Nuclear-cytoplasmic Transport Research Articles

The intron-containing transcript: an evolutionarily conserved characteristic of genes orthologous to nxf1 (Nuclear eXport Factor 1)

Background. The function of nxf1 (Nuclear eXport Factor 1) gene is the nuclear-cytoplasmic transport of most mRNAs. A characteristic feature of nxf1 genes in animals belonging to different taxonomic groups is the existence of an alternative transcript with a homologous intron called a cassette intron. Materials and methods. The following databases were used: Genbank (http://www.ncbi.nlm.nih.gov/); Flybase (http://flybase.org/); UCSC Genome (http://genome.ucsc.edu). To build the secondary structures of nucleotide sequences we used the UNAFold v3.8 suite (http://mfold.rna.albany.edu/). Results. The existence of evolutionarily conserved sequences of intron 10–11 in nxf1 genes in vertebrates, and the presence of two poly(A) sequences of intron 5–6 in nxf1 genes of Drosophilidae, may be adaptive. The nxf1 cassette introns form characteristic secondary structures. Conclusion. The paper discusses the possible functional significance of the intron-retaining transcripts of nxf1 genes.

Subcellular localization and self-interaction of plant-specific Nt-4/1 protein

The Nicotiana tabacum Nt-4/1 protein is a plant-specific protein of unknown function. Analysis of bacterially expressed Nt-4/1 protein in vitro revealed that the protein secondary structure is mostly alpha-helical and suggested that it could consist of three structural domains. Earlier studies of At-4/1, the Arabidopsis thaliana-encoded ortholog of Nt-4/1, demonstrated that GFP-fused At-4/1 was capable of polar localization in plant cells, association with plasmodesmata, and cell-to-cell transport. Together with the At-4/1 ability to interact with a plant virus movement protein, these data supported the hypothesis of the At-4/1 protein involvement in viral transport through plasmodesmata. Studies of the Nt-4/1-GFP fusion protein reported in this paper revealed that the protein was localized to cytoplasmic bodies, which were co-aligned with actin filaments and capable of actin-dependent intracellular movement. The Nt-4/1-GFP bodies, being non-membrane structures, were found in association with the plasma membrane, the tubular endoplasmic reticulum and endosome-like structures. Bimolecular fluorescence complementation experiments and inhibition of nuclear export showed that the Nt-4/1 protein was capable of nuclear-cytoplasmic transport. The nuclear export signal (NES) was identified in the Nt-4/1 protein by site-directed mutagenesis. The Nt-4/1 NES mutant was localized to the nucleoplasm forming spherical bodies. Immunogold labeling and electron microscopy of cytoplasmic Nt-4/1-containing bodies and nuclear structures containing the Nt-4/1 NES mutant revealed differences in their fine structure. In mammalian cells, Nt-4/1-GFP formed cytoplasmic spherical bodies similar to those found for the Nt-4/1 NES mutant in plant cell nuclei. Using dynamic laser light scattering and electron microscopy, the Nt-4/1 protein was found to form multimeric complexes in vitro.

Impaired Cytoplasmic–Nuclear Transport of Hypoxia‐Inducible Factor‐1α in Amyotrophic Lateral Sclerosis

We investigated the mechanisms underlying abnormal vascular endothelial growth factor (VEGF) production in amyotrophic lateral sclerosis (ALS). We immunohistochemically studied VEGF, its receptors VEGFR1 and 2, and hypoxia-inducible factor-1α (HIF-1α) in autopsied ALS spinal cords. We also chronologically assessed the expression of HIF-1α, karyopherin β1, karyopherin β-cargo protein complex inhibitors and nuclear pore complex proteins in G93A mutant superoxide dismutase 1 (mSOD1) transgenic mice at presymptomatic, symptomatic and end stages. In ALS patients, compared with controls, HIF-1α immunoreactivity in the cytoplasm of anterior horn cells (AHCs) was significantly increased, while immunoreactivities for VEGF and VEGFRs were significantly decreased. Similar changes in HIF-1α and VEGF levels were observed in mSOD1 transgenic mice. HIF-1α co-localized with karyopherin β1 in the cytoplasm of AHCs and karyopherin β1 co-localized with nucleoporin 62 (Nup62) on the nuclear envelope. From the presymptomatic stage of mSOD1 transgenic mice, karyopherin β1 immunoreactivity in AHC nuclei significantly decreased and morphological irregularities of the Nup62-immunostained nuclear envelope became more pronounced with disease progression. Thus, in AHCs from mSOD1 transgenic mice, transport of cytoplasmic HIF-1α to the nuclear envelope and into the nucleus is impaired from the presymptomatic stage, suggesting that impaired cytoplasmic-nuclear transport of HIF-1α through the nuclear pore might precede motor neuron degeneration.

