Nuclear transport proteins: structure, function, and disease relevance.

Proper subcellular localization is crucial for the functioning of biomacromolecules, including proteins and RNAs. Nuclear transport is a fundamental cellular process that regulates the localization of many macromolecules within the nuclear or cytoplasmic compartments. In humans, approximately 60 proteins are involved in nuclear transport, including nucleoporins that form membrane-embedded nuclear pore complexes, karyopherins that transport cargoes through these complexes, and Ran system proteins that ensure directed and rapid transport. Many of these nuclear transport proteins play additional and essential roles in mitosis, biomolecular condensation, and gene transcription. Dysregulation of nuclear transport is linked to major human diseases such as cancer, neurodegenerative diseases, and viral infections. Selinexor (KPT-330), an inhibitor targeting the nuclear export factor XPO1 (also known as CRM1), was approved in 2019 to treat two types of blood cancers, and dozens of clinical trials of are ongoing. This review summarizes approximately three decades of research data in this field but focuses on the structure and function of individual nuclear transport proteins from recent studies, providing a cutting-edge and holistic view on the role of nuclear transport proteins in health and disease. In-depth knowledge of this rapidly evolving field has the potential to bring new insights into fundamental biology, pathogenic mechanisms, and therapeutic approaches.

Reversible disruption of XPO1-mediated nuclear export inhibits respiratory syncytial virus (RSV) replication.

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter.

Arabidopsis KETCH1 Is Critical for the Nuclear Accumulation of Ribosomal Proteins and Gametogenesis.

Male and female gametophytes are generated from micro- or megaspore mother cells through consecutive meiotic and mitotic cell divisions. Defects in these divisions often result in gametophytic lethality. Gametophytic lethality was also reported when genes encoding ribosome-related proteins were mutated. Although numerous ribosomal proteins (RPs) have been identified in plants based on homology with their yeast and metazoan counterparts, how RPs are regulated, e.g., through dynamic subcellular targeting, is unknown. We report here that an Arabidopsis (Arabidopsis thaliana) importin ß, KETCH1 (karyopherin enabling the transport of the cytoplasmic HYL1), is critical for gametogenesis. Karyopherins are molecular chaperones mediating nucleocytoplasmic protein transport. However, the role of KETCH1 during gametogenesis is independent of HYPONASTIC LEAVES 1 (HYL1), a previously reported KETCH1 cargo. Instead, KETCH1 interacts with several RPs and is critical for the nuclear accumulation of RPL27a, whose mutations caused similar gametophytic defects. We further showed that knocking down KETCH1 caused reduced ribosome biogenesis and translational capacity, which may trigger the arrest of mitotic cell cycle progression and lead to gametophytic lethality.

Selective Inhibitors of Nuclear Export in the Treatment of Hematologic Malignancies.

The correct localization of molecules between nucleus and cytoplasm is fundamental for cellular homeostasis and is controlled by a bidirectional transport system. Exportin 1 (XPO1) regulates the passage of numerous cancer-related proteins. In this review, we summarize the development of a novel class of antitumor agents, known as selective inhibitors of nuclear export (SINEs). We report results of preclinical studies and clinical trials, and discuss the mechanism of action of SINEs and their effects in multiple myeloma, non-Hodgkin lymphomas, lymphoblastic leukemia, and acute and chronic myeloid leukemia. In the future, the numerous experimental studies currently underway will allow us to define the role of SINEs and will possibly permit these substances to be introduced into daily clinical practice.

Ursolic Acid Isolated from the Leaves of Loquat ( Eriobotrya japonica) Inhibited Osteoclast Differentiation through Targeting Exportin 5.

One of the conventional strategies for treating osteoporosis is to eliminate the multinucleated osteoclasts that are responsible for bone resorption. Our previous study revealed that ursolic acid, isolated from leaves of loquat that is used as tasty tea in Japan, suppressed osteoclastogenesis. We confirmed that ursolic acid exhibited osteoclast differentiation inhibitory activity with an 50% inhibitory concentration (IC50) value of 5.4 ± 0.96 µM. To disclose its mechanism of action, this study first uses polymer-coated magnetic nanobeads to identify potential target proteins. As a result, we identified a nuclear exporter protein named exportin 5 (XPO5). Further studies demonstrated that knockdown of XPO5 significantly blocks osteoclast differentiation ( P < 0.01). Expression profiling of mature microRNAs in the cells revealed that downregulation of XPO5 by small interfering RNA or by ursolic acid could downregulate the expression of mature microRNA let-7g-5p during osteoclast differentiation ( P < 0.01). Collectively, our findings suggest that ursolic acid inhibits osteoclast differentiation through targeting XPO5, which provides further evidence for the healthy function of the tea. This study also provides new insights into the role of XPO5 and its mediated microRNAs in treatment for bone resorption diseases.

