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Results for “Nuclear Localization Signals”

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PubMedDate not supplied

Investigation of nuclear architecture with a domain-presenting expression system.

We have investigated the topogenic properties of the nucleus by ectopic expression of chimeric proteins consisting of a NLS-modified cytoplasmic filament-forming protein, Xenopus laevis vimentin, and domains of inner nuclear membrane proteins. Whereas the "carrier" without cargo, the NLS-vimentin alone, is deposited in a few nuclear body-type structures (J.M. Bridger, H. Herrmann, C. Münkel, P. Lichter, J. Cell Sci., 111, 1241-1253), the distribution is entirely changed upon coupling with the evolutionarily conserved domain of the lamin B tail, the entire lamin B tail, the amino-terminal nucleoplasmic segment of the lamin B receptor (LBR), and the LEM domain of emerin, respectively. Remarkably, every individual chimeric protein exhibits a completely different distribution. Therefore, we assume that the chimeric parts are specifically recognized by factors engaged in nucleus-specific topogenesis. Thus, the conserved domain of the lamin B tail results in the formation of many small accumulations spread all over the nucleus. The chimera with the complete lamin B tail is deposited in short fibrillar aggregates within the nucleus. It does not mediate the integration of the chimeric protein into the nuclear membrane in cultured cells, indicating that the lamin tail alone is not sufficient to direct the integration of a protein into the lamina in vivo. In contrast, in the nuclear assembly system of Xenopus laevis the recombinant NLS-vimentin-lamin tail protein is concentrated at the nuclear membrane. The LBR chimera is arranged in a "beaded-chain"-type fashion, quite different from the more random deposition of NLS-vimentin alone. To our surprise, the LEM domain of emerin induces the retention of most of the chimeric proteins within the cytoplasm. Hence, it appears to be engaged in a strong cytoplasmic interaction that overrides the nuclear localization signal. Finally, the lamin chimera with the conserved part of the lamin B tail is shown to recruit LBR to the nuclear vimentin bodies and, vice versa, the LBR chimera attracts lamin B in transfected cells, thereby demonstrating their bona fide interaction in vivo.

Animals↗

PubMed1998

Angiotensin II-induced nuclear targeting of the angiotensin type 1 (AT1) receptor in brain neurons.

Angiotensin II (Ang II) interaction with the neuronal AT1 receptor results in a chronic stimulation of neuromodulation that involves the expression of norepinephrine transporter (NET) and tyrosine hydroxylase (TH). In view of this unique property and the presence of putative nuclear localization signal (NLS) consensus sequence in the AT1 receptor, this study was conducted to investigate the hypothesis that Ang II would induce nuclear sequestration of this G protein-coupled receptor and that the sequestration may have implications on Ang II-induced expression of NET and TH genes. Incubation of neuronal cultures with Ang II caused a time- and dose-dependent increase in the levels of AT1 receptor immunoreactivity in the nucleus. A 6.7-fold increase was observed with 100 nM Ang II, in 15 min, that was blocked by losartan, an AT1 receptor-specific antagonist. Ang II-induced nuclear sequestration was specific for AT1 receptor, because Ang II failed to produce a similar effect on neuronal AT2 receptors. The presence of the putative NLS sequence in the cytoplasmic tail of the AT1 receptor seems to be the key in nuclear targeting because: 1) nuclear targeting was attenuated by a peptide of the AT1 receptor that contained the putative NLS sequence; and 2) Ang II failed to cause nuclear translocation of the AT2 receptor, which does not contain the putative NLS. Ang II also caused a time- and dose-dependent stimulation of P62 phosphorylation, a glycoprotein of the nuclear pore complex. A 6-fold stimulation of phosphorylation was observed with 100 nM Ang II, in 15 min, that was completely blocked by losartan and not by PD123,319, an AT2 receptor specific antagonist. Preloading of neurons with p62-pep (a peptide containing consenses of mitogen-activated protein kinase in p62) resulted in a loss of Ang II-induced p62 phosphorylation and stimulation of NET and TH messenger RNA levels. In conclusion, these data demonstrate that Ang II induces nuclear sequestration of AT1 receptor involving NLS in the AT1 receptor and p62 of the nuclear pore complex in brain neurons. A possible role of such a nuclear targeting of the AT1 receptor on chronic neuromodulatory actions of Ang II has been discussed.

