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N Imamoto

Publications and source records attributed to N Imamoto.

At least 19 recordsLinked to original sources
PubMed1999

A monoclonal antibody to the COOH-terminal acidic portion of Ran inhibits both the recycling of Ran and nuclear protein import in living cells.

A small GTPase Ran is a key regulator for active nuclear transport. In immunoblotting analysis, a monoclonal antibody against recombinant human Ran, designated ARAN1, was found to recognize an epitope in the COOH-terminal domain of Ran. In a solution binding assay, ARAN1 recognized Ran when complexed with importin beta, transportin, and CAS, but not the Ran-GTP or the Ran-GDP alone, indicating that the COOH-terminal domain of Ran is exposed via its interaction with importin beta-related proteins. In addition, ARAN1 suppressed the binding of RanBP1 to the Ran-importin beta complex. When injected into the nucleus of BHK cells, ARAN1 was rapidly exported to the cytoplasm, indicating that the Ran-importin beta-related protein complex is exported as a complex from the nucleus to the cytoplasm in living cells. Moreover, ARAN1, when injected into the cultured cells induces the accumulation of endogenous Ran in the cytoplasm and prevents the nuclear import of SV-40 T-antigen nuclear localization signal substrates. From these findings, we propose that the binding of RanBP1 to the Ran-importin beta complex is required for the dissociation of the complex in the cytoplasm and that the released Ran is recycled to the nucleus, which is essential for the nuclear protein transport.

Amino Acid Sequence↗
PubMed1999

beta-subunit of nuclear pore-targeting complex (importin-beta) can be exported from the nucleus in a Ran-independent manner.

The nuclear export of importin-alpha is mediated by CAS, which is related to importin-beta, whereas the mechanism for the export of importin-beta remains unclear. In this study, we demonstrate that the nuclear export of importin-beta is mediated by the nuclear pore complex-binding domain of this molecule. Insensitivity to leptomycin B indicates that its export is not mediated by a leucine-rich nuclear export signal-specific receptor, CRM1. Furthermore, the nuclear export of importin-beta was not inhibited by co-injection with a GTPase-deficient Ran mutant (G19V). The cell line tsBN2 contains a temperature-sensitive point mutation in the RCC1 gene, which encodes a guanine nucleotide exchange factor of Ran. At the nonpermissive temperature, importin-beta was exported from the nucleus of these cells, even when RanGAP1, a GTPase-activating protein for Ran, was co-injected. These results not only provide support for the view that Ran-dependent GTP hydrolysis is not required for the nuclear export of importin-beta but also indicate that nuclear RanGTP is not essential for its export. As a result, we propose that importin-beta can be recycled from the nucleus alone in a Ran-independent manner.

Animals↗
PubMed1999

beta-catenin can be transported into the nucleus in a Ran-unassisted manner.

The nuclear accumulation of beta-catenin plays an important role in the Wingless/Wnt signaling pathway. This study describes an examination of the nuclear import of beta-catenin in living mammalian cells and in vitro semi-intact cells. When injected into the cell cytoplasm, beta-catenin rapidly migrated into the nucleus in a temperature-dependent and wheat germ agglutinin-sensitive manner. In the cell-free import assay, beta-catenin rapidly migrates into the nucleus without the exogenous addition of cytosol, Ran, or ATP/GTP. Cytoplasmic injection of mutant Ran defective in its GTP hydrolysis did not prevent beta-catenin import. Studies using tsBN2, a temperature-sensitive mutant cell line that possesses a point mutation in the RCC1 gene, showed that the import of beta-catenin is insensitive to nuclear Ran-GTP depletion. These results show that beta-catenin possesses the ability to constitutively translocate through the nuclear pores in a manner similar to importin beta in a Ran-unassisted manner. We further showed that beta-catenin also rapidly exits the nucleus in homokaryons, suggesting that the regulation of nuclear levels of beta-catenin involves both nuclear import and export of this molecule.

Animals↗
PubMed1998

Nuclear transport factor p10/NTF2 functions as a Ran-GDP dissociation inhibitor (Ran-GDI).