Complexes of Vesicular Stomatitis Virus Matrix Protein with Host Rae1 and Nup98 Involved in Inhibition of Host Transcription

Vesicular stomatitis virus (VSV) suppresses antiviral responses in infected cells by inhibiting host gene expression at multiple levels, including transcription, nuclear cytoplasmic transport, and translation. The inhibition of host gene expression is due to the activity of the viral matrix (M) protein. Previous studies have shown that M protein interacts with host proteins Rae1 and Nup98 that have been implicated in regulating nuclear-cytoplasmic transport. However, Rae1 function is not essential for host mRNA transport, raising the question of how interaction of a viral protein with a host protein that is not essential for gene expression causes a global inhibition at multiple levels. We tested the hypothesis that there may be multiple M protein-Rae1 complexes involved in inhibiting host gene expression at multiple levels. Using size exclusion chromatography and sedimentation velocity analysis, it was determined that Rae1 exists in high, intermediate, and low molecular weight complexes. The intermediate molecular weight complexes containing Nup98 interacted most efficiently with M protein. The low molecular weight form also interacted with M protein in cells that overexpress Rae1 or cells in which Nup98 expression was silenced. Silencing Rae1 expression had little if any effect on nuclear accumulation of host mRNA in VSV-infected cells, nor did it affect VSV's ability to inhibit host translation. Instead, silencing Rae1 expression reduced the ability of VSV to inhibit host transcription. M protein interacted efficiently with Rae1-Nup98 complexes associated with the chromatin fraction of host nuclei, consistent with an effect on host transcription. These results support the idea that M protein-Rae1 complexes serve as platforms to promote the interaction of M protein with other factors involved in host transcription. They also support the idea that Rae1-Nup98 complexes play a previously under-appreciated role in regulation of transcription.

Ligand‐dependent nucleo‐cytoplasmic shuttling of peroxisome proliferator‐activated receptors, PPARα and PPARγ

Peroxisome proliferator-activated receptors (PPARs) play important roles in diverse biological processes including metabolisms of sugars and lipids and differentiation of cells such as adipocytes. PPARs are transcription factors belonging to the ligand-dependent hormone receptor group. To function as transcription factors, PPARs translocate into nucleus where they associate with transcription apparatus. However, mechanisms underlying nuclear transport of PPARs remain enigmatic. We show here that PPARα and PPARγ dynamically shuttle between nucleus and cytoplasm, although they constitutively and predominantly appear in nucleus. With a series of truncation mutants, we identify that PPAR nuclear transport is mediated by at least two nuclear localization signals (NLSs) in DNA-binding domain (DBD)-hinge and activation function 1 (AF1) regions and their respective receptors including importinα/β, importin 7, and an unidentified receptor. PPARs also harbor two nuclear export signals in DBD and ligand-binding domain regions that are recognized by distinct export receptors, calreticulin and CRM1. Moreover, we show that nuclear-cytoplasmic shuttling of PPARs is regulated by respective PPAR ligands and Ca2+ concentration. Taken together, we suggest that the multiple pathways for the nuclear-cytoplasmic transport of PPARs regulate the biological functions of PPARs in response to external signals.

Exploring the Yeast Acetylome Using Functional Genomics

Lysine acetylation is a dynamic posttranslational modification with a well-defined role in regulating histones. The impact of acetylation on other cellular functions remains relatively uncharacterized. We explored the budding yeast acetylome with a functional genomics approach, assessing the effects of gene overexpression in the absence of lysine deacetylases (KDACs). We generated a network of 463 synthetic dosage lethal (SDL) interactions involving class I and II KDACs, revealing many cellular pathways regulated by different KDACs. A biochemical survey of genes interacting with the KDAC RPD3 identified 72 proteins acetylated in vivo. In-depth analysis of one of these proteins, Swi4, revealed a role for acetylation in G1-specific gene expression. Acetylation of Swi4 regulates interaction with its partner Swi6, both components of the SBF transcription factor. This study expands our view of the yeast acetylome, demonstrates the utility of functional genomic screens for exploring enzymatic pathways, and provides functional information that can be mined for future studies.