Amino Acid Sequence↗

PubMed1999

Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase.

Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Nuclear localization is required for induction of apoptotic cell death by the Rb-associated p84N5 death domain protein.

The mechanisms utilized to transduce apoptotic signals that originate from within the nucleus, in response to DNA damage for example, are not well understood. Identifying these mechanisms is important for predicting how tumor cells will respond to genotoxic radiation or chemotherapy. The Rb tumor suppressor protein can inhibit apoptosis triggered by DNA damage, but how it does so is unclear. We have previously characterized a death domain protein, p84N5, that specifically associates with an amino-terminal domain of Rb protein. The p84N5 death domain is required for its ability to trigger apoptotic cell death. Association with Rb protein inhibits p84N5-induced apoptosis suggesting that it may be a mediator of Rb's effects on apoptosis. Unlike other death domain-containing apoptotic signaling proteins, however, p84N5 is localized predominantly within the nucleus of interphase cells. Here we test whether p84N5 requires nuclear localization in order to trigger apoptosis. We identify the p84N5 nuclear localization signal and demonstrate that nuclear localization is required for p84N5-induced apoptosis. To our knowledge, this identifies p84N5 as the first death-domain containing apoptotic signaling protein that functions within the nucleus. By analogy to other death domain containing proteins, p84N5 may play some role in apoptotic signaling within the nucleus. Further, p84N5 is a potential mediator of Rb protein's effects on DNA damage induced apoptosis.

Active Transport, Cell Nucleus↗

PubMed1995

Nuclear multicatalytic proteinase alpha subunit RRC3: differential size, tyrosine phosphorylation, and susceptibility to antisense oligonucleotide treatment.

Multicatalytic proteinases (MCPs) are macromolecular structures involved in intracellular degradation of many types of proteins. MCPs are composed of a 20S "core" which consists of both structural (alpha) and presumed catalytic (beta) subunits in association with complexes of accessory proteins. Immunohistochemical studies have shown MCP subunits to be largely cytoplasmic, although nuclear localization is also observed. Reverse transcription/polymerase chain reaction amplifications were performed with redundant primers to conserved regions within known subunits, in an attempt both to identify potential new subunits and to define the repertoire of subunits expressed in hepatocytes. No new subunits were identified, and we found that RRC3, an alpha subunit of MCPs which contains a putative nuclear localization signal (NLS), was the predominant alpha subunit expressed in hepatocytes and hepatocyte-derived cell lines. Antibodies were developed against a unique C-terminal peptide region of RRC3. Immunohistochemical studies using affinity-purified antibodies showed that RRC3 has both cytoplasmic and nuclear localizations. Immunoprecipitation/immunoblot analyses showed that a significant proportion of nuclear RRC3 was associated with the nuclear scaffold (NS). NS RRC3 showed a significantly smaller M(r) (24,000) than the cytoplasmic form (M(r) 28,000), and only the nuclear form contained phosphotyrosine. In metabolic labeling experiments with [32P]orthophosphate, the major nuclear and NS form observed showed an M(r) of 24,000, whereas no labeling of cytosolic RRC3 was observed. A minor 32P-labeled band of M(r) 28,000 was also observed in nuclei, and this M(r) 28,000 form was found in the soluble nuclear extract within MCP complexes. These results suggest that tyrosine phosphorylation of the cytosolic form (M(r) 28,000) rapidly triggers nuclear import, which is in turn quickly followed by conversion to the major M(r) 24,000 form associated with NS. Treatment with antisense oligonucleotides targeted to the initiation site of RRC3 reduced the growth of a hepatocyte-derived cell line by 95% and produced a marked morphological change (in the absence of overt toxicity). Under these treatment conditions, RRC3 mRNA was dramatically reduced. RRC3 protein was also dramatically reduced in the NS, but showed only a small reduction in cytosol, suggesting that the nuclear RRC3 may be important in cell growth and differentiation.