The cytosolic nuclear transport factor p10/NTF2 is required for the translocation of karyophilic molecules through nuclear pores [1] [2] [3], and the small GTPase Ran is a key regulator of protein transport between the nucleus and cytoplasm [4] [5]. It has been reported that p10/NTF2 interacts directly and specifically with Ran-GDP but not with Ran-GTP [6]. The precise role(s) of p10/NTF2 in the Ran GTP/GDP cycle are thus far unclear, however. In this study, we show that mammalian p10/NTF2 dramatically inhibits the dissociation of [3H]GDP from Ran and the binding of [35S]GTPgammaS to Ran following the dissociation of non-radioactive GDP by RCC1, the only known mammalian guanine nucleotide exchange factor for Ran (Ran-GEF) [7]. In contrast, the dissociation of [35S]GTP gamma S from Ran, which was also catalyzed by RCC1, was not affected by p10/NTF2. Furthermore, the activities of wild-type p10/NTF2 and the mutant forms M84T and D92G in an assay of nuclear protein import in a digitonin-permeabilized cell-free system correlated with their level of inhibition of the dissociation of nucleotide from Ran-GDP. These results suggest that p10/NTF2 acts as a GDP dissociation inhibitor for Ran (Ran-GDI), thereby coordinating the Ran-dependent reactions that underlie nuclear protein import.

Animals↗
PubMed1998

In vitro characterization of rice importin beta1: molecular interaction with nuclear transport factors and mediation of nuclear protein import.

We recently isolated two cDNAs encoding importin 3 homologues (rice importin beta1 and beta2), the first such homologues identified in plants. To address the function of rice importin beta1 in the process of nuclear import of proteins, we carried out in vitro binding and nuclear import assays. Recombinant protein of rice importin beta1 assembled a complex (PTAC) with rice importin alpha1 and NLS protein, and also bound to the nuclear envelope of tobacco BY-2 cells. Ran-GTP, but not Ran-GDP, interacted with rice importin beta1 and dissociated the heterodimer formed between rice importin alpha1 and rice importin beta1. An in vitro nuclear import assay using digitonin-permeabilized HeLa cells revealed that rice importin beta1 can mediate nuclear envelope docking of NLS proteins and their subsequent translocation into the nucleus. These data strongly suggest that rice importin beta1 functions as a component of the NLS receptor in plant cells.

Cell Nucleus↗
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↗
PubMed1998

Mutations in fission yeast Cut15, an importin alpha homolog, lead to mitotic progression without chromosome condensation.

Chromosome condensation is a major mitotic event. Fission yeast mutations in topoisomerase II and condensin subunits produce the characteristic 'cut' phenotypes, in which the septum bisects the nuclear material in the absence of normal condensation and sister chromatid separation. We show here that the same condensation defect is produced in cut15 temperature-sensitive mutants at the restrictive temperature (36 degrees C). The gene product of cut15+ is, surprisingly, very similar to importin alpha, which binds proteins containing a nuclear localization signal (NLS) and forms the heterodimer with importin beta that mediates translocation through the nuclear pore complex. We show that in a nuclear import assay, purified Cut15 protein behaved identically to mammalian importin alpha but mutant Cut15 did not. Mutant Cut15 failed to bind an NLS-containing protein in vitro but could still bind importin beta. Unexpectedly, however, NLS proteins were imported into the nucleus in cut15 mutants. Cut15 is thus essential for mitotic chromosome condensation, but its role in nuclear import might be dispensable. Green fluorescent protein (GFP)-tagged Cut15 was enriched within the nucleus specifically during prometaphase-metaphase, so the interaction of Cut15 with nuclear NLS proteins during mitosis might be important for condensation.

Animals↗
PubMed1998

Familial occurrence of moyamoya disease in the mother and four daughters including identical twins.

Familial occurrence of moyamoya disease is described in the mother and four daughters, including identical twins. Physical examination findings on admission were all normal and no mental retardation was observed. The third daughter had suffered from a ventricular septal defect when aged 6 years, but the others all had unremarkable past histories. Four of the five patients presented with transient ischemic attack as the initial symptoms, but one patient remains asymptomatic. Two patients had had repeated transient ischemic attacks. Cerebral angiography revealed either stenosis or occlusion of the intracranial portion of the bilateral internal carotid arteries associated with moyamoya vessels in all patients. The findings of moyamoya disease in a parent and four siblings including identical twins suggest that genetic factors are important in the pathogenesis of moyamoya disease.

Adolescent↗
PubMed1997

Extracellular signal-dependent nuclear import of Stat1 is mediated by nuclear pore-targeting complex formation with NPI-1, but not Rch1.