Traditional herbal medicine-derived sulforaphene promotes mitophagic cell death in lymphoma cells through CRM1-mediated p62/SQSTM1 accumulation and AMPK activation.

Sulforaphene (LFS-01) is the major chemical constituent of Raphanus sativus, a medicinal herb used for over a thousand years in traditional Chinese medicine. Here we identified that LFS-01 can selectively eradicate lymphoma cells while sparing normal lymphocytes by triggering concomitant mitophagy and apoptosis. We demonstrated that LFS-01 can retain Nrf2 in the nucleus by covalently modulating CRM1 and consequently upregulate p62/SQSTM1, an essential structural component of the autophagosomes during mitophagic process. We found that LFS-01 treatment also stimulated AMPK and thereby inhibited the mTOR pathway. On the contrary, we revealed that AMPK inhibition can severely impair the LFS-01-mediated mitophagy. Transcriptomic studies confirmed that 15 autophagy-associated genes such as p62/SQSTM1, VCP and BCL2 were differentially expressed after LFS-01 treatment. Furthermore, protein interactome network analysis revealed that the events of apoptosis and the assembly of autophagy vacuole were significant upon LFS-01 exposure. Lastly, we found that LFS-01 exhibited strong efficacy in xenograft mouse model yet with the lack of apparent toxicity to animals. We concluded that LFS-01 triggered mitophagic cell death via CRM1-mediated p62 overexpression and AMPK activation. Our findings provide new insights into the mechanism of action for LFS-01 and highlight its potential applications in treating major human diseases.

Downregulation of importin-9 protects MCF-7 cells against apoptosis induced by the combination of garlic-derived alliin and paclitaxel.

Numerous studies on the biological mechanism of breast cancer have identified a number of potential therapeutic molecular targets. In this context, one type of potential candidates appears to be agents that target the actin cytoskeleton of cancer cells or regulate actin cytoskeleton dynamics. The aim of the present study was to study the impact of altered actin transport between the cytoplasm and nucleus by the downregulation of importin-9 (IPO9) in breast adenocarcinoma MCF-7 cells exposed to an apoptosis-inducing combination of garlic-derived S-allyl-L-cysteine sulfoxide (alliin) and paclitaxel (PTX). The expression of IPO9 was downregulated by the transfection of non-aggressive breast cancer MCF-7 cells with siRNA against IPO9. The altered expression of IPO9 and cofilin-1 (CFL1) was examined using western blotting. Moreover, the effect of the downregulation of IPO9 on cell death induced by the combination of PTX and alliin was also investigated. The alterations of IPO9 and CFL1 levels were also related with F-actin organizational changes and F-actin fluorescence intensity in the nuclear/perinuclear area of the cells. The results presented here indicate that alliin and PTX act synergistically to promote and potentiate apoptosis in MCF-7 cells. Furthermore, using RNA interference technique, we showed that downregulation of IPO9 expression was correlated with a significant reduction in the apoptotic cell population as well as with a decrease in F-actin content in whole cells, and in the cortical and nuclear/perinuclear areas of the cells. Simultaneously, the downregulation of IPO9 was also accompanied by the increased post-translational expression of CFL1. Furthermore, the data obtained in the present study allow us to conclude that CFL1 itself does not translocate actin into the cell nucleus but this transport requires the functional expression of IPO9.

A method for quantification of exportin-1 (XPO1) occupancy by Selective Inhibitor of Nuclear Export (SINE) compounds.