Schizosaccharomyces pombe, unlikeSaccharomyces cerevisiae, may not directly regulate nuclear-cytoplasmic transport of spliced tRNAs in response to nutrient availability

Eukaryotic cells adapt to changes in nutrient levels by regulating key processes, such as gene transcription, ribosome biogenesis, and protein translation. Several studies have shown that nuclear export of tRNAs is also regulated in Saccharomyces cerevisiae and rat hepatoma H4IIE cells during nutrient stress. However, recent studies suggest that nutrient stress does not affect nuclear tRNA export in several mammalian cell lines, including rat hepatoma H4IIE. Furthermore, in contrast to previous studies, data reported more recently established that nuclear export of mature tRNAs derived from intron-containing pre-tRNAs, but not mature tRNAs made from intronless precursors, is affected by nutrient stress in several species of Saccharomyces, but not in the yeast Kluyveromyces lactis . Here, we provide evidence suggesting that Schizosaccharomyces pombe, like mammalian cells and K. lactis, but unlike Saccharomyces, do not directly regulate nuclear export of mature tRNAs made from intron-containing pre-tRNAs in response to nutrient stress. These studies collectively suggest that regulation of nuclear export of spliced tRNAs to the cytoplasm in response to nutrient availability may be limited to the genus Saccharomyces, which unlike other yeasts and higher eukaryotes produce energy for fermentative growth using respiration-independent pathways by downregulating the citric acid cycle and the electron transport chain.

Differential Processing of Nuclear Pore Complex Proteins by Rhinovirus 2A Proteases from Different Species and Serotypes

Human rhinoviruses (HRVs) from the HRV-A, HRV-B, and HRV-C species use encoded proteases, 2A(pro) and 3C(pro), to process their polyproteins and shut off host cell activities detrimental to virus replication. Reactions attributed to 2A(pro) include cleavage of eIF4G-I and -II to inhibit cellular mRNA translation and cleavage of select nucleoporin proteins (Nups) within nuclear pore complexes (NPCs) to disrupt karyopherin-dependent nuclear-cytoplasmic transport and signaling. Sequence diversity among 2A(pro) proteases from different HRV clades, even within species, suggested individual viruses might carry out these processes with unique mechanistic signatures. Six different recombinant 2A(pro) proteases (A16, A89, B04, B14, Cw12, and Cw24) were compared for their relative substrate preferences and cleavage kinetics using eIF4G from cellular extracts and Nups presented in native (NPC) or recombinant formats. The enzyme panel attacked these substrates with different rates or processing profiles, mimicking the preferences observed during natural infection (A16 and B14). For eIF4G, all 2A(pro) proteases cleaved at similar sites, but the comparative rates were species specific (HRV-A > HRV-C ≫ HRV-B). For Nup substrates, 5 of the 6 enzymes had unique product profiles (order of Nup selection) or reacted at different sites within Nup62, Nup98, and Nup153. Only A16 and A89 behaved similarly in most assays. Since each type of karyopherin receptor prefers particular Nups or uses a limited cohort of binding motifs within those Nups, the consequences of individual 2A(pro) avidities could profoundly affect relative viral replication levels, intracellular signaling, or extracellular signaling, all of which are underlying triggers for different host immune responses.

Plants, like mammals, but unlike Saccharomyces, do not regulate nuclear-cytoplasmic tRNA trafficking in response to nutrient stress

Cells respond to nutrient stress by regulating gene transcription and various key metabolic processes, including ribosome biogenesis and protein synthesis. Several studies have shown that yeasts and mammalian cells also regulate export of tRNAs from the nucleus to the cytosol in response to nutrient stress. However, nuclear export of tRNA in mammalian cells during nutrient stress is controversial, as it has been recently shown that nuclear-cytoplasmic transport of tRNAs in several mammalian cell lines is not affected by nutrient deprivation. Furthermore, contrary to previous studies, data reported recently indicate that nuclear export of mature tRNAs derived from intron-containing precursor tRNAs, but not tRNAs made from intronless precursors, is affected by nutrient availability in several Saccharomyces species, although not in Kluyveromyces lactis and Schizosaccharomyces pombe. Here, we report that plants, like mammals and some yeasts, but unlike Saccharomyces, do not directly regulate nuclear export of tRNA in response to nutrient stress, indicating that this process is not entirely conserved among evolutionarily diverse organisms.