In response to interferon-gamma (IFN-gamma), Stat1 is tyrosine phosphorylated and translocates to the nucleus where it activates transcription. In this study, we identified factors which mediate the nuclear import of Stat1. Tyrosine-phosphorylated Stat1 associated with the beta subunit (a 97 kDa component) of the nuclear pore-targeting complex via the NPI-1 family, but not the Rch1 family, of alpha subunit (a 58 kDa component) as a result of IFN-gamma stimulation. Antibodies against NPI-1 or beta subunit consistently inhibited the IFN-gamma-dependent nuclear import of Stat1 in living cells, although antibodies reactive to Rch1 had no effect. Solution binding assays with deletion mutants of NPI-1 showed that the Stat1-binding domain of NPI-1 was located in the carboxy-terminal region, which is clearly distinct from the SV40 large T antigen nuclear localization signal (NLS)-binding region. These results indicate that the extracellular signal-dependent nuclear transport of Stat1 is mediated by NPI-1, but not Rch1, in conjunction with beta subunit, and that these factors participate in, not only constitutive, but also the conditional nuclear import of proteins.

Binding Sites↗
PubMed1997

Ran-unassisted nuclear migration of a 97-kD component of nuclear pore-targeting complex.

A 97-kD component of nuclear pore-targeting complex (the beta-subunit of nuclear pore-targeting complex [PTAC]/importin/karyopherin) mediates the import of nuclear localization signal (NLS)-containing proteins by anchoring the NLS receptor protein (the alpha-subunit of PTAC/importin/karyopherin) to the nuclear pore complex (NPC). The import requires a small GTPase Ran, which interacts directly with the beta-subunit. The present study describes an examination of the behavior of the beta-subunit in living cells and in digitonin-permeabilized cells. In living cells, cytoplasmically injected beta-subunit rapidly migrates into the nucleus. The use of deletion mutants reveals that nuclear migration of the beta-subunit requires neither Ran- nor alpha-subunit-binding but only the NPC-binding domain of this molecule, which is also involved in NLS-mediated import. Furthermore, unlike NLS-mediated import, a dominant-negative Ran, defective in GTP-hydrolysis, did not inhibit nuclear migration of the beta-subunit. In the digitonin-permeabilized cell-free import assay, the beta-subunit transits rapidly through the NPC into the nucleus in a saturating manner in the absence of exogenous addition of soluble factors. These results show that the beta-subunit undergoes translocation at the NPC in a Ran-unassisted manner when it does not carry alpha-subunit/NLS substrate. Therefore, a requirement for Ran arises only when the beta-subunit undergoes a translocation reaction together with the alpha-subunit/NLS substrate. The results provide an insight to the yet unsolved question regarding the mechanism by which proteins are directionally transported through the NPC, and the role of Ran in this process.

Animals↗
PubMed1997

Differential modes of nuclear localization signal (NLS) recognition by three distinct classes of NLS receptors.

The targeting of karyophilic proteins to nuclear pores is mediated via the formation of a nuclear pore-targeting complex, through the interaction of nuclear localization signal (NLS) with its NLS receptor. Recently, a novel human protein, Qip1, was identified from a yeast two-hybrid system with DNA helicase Q1. This study demonstrates that Qip1 is a novel third class of NLS receptor that efficiently recognizes the NLS of the helicase Q1. Moreover, the data obtained in this study show that the specific interaction between Qip1 and the NLS of the helicase Q1 requires its upstream sequence of the minimal essential NLS. By using purified recombinant proteins alone in the digitonin-permeabilized cell-free transport system, it was demonstrated that the two known human NLS receptors, Rch1 and NPI-1, are able to transport all the tested NLS substrates into the nucleus, while Qip1 most efficiently transports the helicase Q1-NLS substrates, which contain its upstream sequence in so far as we have examined the system. Furthermore, in HeLa cell crude cytosol, it was found that endogenous Rch1 binds to all the tested NLS substrates, while the binding of endogenous NPI-1 is restricted to only some NLSs, despite the fact that NPI-1 itself shows binding activity to a variety of NLSs. These results indicate that at least three structurally and functionally distinct NLS receptors exist in the human single cell population, and suggest that the nuclear import of karyophilic proteins may be controlled in a complex manner at the NLS recognition step by the existence of a variety of NLS receptors with various specificities to each NLS.

Adenosine Triphosphatases↗
PubMed1997

Identification of novel homologues of mouse importin alpha, the alpha subunit of the nuclear pore-targeting complex, and their tissue-specific expression.