Selective Inhibitor of Nuclear Export (SINE) compounds are a family of small-molecules that inhibit nuclear export through covalent binding to cysteine 528 (Cys528) in the cargo-binding pocket of Exportin 1 (XPO1/CRM1) and promote cancer cell death. Selinexor is the lead SINE compound currently in phase I and II clinical trials for advanced solid and hematological malignancies. In an effort to understand selinexor-XPO1 interaction and to establish whether cancer cell response is a function of drug-target engagement, we developed a quantitative XPO1 occupancy assay. Biotinylated leptomycin B (b-LMB) was utilized as a tool compound to measure SINE-free XPO1. Binding to XPO1 was quantitated from SINE compound treated adherent and suspension cells in vitro, dosed ex vivo human peripheral blood mononuclear cells (PBMCs), and PBMCs from mice dosed orally with drug in vivo. Evaluation of a panel of selinexor sensitive and resistant cell lines revealed that resistance was not attributed to XPO1 occupancy by selinexor. Administration of a single dose of selinexor bound XPO1 for minimally 72 hours both in vitro and in vivo. While XPO1 inhibition directly correlates with selinexor pharmacokinetics, the biological outcome of this inhibition depends on modulation of pathways downstream of XPO1, which ultimately determines cancer cell responsiveness.

Arsenite Disrupts Zinc-Dependent TGFß2-SMAD Activity During Murine Cardiac Progenitor Cell Differentiation.

TGFß2 (transforming growth factor-ß2) is a key growth factor regulating epithelial to mesenchymal transition (EMT). TGFß2 triggers cardiac progenitor cells to differentiate into mesenchymal cells and give rise to the cellular components of coronary vessels as well as cells of aortic and pulmonary valves. TGFß signaling is dependent on a dynamic on and off switch in Smad activity. Arsenite exposure of 1.34 µM for 24-48 h has been reported to disrupt Smad phosphorylation leading to deficits in TGFß2-mediated cardiac precursor differentiation and transformation. In this study, the molecular mechanism of acute arsenite toxicity on TGFß2-induced Smad2/3 nuclear shuttling and TGFß2-mediated cardiac EMT was investigated. A 4-h exposure to 5 µM arsenite blocks nuclear accumulation of Smad2/3 in response to TGFß2 without disrupting Smad phosphorylation or nuclear importation. The depletion of nuclear Smad is restored by knocking-down Smad-specific exportins, suggesting that arsenite augments Smad2/3 nuclear exportation. The blockage in TGFß2-Smad signaling is likely due to the loss of Zn(2+) cofactor in Smad proteins, as Zn(2+) supplementation reverses the disruption in Smad2/3 nuclear translocation and transcriptional activity by arsenite. This coincides with Zn(2+) supplementation rescuing arsenite-mediated deficits in cardiac EMT. Thus, zinc partially protects cardiac EMT from developmental toxicity by arsenite.

Compound Astragalus and Salvia miltiorrhiza extracts modulate MAPK-regulated TGF-ß/Smad signaling in hepatocellular carcinoma by multi-target mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Astragalus membranaceus Bunge (Leguminosae) and Salvia miltiorrhiza Bunge (Lamiaceae) are two important Chinese herbs with a long history of extensive ethnobotanical usage in the treatment of liver-related diseases over many centuries. Presently, these two herbs are being used either as a single herbal formulation or a composite formula for the treatment of liver related conditions. In response, recent studies on these two herbs have focused on elucidating their mechanisms of action, particularly with regards to their anti-hepatocarcinogenic effects. Previously, we have reported that Compound Astragalus and Salvia miltiorrhiza extract (CASE), a synergized composite extract from Astragalus membranaceus and Salvia miltiorrhiza ameliorates liver fibrosis and hepatocellular carcinoma (HCC) by modulating the TGF-ß/Smad pathway. Meanwhile, MAPK activation and MAPK-dependent linker phosphorylation of Smad2/3 and their preferential nuclear import are crucial for overall oncogenic role of TGF-ß/Smad signaling in HCC. To elucidate further, we studied the effect of CASE on the MAPK pathway and how it affects MAPK-dependent regulation of TGF-ß/Smad signaling using both cell and animal models of HCC. MATERIALS AND METHODS: We used immunofluorescence and western blot techniques to monitor effect of CASE on the activation of the MAPKs (pERK, pJNK and pp38) in TGF-ß1-stimulated hepatic stellate cells (HSCs), HepG2 cells and also diethylnitrosamine (DEN)-induced HCC in rats. Also phosphorylation and subcellular distribution of pSmad2/3, Smad4 and Imp7/8 in TGF-ß1-stimulated HSC and HepG2 cells were monitored. The expression of pERK, pJNK, pp38 and PAI-1 gene were monitored by using western blot technique. The effect of CASE on domain-specific phosphorylation of Smad2/3 and their subcellular distribution, and the expression of Smad4 and its subcellular distribution in TGF-ß1-stimulated HSCs and HepG2 cells were evaluated by using immunofluorescence technique. And the expression of Imp7/8 and their subcellular distribution were assessed by both immunofluorescence and western blot techniques, while PAI-1 gene expression was assessed by western blot RESULTS: In vitro, CASE in a concentration-dependent manner increased the expression of pp38 but decreased the expression of pERK and pJNK; however, in vivo, CASE in a dose dependent manner decreased the expression of pERK, pJNK as well as pp38. Also, CASE concentration dependently inhibited pSmad2C/L, pSmad3L, Smad4, Imp7/8 and their nuclear import; it had no effect on pSmad3C in HepG2 cells; significantly decreased PAI-1 gene expression in both in vitro and in vivo. CONCLUSIONS: CASE blocked MAPK activation, MAPK-dependent linker phosphorylation of Smad2/3, Smad4 expression, Imp7 expression and their nuclear import leading to significant down-regulation of PAI-1 gene expression; further highlighting the multi-target anti-HCC effect of CASE and its potential drug candidature.