Amino Acid Sequence↗

PubMed2000

Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin alpha/beta-mediated process.

Histone 2b nuclear transport was investigated using the digitonin-permeabilized cell system and the rat liver resealed nuclear envelope system. In permeabilized cells, maximal uptake of histone 2b is dependent on cytosolic components and an appropriate energy source. Addition of the recombinant proteins importin alpha/beta, and Ran, as well as ATP and GTP, to cytosol-depleted permeabilized cells does not enhance the uptake of histone 2b in contrast to that of nucleoplasmin serving as a control. Nuclear import of histone 2b cannot be blocked by addition of an excess of a nuclear localization signal-bearing peptide or nucleoplasmin. Similar results were obtained with resealed nuclear envelopes. As shown previously, resealed vesicles respond to the importin signal for the uptake of nuclear localization signal-bearing proteins which allows investigation of the import mechanism independent of intranuclear binding to chromatin. Uptake of histone 2b therefore seems to be an energy-requiring transport mechanism different from the import of proteins bearing a typical nuclear localization signal.

Active Transport, Cell Nucleus↗

PubMed1998

Cytoplasmic localization of LIM-kinase 1 is directed by a short sequence within the PDZ domain.

LIM-containing protein kinase 1 (LIMK1) is a serine/threonine kinase with a structure composed of two LIM domains, a PDZ domain, and a protein kinase domain. We examined the subcellular localization of LIMK1 and its variously deleted mutants in HeLa cells by transfection with these cDNAs. Immunofluorescence analysis revealed that the full-length LIMK1 and its mutants deleted with LIM domain or protein kinase domain preferentially localized in the cytoplasm, while the mutants deleted with the PDZ domain or a 52 amino acid region (B region) within the PDZ domain localized mainly in the nucleus. When the normally nuclear cyclin A was fused with the PDZ domain or the B region of LIMK1, it was localized in the cytoplasm of transfected cells. The corresponding region of the PDZ domain of postsynaptic density protein (PSD)-95 had no such function. Additionally, the PDZ domain of LIMK1 had no potential to bind to the C-terminal S/TXV peptides, to which the PSD-95 PDZ domain can bind. Taken together these results suggest that the PDZ domain, particularly the B region, of LIMK1 has a specific function to localize the protein in the cytoplasm. When glutathione S-transferase (GST) fused with the PDZ domain of LIMK1 (GST-PDZ) or GST-PDZ deleted with the B region (GST-PDZ delta B) was microinjected into the nucleus of COS cells, GST-PDZ was almost completely excluded from the nucleus within 30 min, whereas GST-PDZ delta B remained in the nucleus. These findings suggest that the B region of LIMK1 probably has nuclear export signal activity.

Amino Acid Sequence↗

PubMed1992

Mitogenic signaling by colony-stimulating factor 1 and ras is suppressed by the ets-2 DNA-binding domain and restored by myc overexpression.

The activity of p21ras is required for the proliferative response to colony-stimulating factor 1 (CSF-1), and signals transduced by both the CSF-1 receptor (CSF-1R) and p21ras stimulate transcription from promoter elements containing overlapping binding sites for Fos/Jun- and Ets-related proteins. A sequence encoding the DNA-binding domain and nuclear localization signal of human c-ets-2, which lacked portions of the c-ets-2 gene product necessary for trans activation, was fused to the bacterial lacZ gene and expressed from an actin promoter in NIH 3T3 cells expressing either the v-ras oncogene or human CSF-1R. Nuclear expression of the Ets-LacZ protein, confirmed by histochemical staining of beta-galactosidase, inhibited the activity of ras-responsive enhancer elements and suppressed morphologic transformation by v-ras as well as CSF-1R-dependent colony formation in semisolid medium. When CSF-1R-bearing cells expressing the Ets-LacZ protein were stimulated by CSF-1, induction of c-ets-2, c-jun, and c-fos ensued, but the c-myc response was impaired. Enforced expression of the c-myc gene overrode the suppressive effect of ets-lacZ and restored the ability of these cells to form colonies in response to CSF-1. NIH 3T3 cells engineered to express a CSF-1R (Phe-809) mutant similarly cannot form CSF-1-dependent colonies in semisolid medium and exhibit an impaired c-myc response, but expression of an exogenous myc gene resensitizes these cells to CSF-1 [M. F. Roussel, J. L. Cleveland, S. A. Shurtleff, and C. J. Sherr, Nature (London) 353:361-363, 1991]. The ability of these cells to respond to CSF-1 was also rescued by enforced expression of an endogenous c-ets-2 gene. The ets family of transcription factors therefore plays a central role in integrating both CSF-1R and ras-induced mitogenic signals and in modulating the myc response to CSF-1 stimulation.