Inhibition of POM152 and POM34 expression in budding yeast arrests the assembly of the nuclear pore complex and increases its inner diameter

The central compartment of the nuclear pore complex (NPC), allowing the nuclear-cytoplasmic transport of molecules, consists of transmembrane proteins which anchor the pore in the nuclear envelope. Morphological and morphometric analyses of nuclear pore organization in Saccharomyces cerevisiae with mutations in the POM152 and POM34 genes have been performed. The absence of Pom152 or Pom34 initiates the disruption of the nuclear pore complex assembly. As a result, their quantity in the nuclear envelope decreases. Electron-microscopic examination shows that the inhibition of POM152 expression is also accompanied by an increase in the NPC inner diameter not observed in mutant cells with Pom34 depletion. Interactions of the transmembrane proteins with other nucleoporins and their role in pore reconstruction during cell division are discussed.

Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during skeletal myogenesis

The nuclear pore complex (NPC) is the only gateway for molecular trafficking across the nuclear envelope. The NPC is not merely a static nuclear-cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification. However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation. In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358/RanBP2 within individual NPCs increases during muscle differentiation in C2C12 cells. Using atomic force microscopy, we demonstrate structural differences between the cytoplasmic surfaces of myoblast and myotube NPCs and a correlation between the copy number of Nup358 and the NPC structure. Furthermore, small interfering RNA-mediated depletion of Nup358 in myoblasts suppresses myotube formation without affecting cell viability, suggesting that NUP358 plays a role in myogenesis. These findings indicate that the NPC undergoes dynamic remodeling during muscle cell differentiation and that Nup358 is prominently involved in the remodeling process.

All Hands on Deck—The Role of Chloroplasts, Endoplasmic Reticulum, and the Nucleus in Driving Plant Innate Immunity

Plant innate immunity is mediated by cell membrane and intracellular immune receptors that function in distinct and overlapping cell-signaling pathways to activate defense responses. It is becoming increasingly evident that immune receptors rely on components from multiple organelles for the generation of appropriate defense responses. This review analyzes the defense-related functions of the chloroplast, nucleus, and endoplasmic reticulum (ER) during plant innate immunity. It details the role of the chloroplasts in synthesizing defense-specific second messengers and discusses the retrograde signal transduction pathways that exist between the chloroplast and nucleus. Because the activities of immune modulators are regulated, in part, by their subcellular localization, the review places special emphasis on the dynamics and nuclear–cytoplasmic transport of immune receptors and regulators and highlights the importance of this process in generating orderly events during an innate immune response. The review also covers the recently discovered contributions of the ER quality-control pathways in ensuring the signaling competency of cell surface immune receptors or immune regulators.

Self-organization of endoplasmic reticulum aggregates in cells with overexpression of nucleoporin POM121

The nuclear pore complexes are complex protein structures located in the nuclear envelope, where they control the nuclear-cytoplasmic transport, and inside the stacks of endoplasmic reticulum cisternae, annulate lamellae. After overexpression of some nucleoporins, numerous granules are visible in the cytoplasm. According to the published data, these granules are the annulate lamellae. In the current paper, the structural organization of POM121-containing granules was analyzed using correlative light and electron microscopy. The ultrastructural study demonstrates that POM121-containing granules are not annulate lamellae but aggregates of endoplasmic reticulum membranes. Thus, overexpressed POM121 is not able to induce the annulate lamella formation. The mechanisms of self-organization of non-functional structures (such as the aggregates of endoplasmic reticulum membranes described here) and possible involvement of these mechanisms in the formation of cellular structures are discussed.

Subcellular localization of the new plant protein 4/1 and analysis of heterologous protein-protein interactions indicate its ability for nuclear-cytoplasmic transport

Subcellular localization of the new plant protein 4/1 and analysis of heterologous protein-protein interactions indicate its ability for nuclear-cytoplasmic transport

The Arabidopsis CBP20 targets the cap‐binding complex to the nucleus, and is stabilized by CBP80