Nuclear export inhibitors avert progression in preclinical models of inflammatory demyelination.

Axonal damage has been associated with aberrant protein trafficking. We examined a newly characterized class of compounds that target nucleo-cytoplasmic shuttling by binding to the catalytic groove of the nuclear export protein XPO1 (also known as CRM1, chromosome region maintenance protein 1). Oral administration of reversible CRM1 inhibitors in preclinical murine models of demyelination significantly attenuated disease progression, even when started after the onset of paralysis. Clinical efficacy was associated with decreased proliferation of immune cells, characterized by nuclear accumulation of cell cycle inhibitors, and preservation of cytoskeletal integrity even in demyelinated axons. Neuroprotection was not limited to models of demyelination, but was also observed in another mouse model of axonal damage (that is, kainic acid injection) and detected in cultured neurons after knockdown of Xpo1, the gene encoding CRM1. A proteomic screen for target molecules revealed that CRM1 inhibitors in neurons prevented nuclear export of molecules associated with axonal damage while retaining transcription factors modulating neuroprotection.

CRM1 is a direct cellular target of the natural anti-cancer agent plumbagin.

Plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, has been shown to exert anti-cancer and anti-proliferative activities in vitro as well as in animal tumor models. However, the mechanism underlying its anti-tumor action still remains unclear. CRM1 is a nuclear export receptor involved in the active transport of tumor suppressors whose function is altered in cancer due to increased expression and overactive transport. We showed that CRM1 is a direct cellular target of plumbagin. The nuclei of cells incubated with plumbagin accumulated tumor-suppressor proteins and inhibited the interactions between CRM1 and these proteins. Particularly, we demonstrated that plumbagin could specifically react with the conserved Cys(528) of CRM1 but not with a Cys(528) mutant peptide through Mass spectrometric analysis. More importantly, cancer cells that are transfected with mutant CRM1 (C528S) are resistant to the inhibitory effects of plumbagin, demonstrating that the inhibition is through direct interaction with Cys(528) of CRM1. The inhibition of nuclear traffic by plumbagin may account for its therapeutic properties in cancer and inflammatory diseases. Our findings could contribute to the development of a new class of CRM1 inhibitors.

Preclinical and clinical efficacy of XPO1/CRM1 inhibition by the karyopherin inhibitor KPT-330 in Ph+ leukemias.