3T3 Cells↗

PubMed1999

Overexpressed cyclin D3 contributes to retaining the growth inhibitor p27 in the cytoplasm of thyroid tumor cells.

The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.

CDC2-CDC28 Kinases↗

PubMed1997

Multiple redox regulation in NF-kappaB transcription factor activation.

The well-known Rel/NF-kappaB family of vertebrate transcription factors comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or hetero-dimers. All Rel proteins contain a highly conserved domain of approximately 300 amino-acids, called the Rel homology domain (RH), which contains sequences necessary for the formation of dimers, nuclear localization, DNA binding and IkappaB binding. Nuclear expression and consequent biological action of the eukaryotic NF-kappaB transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as IkappaB. The IkappaB proteins include a group of related proteins that interact with Rel dimers and regulate their activities. The interaction of a given IkappaB protein with a Rel complex can affect the Rel complex in distinct ways. In the best characterized example, IkappaB-alpha interacts with a p50/RelA (NF-kappaB) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. The NF-kappaB/IkappaB-alpha complex is located in the cytoplasm of most resting cells, but can be rapidly induced to enter the cell nucleus. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), IkappaB-alpha undergoes phosphorylation at serine residues by a ubiquitin-dependent protein kinase, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, probably while still complexed with NF-kappaB. Removal of IkappaB-alpha uncovers the nuclear localization signals on subunits of NF-kappaB, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. Like proinflammatory cytokines (e.g. IL-1, TNF), various ROS (peroxides, singlet oxygen, ...) as well as UV (C to A) light are capable of mediating NF-kappaB nuclear translocation, while the sensor molecules which are sensitive to these agents and trigger IkappaB-alpha proteolysis are still unidentified. We also show that a ROS-independent mechanism is activated by IL-1beta in epithelial cells and seems to involve the acidic sphingomyelinase/ceramide transduction pathway.

Animals↗

PubMed2000

Neuroprotective effect of AIP on N-methyl-D-aspartate-induced cell death in retinal neurons.

Excessive activation of glutamate receptors mediates neuronal death, but the intracellular signaling pathways that mediate this type of neuronal death are only partly understood. Previously, we have demonstrated that calcium/calmodulin-dependent protein kinase II-alpha(B) (CaMKII-alpha(B)) containing a nuclear localizing signal but not CaMKII-alpha is altered in retinal neurons exposed to N-methyl-D-aspartate (NMDA). The present study describes a prospective function of CaMKII-alpha(B) in signal transduction leading to apoptosis. The terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) method was used to detect fragmented DNA in fixed tissue sections of rat retina. The TUNEL assay confirmed that cell death occurs in the inner nuclear and ganglion cell layers following injection of 4 mM NMDA. A specific AIP (myristoylated autocamtide-2-related inhibitory peptide) with proven cell permeability inhibits CaMKII activity in vivo. Neuroprotection achieved by 500 microM AIP was complete when administered 2 h before and coincident with the NMDA application. Additionally, 100 microM of AIP protects only partially against the NMDA-induced excitotoxicity. The conformationally active fragment of caspase-3 (17 kDa), known to be involved in neuronal apoptosis was apparent within 30 min and at 2 h postinjection with NMDA. This activation was inhibited by 500 microM AIP when administered 2 h before and coincident with the NMDA application. The results suggest that CaMKII-alpha(B) isoform plays a role in excitotoxicity-induced neuronal apoptosis.

Animals↗

PubMed2000

Regenerating motor neurons express Nna1, a novel ATP/GTP-binding protein related to zinc carboxypeptidases.