The cap-binding protein complex (CBC) binds to the caps of all RNA polymerase II transcripts, and plays an important role in RNA metabolism. We characterized interactions, localization and nuclear-cytoplasmic transport of two subunits of the Arabidopsis thaliana cap-binding protein complex (AtCBC): AtCBP20 and AtCBP80. Using CFP/YFP-tagged proteins, we show that transport of AtCBC from the cytoplasm to the nucleus in the plant cell is different from that described in other eukaryotic cells. We show that the smaller subunit of the complex, AtCBP20, plays a crucial role in the nuclear import of AtCBC. The C-terminal part of AtCBP20 contains two functionally independent nuclear localization signals (NLSs). At least one of these two NLSs is required for the import of CBC into the plant nucleus. The interaction between the A. thaliana CBP20 and CBP80 was also analyzed in detail, using the yeast two-hybrid system and fluorescence resonance energy transfer (FRET) assays. The N-terminal part of AtCBP20 is essential for interaction with AtCBP80. Furthermore, AtCBP80 is important for the protein stability of the smaller subunit of CBC. Based on these data, we propose a model for the nuclear-cytoplasmic trafficking of the CBC complex in plants.

Screening for TARDBP mutations in Japanese familial amyotrophic lateral sclerosis

TAR-DNA-binding protein 43 (TDP-43), encoded by the TARDBP gene on chromosome 1p36.22, has been identified as the major pathological protein in abnormal inclusions in neurons and glial cells in sporadic amyotrophic lateral sclerosis (SALS), SOD1-negative familial ALS (FALS) and frontotemporal lobar dementia (FTLD). Twenty mutations of TARDBP in SOD1-negative FALS and SALS cases have been reported so far. To investigate the presence and frequency of TARDBP mutations in Japanese SOD1-negative FALS patients, we performed mutational screening of TARDBP in 30 SOD1-negative FALS patients. An N352S mutation was found in one case of FALS, but no TARDBP mutations were found in cases of SALS. It was thought that this mutation increases TDP-43 phosphorylation. This might lead to impaired nuclear cytoplasmic transport or protein–protein interaction, thereby leading to TDP-43 accumulation.

Human Nucleophosmin (NPM1) Perturbs Myeloid Development in Zebrafish in Vivo

The most frequent genetic alterations in adult cases of Acute Myeloid Leukemia (AML) are mutations in the human nucleophosmin (hNPM1) gene. In about 30% of AMLs this nucleolar phosphoprotein is aberrantly localized to the cytoplasm (hNPMc) due to these mutations affecting the protein's nuclear shuttling. hNPMc AMLs exhibit distinctive clinical and biological features that have led to its WHO classification as a distinct myeloid neoplasm. Despite its prominent association with AML, in vitro and in vivo models of hNPMc transformation in myeloid cells are lacking and its role in this process remains poorly understood. To further our understanding of hNPM function we are using the zebrafish vertebrate model system that is ideally suited for the in vivo analysis of cellular function and development during embryonic hematopoiesis. Importantly, the wide variety of blood cell types and key cellular factors regulating hematopoiesis are highly conserved between mammals and zebrafish. To investigate the in vivo role of hNPMc in hematopoiesis, we injected mRNAs encoding hNPM1wt and the leukemia-associated mutant hNPMc into one-cell stage zebrafish embryos. By fusing GFP to these hNPM proteins, we were able to follow the developmental expression of hNPM1 and its subcellular localization during embryogenesis. Analysis using confocal microscopy showed that NPMc is localized to the cell cytoplasm, while NPM1wt is found in nucleoli, as in human and mouse cells. These studies demonstrate the conservation of the nuclear-cytoplasmic transport functions of the human proteins in the zebrafish and further support its validity as a model to analyze and determine hNPM function. We also observed that hNPMc is expressed at far lower levels than its wild-type counterpart and is almost undetectable at 36hpf while hNPMwt continues to be expressed. Unlike mammals, two endogenous zebrafish NPM1 proteins were identified and named, zNpm1a and zNpm1b. Both zNpm1a and zNpm1b proteins are ubiquitously expressed in the embryo and demonstrated nucleolar localization. Expression of hNPMwt resulted in its colocalization with endogenous zNpm while hNPMc was able to bring about the export of both zebrafish proteins to the cytoplasm through heterotypic interactions. Co-immunoprecipitation experiments confirmed the interaction between human and zebrafish NPM1 proteins and zNpm1a and zNpm1b were both able to bind and co-immunoprecipitate with hNPM1 and hNPMc. These experiments suggest that transient hNPMc expression during zebrafish hematopoiesis may mimic its function in human leukemic blasts and provide clues to its functional role in AML. Comparable protein levels of either hNPMwt or hNPMc were expressed in embryos, confirmed by western blot at 22–24 hpf, and analyzed by whole mount in situ hybridization (WISH) using antisense RNA markers of specific hematopoietic lineages. Expression of hNPMc caused an increase in cMYB expression at 36 hpf, indicating an increase in the hematopoietic stem cell compartment. Furthermore, myeloid precursors (PU.1 at 22 hpf) also showed an increase upon hNPMc expression; however, mature myeloid cell (MPO and L-plastin at 26 hpf) levels were not increased relative to those in control hNPMwt injected embryos. Interestingly, the expression of hNPMc in p53 mutant embryos resulted in elevated levels of both PU.1 and MPO expressing cells, suggesting that hNPMc in zebrafish can activate p53 dependent cell cycle arrest, senescence or cell death in PU.1 cells prior to differentiation. These in vivo studies of hNPMc function during zebrafish hematopoietic differentiation suggest that hNPMc expression may increase the stem cell/ myeloid precursor compartment and can activate a p53 dependent cell death response in myeloid cells. Taking advantage of the zebrafish system in these continuing studies will further address how hNPMc expression may contribute to leukemogenesis.