As tyrosine kinase inhibitors (TKIs) fail to induce long-term response in blast crisis chronic myelogenous leukemia (CML-BC) and Philadelphia chromosome-positive (Ph(+)) acute lymphoblastic leukemia (ALL), novel therapies targeting leukemia-dysregulated pathways are necessary. Exportin-1 (XPO1), also known as chromosome maintenance protein 1, regulates cell growth and differentiation by controlling the nucleocytoplasmic trafficking of proteins and RNAs, some of which are aberrantly modulated in BCR-ABL1(+) leukemias. Using CD34(+) progenitors from CML, B-ALL, and healthy individuals, we found that XPO1 expression was markedly increased, mostly in a TKI-sensitive manner, in CML-BC and Ph(+) B-ALL. Notably, XPO1 was also elevated in Ph(-) B-ALL. Moreover, the clinically relevant XPO1 inhibitor KPT-330 strongly triggered apoptosis and impaired the clonogenic potential of leukemic, but not normal, CD34(+) progenitors, and increased survival of BCR-ABL1(+) mice, 50% of which remained alive and, mostly, became BCR-ABL1 negative. Moreover, KPT-330 compassionate use in a patient with TKI-resistant CML undergoing disease progression significantly reduced white blood cell count, blast cells, splenomegaly, lactate dehydrogenase levels, and bone pain. Mechanistically, KPT-330 altered the subcellular localization of leukemia-regulated factors including RNA-binding heterogeneous nuclear ribonucleoprotein A1 and the oncogene SET, thereby inducing reactivation of protein phosphatase 2A tumor suppressor and inhibition of BCR-ABL1 in CML-BC cells. Because XPO1 is important for leukemic cell survival, KPT-330 may represent an alternative therapy for TKI-refractory Ph(+) leukemias.

CRM1 is a cellular target of curcumin: new insights for the myriad of biological effects of an ancient spice.

Curcumin is the major constituent of turmeric plant, an ancient spice widely used in Indian cuisine and traditional herbal medicine. Recently, the potential medical use of curcumin as anti-cancer and anti-inflammatory agent has set off an upsurge in research into the mechanism for its broad biological effects. We showed that CRM1, an important nuclear exportin, is a cellular target of curcumin by serious experimental and theoretical investigation. Using a nuclear export functional assay, we observed a clear and rapid shift of cargo proteins from a cytoplasmic localization to the nucleus when treated with curcumin or its structural analogue dibenzylideneacetone (DBA). We demonstrated that curcumin could specifically target the conserved Cys(528) of CRM1 through mass spectrometric analysis and in vivo experiments. Furthermore, computational modeling has revealed that curcumin could be correctly docked into the hydrophobic pocket of CRM1 judged from shape complementarity and putative molecular interactions. The Michael acceptor moiety on curcumin is within the appropriate distance to enable Michael reaction with Cys residue of CRM1. More importantly, we showed that nuclear retention of FOXO1 could be observed in the presence of Leptomycin B (LMB) or curcumin whereas in cells expressing the CRM1-Cys(528) mutant, only a cytoplasmic localization was observed. The inhibition of nuclear traffic by curcumin may account for its myriad of biological effects, particularly for its therapeutic properties in cancer and inflammatory diseases. Our findings may have important implications for further clinical investigation of curcumin.

Preclinical activity of a novel CRM1 inhibitor in acute myeloid leukemia.

Chromosome maintenance protein 1 (CRM1) is a nuclear export receptor involved in the active transport of tumor suppressors (e.g., p53 and nucleophosmin) whose function is altered in cancer because of increased expression and overactive transport. Blocking CRM1-mediated nuclear export of such proteins is a novel therapeutic strategy to restore tumor suppressor function. Orally bioavailable selective inhibitors of nuclear export (SINE) that irreversibly bind to CRM1 and block the function of this protein have been recently developed. Here we investigated the antileukemic activity of KPT-SINE (KPT-185 and KPT-276) in vitro and in vivo in acute myeloid leukemia (AML). KPT-185 displayed potent antiproliferative properties at submicromolar concentrations (IC50 values; 100-500 nM), induced apoptosis (average 5-fold increase), cell-cycle arrest, and myeloid differentiation in AML cell lines and patient blasts. A strong down-regulation of the oncogene FLT3 after KPT treatment in both FLT3-ITD and wild-type cell lines was observed. Finally, using the FLT3-ITD-positive MV4-11 xenograft murine model, we show that treatment of mice with oral KPT-276 (analog of KPT-185 for in vivo studies) significantly prolongs survival of leukemic mice (P < .01). In summary, KPT-SINE are highly potent in vitro and in vivo in AML. The preclinical results reported here support clinical trials of KPT-SINE in AML.

The Agrobacterium VirE3 effector protein: a potential plant transcriptional activator.