To identify genes involved in axon regeneration, differential screening was applied to RNA isolated from spinal cord of mice subjected to sciatic nerve transection or crush injury. A 4-kb transcript, termed nna1, was identified that was rapidly induced in affected motor neurons in both paradigms. The levels of nna1 transcript levels declined in motor neurons within 1-2 weeks after nerve crush, coincident with target reinnervation. If reinnervation was blocked by nerve cut and ligation, nna1 was continuously expressed in motor neurons. In addition, in situ analysis of developing embryonic nervous tissue showed nna1 was highly expressed in differentiating neurons, but not proliferating populations. Nna1 is predicted to be a zinc carboxypeptidase that contains nuclear localization signals and an ATP/GTP binding motif. Cultured neurons transfected with green fluorescent protein (GFP)-nna1 expressed GFP-Nna1 in cytoplasmic and nuclear compartments. Thus, Nna1 may contribute to nuclear signaling events in differentiating and regenerating neurons.

Adenosine Triphosphate↗

PubMed2003

Recruitment of Cdc28 by Whi3 restricts nuclear accumulation of the G1 cyclin-Cdk complex to late G1.

The G1 cyclin Cln3 is a key activator of cell-cycle entry in budding yeast. Here we show that Whi3, a negative G1 regulator of Cln3, interacts in vivo with the cyclin-dependent kinase Cdc28 and regulates its localization in the cell. Efficient interaction with Cdc28 depends on an N-terminal domain of Whi3 that is also required for cytoplasmic localization of Cdc28, and for proper regulation of G1 length and filamentous growth. On the other hand, nuclear accumulation of Cdc28 requires the nuclear localization signal of Cln3, which is also found in Whi3 complexes. Both Cln3 and Cdc28 are mainly cytoplasmic during early G1, and become nuclear in late G1. However, Whi3-deficient cells show a distinct nuclear accumulation of Cln3 and Cdc28 already in early G1. We propose that Whi3 constitutes a cytoplasmic retention device for Cln3-Cdc28 complexes, thus defining a key G1 event in yeast cells.

Blotting, Western↗

PubMed2006

An unconventional human Ccr4-Caf1 deadenylase complex in nuclear cajal bodies.

mRNA deadenylation is a key process in the regulation of translation and mRNA turnover. In Saccharomyces cerevisiae, deadenylation is primarily carried out by the Ccr4p and Caf1p/Pop2p subunits of the Ccr4-Not complex, which is conserved in eukaryotes including humans. Here we have identified an unconventional human Ccr4-Caf1 complex containing hCcr4d and hCaf1z, distant human homologs of yeast Ccr4p and Caf1p/Pop2p, respectively. The hCcr4d-hCaf1z complex differs from conventional Ccr4-Not deadenylase complexes, because (i) hCaf1z and hCcr4d concentrate in nuclear Cajal bodies and shuttle between the nucleus and cytoplasm and (ii) the hCaf1z subunit, in addition to rapid deadenylation, subjects substrate RNAs to slow exonucleolytic degradation from the 3' end in vitro. Exogenously expressed hCaf1z shows both of those activities on reporter mRNAs in human HeLa cells and stimulates general mRNA decay when restricted to the cytoplasm by deletion of its nuclear localization signal. These observations suggest that the hCcr4d-hCaf1z complex may function either in the nucleus or in the cytoplasm after its nuclear export, to degrade polyadenylated RNAs, such as mRNAs, pre-mRNAs, or those RNAs that are polyadenylated prior to their degradation in the nucleus.

Animals↗

PubMed2005

Subunits of the heterotrimeric transcription factor NF-Y are imported into the nucleus by distinct pathways involving importin beta and importin 13.