HnRNPs Relocalize to the Cytoplasm following Infection with Vesicular Stomatitis Virus

Vesicular stomatitis virus (VSV) matrix protein inhibits nuclear-cytoplasmic mRNA transport. The goal of this work is to determine whether VSV inhibits the nuclear-cytoplasmic transport of heterogeneous ribonucleoproteins (hnRNPs), which are thought to serve as mRNA export factors. Confocal microscopy experiments showed that hnRNPA1, hnRNPK, and hnRNPC1/C2, but not hnRNPB1 or lamin A/C, are relocalized to the cytoplasm during VSV infection. We determined whether protein import is inhibited by VSV by transfecting cells with a plasmid encoding enhanced green fluorescent protein (EGFP) tagged with either the M9 nuclear localization sequence (NLS) or the classical NLS. These experiments revealed that both the M9 NLS and the classical NLS are functional during VSV infection. These data suggest that the inhibition of protein import is not responsible for hnRNP relocalization during VSV infection but that hnRNP export is enhanced. We found that hnRNPA1 relocalization was significantly reduced following the silencing of the mRNA export factor Rae1, indicating that Rae1 is necessary for hnRNP export. In order to determine the role of hnRNPA1 in VSV infection, we silenced hnRNPA1 in HeLa cells and assayed three aspects of the viral life cycle: host protein synthesis shutoff concurrent with the onset of viral protein synthesis, replication by plaque assay, and cell killing. We observed that host shutoff and replication are unaffected by the reduction in hnRNPA1 but that the rate of VSV-induced apoptosis is slower in cells that have reduced hnRNPA1. These data suggest that VSV promotes hnRNPA1 relocalization in a Rae1-dependent manner for apoptotic signaling.

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.

Type 1 Parathyroid Hormone Receptor (PTH1R) Nuclear Trafficking: Regulation of PTH1R Nuclear-Cytoplasmic Shuttling by Importin-α/β and Chromosomal Region Maintenance 1/Exportin 1

The type 1 PTH/PTH-related peptide receptor (PTH1R) is a class B G protein-coupled receptor that demonstrates immunoreactivity in the nucleus as well as cytoplasm of target cells. Our previous studies on the PTH1R have shown that it associates with the importin family of transport regulatory proteins. To investigate the role of the importins in PTH1R nuclear import, we used small interfering (si)RNA technology to knock down the expression of importin-beta in the mouse osteoblast-like cell line, MC3T3-E1. Immunofluorescence microscopy as well as ligand blotting for PTH1R in nuclear fractions of importin-beta siRNA-treated cells demonstrated a decrease in nuclear localization of the PTH1R in comparison with control cells. Under normal culture conditions, PTH1R is present in both the nucleus and cytoplasm of cells. Serum starvation favors nuclear localization of PTH1R, whereas returning cells to serum or treatment with PTH-related peptide induced its cytoplasmic localization. To address the nuclear export of PTH1R, interactions between PTH1R and chromosomal region maintenance 1 (CRM1) were investigated. PTH1R and CRM1 coimmunoprecipitated from MC3T3-E1 cells, suggesting that CRM1 and PTH1R form a complex in vivo. After treatment with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, PTH1R accumulated in the nucleus. Taken together, our studies show that PTH1R shuttles from the nucleus to the cytoplasm under normal physiological conditions and that this nuclear-cytoplasmic transport is dependent upon importin-alpha/beta and CRM1.