus and that 20S proteasome components are the dominant ADM-binding sites. The nuclear transport of ADM-proteasome complex is regulated by an ATP-dependent nuclear pore-mediated mechanism.

Active Transport, Cell Nucleus↗
PubMed1988

Effect of basic and nonbasic amino acid substitutions on transport induced by simian virus 40 T-antigen synthetic peptide nuclear transport signals.

A previous study demonstrated the ability of a synthetic peptide homologous to the simian virus 40 T-antigen nuclear transport signal to induce the nuclear transport of carrier proteins and the dependence of peptide-induced transport on a positive charge at the lysine corresponding to amino acid 128 of T antigen. In this investigation synthetic peptides were utilized to examine the effect on transport of amino acid substitutions within the T-antigen nuclear transport signal. Nuclear transport was evaluated by immunofluorescence after microinjection of protein-peptide conjugates into the cytoplasm of mammalian cells. Substitution of other basic amino acids at position 128 revealed a hierarchy for nuclear transport. The rate of nuclear transport was most rapid when a lysine was at position 128 followed in descending order by arginine, D-lysine, ornithine, and p-aminophenylalanine. Peptide-induced nuclear transport was dependent upon a positively charged amino acid at positions 128 and 129, since substitutions of neutral asparagines at these positions abolished transport. However, partial transport was observed with the peptide having an asparagine at position 128 when a high number of peptides were conjugated to the carrier protein.

Amino Acids↗
PubMed1990

Comparison of diverse transport signals in synthetic peptide-induced nuclear transport.

Several investigations have demonstrated the ability of synthetic peptides homologous to the nuclear transport signal of simian virus 40 large T antigen to induce the nuclear transport of nonnuclear carrier proteins. To determine the generality of peptide-induced transport, six peptides with sequences derived from four previously identified nuclear transport signals were synthesized and examined for their ability to induce the transport of mouse immunoglobulin G following microinjection into the cytoplasm of mammalian cells. Peptides containing transport signals from simian virus 40 T antigen, Xenopus nucleoplasmin, and adenovirus E1A proteins were highly efficient at peptide-induced transport, while a peptide homologous to yeast MAT alpha 2 protein was incapable of inducing transport. A short nucleoplasmin peptide that contained only the basic amino acid domain was capable of inducing transport but yielded a much slower rate of transport than a long nucleoplasmin peptide encompassing the previously identified minimal transport signal. The short nucleoplasmin signal exhibited a greater capacity for transport than a peptide homologous to the cytoplasmic mutant T antigen signal when conjugates with a low number of signals coupled per carrier protein were examined. However, the short nucleoplasmin peptide was only marginally more effective than the T antigen mutant peptide when conjugates with a high number of signals coupled per carrier protein were examined.

Adenovirus Early Proteins↗
PubMed2006

Identification and characterization of the Arabidopsis orthologs of nuclear transport factor 2, the nuclear import factor of ran.

Ran is a multifunctional small GTPase that is involved in nucleocytoplasmic transport, mitotic spindle assembly, and nuclear envelope formation. Nuclear import of Ran relies on a small RanGDP-binding protein, Nuclear Transport Factor 2 (NTF2). Three proteins are expressed in Arabidopsis (Arabidopsis thaliana) that show significant sequence similarity to human and yeast (Saccharomyces cerevisiae) NTF2. Here, we demonstrate that two of them, AtNTF2a and AtNTF2b, can functionally replace the essential NTF2 gene in yeast. Consistent with this finding, both AtNTF2a and AtNTF2b interact with yeast and Arabidopsis Ran. The third NTF2-related protein, AtNTL, does not functionally replace NTF2 in yeast. Similar to yeast NTF2-green fluorescent protein (GFP), AtNTF2a-GFP and AtNTF2b-GFP accumulate at the nuclear rim. The AtNTF2a E38K and E91K mutants, which fail to bind Ran, are not functional in yeast, indicating conservation of the requirement for these key amino acids in plants and yeast. AtNTF2a overexpression, but not AtNTF2aE38K overexpression, blocks nuclear import of a plant transcription factor in Nicotiana benthamiana leaves, indicating that excess AtNTF2a disrupts nuclear import in a Ran-binding-dependent manner. On the basis of these results, we propose that AtNTF2a and AtNTF2b function in Ran import in Arabidopsis and that nuclear import of Ran is functionally conserved in plants.

Active Transport, Cell Nucleus↗