Transport of karyophilic proteins into the nucleus is mediated by nuclear localization signals (NLSs) via a multistep process. The karyophiles are recognized by the importin alpha subunit in the cytoplasm to form a stable complex, termed the nuclear pore-targeting complex (PTAC). To date, three different mammalian alpha subunits (mSRP1/NPI-1, PTAC58/mPendulin/Rch1 and Qip1) have been identified. In this study, we report the identification of three additional mouse genes homologous to the known alpha subunits using RT-PCR methodology and show that the mouse alpha subunits can be classified into at least three subfamilies, alpha-P, alpha-Q and alpha-S families, each composed of closely related members (more than 80% amino acid sequence identity). These three subfamilies, however, have approximately 50% amino acid identity to one another. Northern blot analysis showed that all were differentially expressed in various mouse tissues. These results suggest that the function of these proteins may be controlled in a tissue-specific manner and that their combinatorial expression may play a role in differentiation and organogenesis.

Amino Acid Sequence↗
PubMed1997

Essential role of active nuclear transport in apoptosis.

BACKGROUND: Apoptosis is defined by chromatin condensation, nuclear fragmentation and the formation of apoptotic bodies. Because apoptotic signals are transmitted through a common pathway that includes the target steps of death-driving ICE-family proteases and anti-cell death protein Bcl-2 in the cytoplasm, the signals must be transferred from the cytoplasm to the nucleus, at least to induce the apoptotic manifestation of the nucleus. Small signal molecules might diffuse across nuclear pores, but larger molecules are transported by active mechanisms requiring ATP and GTP hydrolysis. It is not known whether apoptotic signals are transmitted into the nucleus by the mechanisms of active nuclear transport. RESULTS: To test the possibility that active nuclear transport is involved in apoptotic signal transmission, we have analysed the effects of molecules that inhibit active nuclear transport on apoptosis. Wheat germ agglutinin (WGA), excess amounts of p10 protein, Ran-GTPgammaS complex, and anti-PTAC58 antibody, which all inhibit active nuclear transport when exogenously microinjected, prevent Fas-induced apoptotic nuclear manifestation. WGA also prevents apoptotic nuclear change promoted by microinjected active CPP32beta/Yama protease (an ICE family member), which plays an essential role in most apoptosis. CONCLUSIONS: The results presented here strongly suggest that active nuclear transport is essential for apoptotic signal transduction.

Antibodies↗
PubMed1997

[Nuclear pore-targeting complex/importin family].

Transport of proteins into the nucleus is essential for many cellular functions to proceed. Nuclear import of proteins is directed by short amino acid sequence termed nuclear localization signals (NLS). The process of nuclear import is highly selective, requires energy, and is mediated by several soluble/cytoplasmic factors. At the entry to the import pathway, nuclear proteins form a stable complex, termed nuclear pore-targeting complex (PTAC). The complex consists of a nuclear protein and two cytosolic factors termed PTAC58 (importin alpha) and PTAC97 (importin beta). This report describes the function of PTAC/importin as well as their recently identified family proteins.

Biological Transport↗
PubMed1996

Magnetoencephalographic features in neurocysticercosis.

BACKGROUND: Magnetoencephalography (MEG) is a method of determining the brain activity noninvasively be detecting the magnetic fields associated with neuronal electrical activities. METHODS: By using 37-channel DC-superconducting quantum interference devices, MEG activity was recorded in a patient with neurocysticerosis, who had a long-term history of epilepsy. RESULTS: MEG clearly demonstrated accumulation of current dipoles originating from high-frequency waves around the cysticercal cyst, while scalp electroencephalogram failed to reveal paroxysmal discharge. Intraoperative electrocorticography revealed multiple spike activities around the lesion, consistent with MEG findings. CONCLUSIONS: We discussed the application of MEG to the patients with neurocysticercosis in estimating epileptogenic sources.

Adult↗
PubMed1995

In vivo evidence for involvement of a 58 kDa component of nuclear pore-targeting complex in nuclear protein import.

We recently showed that a nuclear location signal (NLS)-containing karyophile forms a stable complex with cytoplasmic components for nuclear pore-targeting The complex, termed nuclear pore-targeting complex (PTAC), contained two essential proteins of 54 and 90 kDa, respectively, as estimated by electrophoresis. In this study, we found that the 54 kDa component of PTAC is the mouse homologue of Xenopus importin (m-importin). Cytoplasmic injection of the antibodies raised against recombinant m-importin showed an inhibitory effect on nuclear import of a karyophile in living mammalian cells. A portion of cytoplasmically injected antibodies migrated rapidly into the nucleus, indicating dynamic movement of this protein across the nuclear envelope. Moreover, the injected antibodies co-precipitated the karyophile, in an NLS-dependent manner, with endogenous m-importin in the cytoplasm. These results provide in vivo evidence that m-importin is involved in nuclear protein import through association with a NLS in the cytoplasm before nuclear pore binding.

Amino Acid Sequence↗