During the infection of plants, Agrobacterium tumefaciens introduces several Virulence proteins including VirE2, VirF, VirD5 and VirE3 into plant cells in addition to the T-DNA. Here, we report that double mutation of virF and virE3 leads to strongly diminished tumor formation on tobacco, tomato and sunflower. The VirE3 protein is translated from a polycistronic mRNA containing the virE1, virE2 and virE3 genes, in Agrobacterium. The VirE3 protein has nuclear localization sequences, which suggests that it is transported into the plant cell nucleus upon translocation. Indeed we show here that VirE3 interacts in vitro with importin-alpha and that a VirE3-GFP fusion protein is localized in the nucleus. VirE3 also interacts with two other proteins, viz. pCsn5, a component of the COP9 signalosome and pBrp, a plant specific general transcription factor belonging to the TFIIB family. We found that VirE3 is able to induce transcription in yeast when bound to DNA through the GAL4-BD. Our data indicate that the translocated effector protein VirE3 is transported into the nucleus and there it may interact with the transcription factor pBrp to induce the expression of genes needed for tumor development.

Activation of cAMP response element-mediated gene expression by regulated nuclear transport of TORC proteins.

The CREB family of proteins are critical mediators of gene expression in response to extracellular signals and are essential regulators of adaptive behavior and long-term memory formation. The TORC proteins were recently described as potent CREB coactivators, but their role in regulation of CREB activity remained unknown. TORC proteins were found to be exported from the nucleus in a CRM1-dependent fashion. A high-throughput microscopy-based screen was developed to identify genes and pathways capable of inducing nuclear TORC accumulation. Expression of the catalytic subunit of PKA and the calcium channel TRPV6 relocalized TORC1 to the nucleus. Nuclear accumulation of the three human TORC proteins was induced by increasing intracellular cAMP or calcium levels. TORC1 and TORC2 translocation in response to calcium, but not cAMP, was mediated by calcineurin, and TORC1 was shown to be directly dephosphorylated by calcineurin. TORC function was shown to be essential for CRE-mediated gene expression induced by cAMP, calcium, or GPCR activation, and nuclear transport of TORC1 was sufficient to activate CRE-dependent transcription. Drosophila TORC was also shown to translocate in response to calcineurin activation in vivo. Thus, TORC nuclear translocation is an essential, conserved step in activation of cAMP-responsive genes.

Nucleo-cytoplasmic shuttling of the beet necrotic yellow vein virus RNA-3-encoded p25 protein.

The protein p25 encoded by beet necrotic yellow vein virus (BNYVV) RNA-3 is involved in symptom expression of infected plants. Confocal microscopy analysis of wild-type and mutated p25 fused to GFP and transiently expressed in BY-2 tobacco suspension cells identified a nuclear localization signal (NLS) in the N-terminal part of the protein. Functionality of the NLS was confirmed by pull-down assays using rice and pepper importin-alpha. Furthermore, it was demonstrated that p25 contains a nuclear export sequence sensitive to leptomycin B. The nuclear export signal (NES) was characterized by mutagenesis. A GFP-p25 fusion protein expressed during a BNYVV infection of Chenopodium quinoa leaves had the same subcellular localization as observed during transient expression in BY-2 cells. The symptom phenotype induced by expression of GFP-p25 during infection was similar to that induced by wild-type virus. Studies with mutated derivatives of GFP-p25 revealed that symptom phenotype was altered when the subcellular localization of GFP-p25 was modified.

Identification of nuclear import and export signals within the structure of the zinc finger protein TIS11.

TIS11, a member of the CCCH zinc finger protein family, functions as a positive transcriptional regulator. TIS11 was localized in both the cytoplasm and nucleus when transiently expressed in COS-7 cells. Upon treatment with leptomycin B, a specific inhibitor of the nuclear export receptor CRM1, a marked nuclear accumulation of TIS11 was observed, indicating that TIS11 shuttles between the nucleus and the cytoplasm. By deletion studies using a green fluorescent protein fusion system, we have mapped a functional nuclear localization signal (NLS) to a region containing two tandem repeats of the zinc finger motif of TIS11. A site-directed mutagenesis analysis of TIS11 NLS has revealed the critical importance of two arginine residues (Arg127 and Arg131 in the rat TIS11). Furthermore, we demonstrated that the N-terminal Leu-rich region of TIS11 serves as an LMB-sensitive nuclear export signal (NES), indicating that TIS11 follows a CRM1-mediated export pathway. These results suggest that TIS11 is subject to constant nucleocytoplasmic shuttling due to its NLS and NES.