The transcriptional activator NF-Y is a heterotrimeric complex composed of NF-YA, NF-YB, and NF-YC, which specifically binds the CCAAT consensus present in about 30% of eukaryotic promoters. All three subunits contain evolutionarily conserved core regions, which comprise a histone fold motif (HFM) in the case of NF-YB and NF-YC. Our results of in vitro binding studies and nuclear import assays reveal two different transport mechanisms for NF-Y subunits. While NF-YA is imported by an importin beta-mediated pathway, the NF-YB/NF-YC heterodimer is translocated into the nucleus in an importin 13-dependent manner. We define a nonclassical nuclear localization signal (ncNLS) in NF-YA, and mutational analysis indicates that positively charged amino acid residues in the ncNLS are required for nuclear targeting of NF-YA. Importin beta binding is restricted to the monomeric, uncomplexed NF-YA subunit. In contrast, the nuclear import of NF-YB and NF-YC requires dimer formation. Only the NF-YB/NF-YC dimer, but not the monomeric components, are recognized by importin 13 and are imported into the nucleus. Importin 13 competes with NF-YA for binding to the NF-YB/NF-YC dimer. Our data suggest that a distinct binding platform derived from the HFM of both subunits, NF-YB/NF-YC, mediates those interactions.

Amino Acid Motifs↗

PubMed1988

Identification of the human c-myc protein nuclear translocation signal.

We identified and characterized two regions of the human c-myc protein that target proteins into the nucleus. Using mutant c-myc proteins and proteins that fuse portions of c-myc to chicken muscle pyruvate kinase, we found that residues 320 to 328 (PAAKRVKLD; peptide M1) induced complete nuclear localization, and their removal from c-myc resulted in mutant proteins that distributed in both the nucleus and cytoplasm but retained rat embryo cell cotransforming activity. Residues 364 to 374 (RQRRNELKRSP; peptide M2) induced only partial nuclear targeting, and their removal from c-myc resulted in mutant proteins that remained nuclear but were cotransformationally inactive. We conjugated synthetic peptides containing M1 or M2 to human serum albumin and microinjected the conjugate into the cytoplasm of Vero cells. The peptide containing M1 caused rapid and complete nuclear accumulation, whereas that containing M2 caused slower and only partial nuclear localization. Thus, M1 functions as the nuclear localization signal of c-myc, and M2 serves some other and essential function.

Amino Acid Sequence↗

PubMed1991

Multiple pathways in nuclear transport: the import of U2 snRNP occurs by a novel kinetic pathway.

Protein import to the nucleus is a signal-mediated process that exhibits saturation kinetics. We investigated whether signal bearing proteins compete with U2 and U6 snRNPs during import. When injected into Xenopus oocytes, saturating concentrations of P(Lys)-BSA, a protein bearing multiple nuclear localization signals from SV40 large T-antigen, reduce the rate of [125I]P(Lys)-BSA and of [125I]nucleoplasmin import, consistent with their competing for and sharing the same limiting component of the import apparatus. In contrast, saturating concentrations of P(Lys)-BSA do not reduce the rate of HeLa [32P]U2 snRNP assembly or import. The import of U6 snRNP is also competed by P(Lys)-BSA. We conclude that U2 snRNP is imported into oocyte nuclei by a kinetic pathway that is distinct from the one followed by P(Lys)-BSA, nucleoplasmin, and U6 snRNP.

Animals↗

PubMed1990

Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes.

Bacteriophage T7 RNA polymerase and derivatives that contain the nuclear localization signal (NLS) from simian virus 40 T antigen (J. J. Dunn, B. Krippl, K. Bernstein, H. Westphal, and F. W. Studier, Gene 68:259-266, 1988) were expressed in Saccharomyces cerevisiae under the control of the inducible GAL1 promoter. As determined by indirect immunofluorescence, T7 RNA polymerase lacking the NLS remained mostly in the cytoplasm, whereas the protein containing the NLS localized to the nucleus. T7 RNA polymerase containing a mutated NLS remained mostly cytoplasmic. Hybrid proteins containing the NLS near the amino terminus were enzymatically active in the yeast cell, initiating transcription selectively at a T7 promoter placed in yeast chromosomal or plasmid DNA and stopping at a specific T7 terminator. At limiting enzyme concentrations, 5 to 10 times as much target RNA was produced when the polymerase contained the NLS, presumably because more enzyme reached the nucleus. Although substantial amounts of intact mRNA accumulated, no translation of target mRNAs in yeast cells was detected.

Base Sequence↗