Proteomics of nucleocytoplasmic partitioning.

The partitioning of the proteome between nucleus and cytoplasm affects nearly every aspect of eukaryotic biology. Despite this central role, we still have a poor understanding of which proteins localize in the nucleus and how this varies in different cell types and conditions. Recent advances in quantitative proteomics and high-throughput imaging are starting to close this knowledge gap. Studies on protein interaction are beginning to reveal the spectrum of cargos of nuclear import and export receptors. We anticipate that it will soon be possible to predict each protein's nucleocytoplasmic localization based on its importin/exportin interactions and its estimated diffusion rate through the nuclear pore. This insight is likely to provide us with a fundamental understanding of how cells use nucleocytoplasmic partitioning to encode and relay information.

Increased expression of importin-ß, exportin-5 and nuclear transportable proteins in Alzheimer's disease aids anatomic pathologists in its diagnosis.

Understanding the metabolic profile of neurons with the hyperphosphorylated tau protein characteristic of Alzheimer's disease is essential to unraveling new potential therapies and diagnostics for the surgical pathologist. We stratified 75 brain tissues from Alzheimer's disease into hyperphosphorylated tau positive or negative and did co-expression analyses and qRTPCR for importin-ß and exportin-5 plus several bcl2 family members and compared the data to controls, Down's dementia and Parkinson's disease. There was a significant increase in the expression of importin-ß and exportin-5 in Alzheimer's disease relative to the three other categories (each p value<0.0001) where each protein co-localized with hyperphosphorylated tau. Both apoptotic and anti-apoptotic proteins were each significantly increased in Alzheimer's disease relative to the three other groups. Neurons with hyperphosphorylated tau in Alzheimer's disease have the profile of metabolically active cells including increased exportin-5 and importin-ß mRNA and proteins which indicates that immunohistochemistry testing of these proteins may aid the surgical pathologist in making a definitive diagnosis.

Expression of CRM1 and CDK5 shows high prognostic accuracy for gastric cancer.

AIM: To evaluate the predictive value of the expression of chromosomal maintenance (CRM)1 and cyclin-dependent kinase (CDK)5 in gastric cancer (GC) patients after gastrectomy. METHODS: A total of 240 GC patients who received standard gastrectomy were enrolled in the study. The expression level of CRM1 and CDK5 was detected by immunohistochemistry. The correlations between CRM1 and CDK5 expression and clinicopathological factors were explored. Univariate and multivariate survival analyses were used to identify prognostic factors for GC. Receiver operating characteristic analysis was used to compare the accuracy of the prediction of clinical outcome by the parameters. RESULTS: The expression of CRM1 was significantly related to size of primary tumor (P = 0.005), Borrmann type (P = 0.006), degree of differentiation (P = 0.004), depth of invasion (P = 0.008), lymph node metastasis (P = 0.013), TNM stage (P = 0.002) and distant metastasis (P = 0.015). The expression of CDK5 was significantly related to sex (P = 0.048) and Lauren's classification (P = 0.011). Multivariate Cox regression analysis identified that CRM1 and CDK5 co-expression status was an independent prognostic factor for overall survival (OS) of patients with GC. Integration of CRM1 and CDK5 expression could provide additional prognostic value for OS compared with CRM1 or CDK5 expression alone (P = 0.001). CONCLUSION: CRM1 and CDK5 co-expression was an independent prognostic factors for GC. Combined CRM1 and CDK5 expression could provide a prognostic model for OS of GC.

Both high expression of nucleophosmin/B23 and CRM1 predicts poorer prognosis in human gastric cancer.

Nucleophosmin/B23 and CRM1 are molecular markers which play an important role in tumorigenesis and tumor progression in gastric cancer (GC). However, the association between the two remains unclear. This study evaluated the expression and the correlation of B23 and CRM1 in GC. B23 and CRM1 expression in GC and adjacent noncancerous tissues (ANCT) of gastrectomy specimens from 131 GC patients was measured by immunohistochemistry. Positive expression rates of B23 and CRM1 were significantly higher in GC tissues than in ANCT. The high expression rates of B23 and CRM1 were significantly higher in patients with more advanced tumor stages and distant metastasis (all p < 0.05). Only high expression of CRM1was correlated with positive Her2 status (p = 0.01). B23 expression was positively correlated with CRM1expression in GC tissues (p = 0.038). Univariate analysis showed that TNM stage (p = 0.0001), metastasis (p = 0.027), B23 (p = 0.0111), and CRM1 expression (p = 0.0019) were significant risk factors affecting overall survival. Both high expression of B23 and CRM1 in GC patients suggests poor prognosis, co-expression of the two (p = 0.043) even worse. Cox multivariate analysis showed that positive B23 (p = 0.0231) and CRM1 (p = 0.0048) expression were both independent prognostic factors that negatively correlated with survival. We revealed the co-expression of B23 or CRM1 in GC. The expression levels of B23 or CRM1 were closely related to poor prognosis in GC, and both B23 or CRM1 were independent risk factor.

Overexpression of CRM1: A Characteristic Feature in a Transformed Phenotype of Lung Carcinogenesis and a Molecular Target for Lung Cancer Adjuvant Therapy.

Our previous study showed that chromosome region maintenance 1 (CRM1), a nuclear export receptor for various cancer-associated "cargo" proteins, was important in regulating lung carcinogenesis in response to a tobacco carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The objectives of this study are to comprehensively evaluate the significance of CRM1 in lung cancer development and investigate the therapeutic potential of targeting CRM1 for lung cancer treatment using both in vitro and in vivo models. We showed that CRM1 was overexpressed not only in lung tumor tissues from both lung cancer patients and mice treated with NNK but also in NNK-transformed BEAS-2B human bronchial epithelial cells. Furthermore, stable overexpression of CRM1 in BEAS-2B cells by plasmid vector transfection led to malignant cellular transformation. Moreover, a decreased CRM1 expression level in A549 cells by short hairpin siRNA transfection led to a decreased tumorigenic activity both in vitro and in nude mice, suggesting the potential to target CRM1 for lung cancer treatment. Indeed, we showed that the cytotoxic effects of cisplatin on A549 cells with CRM1 down-regulated by short hairpin siRNA were significantly increased, compared with A549 cells, and the cytotoxic effects of cisplatin became further enhanced when the drug was used in combination with leptomycin B, a CRM1 inhibitor, in both in vitro and in vivo models. Cancer target genes were significantly involved in these processes. These data suggest that CRM1 plays an important role in lung carcinogenesis and provides a novel target for lung cancer adjuvant therapy.

Involvement of chromosome region maintenance 1 (CRM1) in the formation and progression of esophageal squamous cell carcinoma.

Chromosome region maintenance 1 (CRM1) has been related to several malignancies. The predictive value of CRM1 in the malignance and prognosis of esophageal squamous cell carcinoma (ESCC), however, is not clear yet. In this study, we displayed that CRM1 expression was up-regulated in ESCC using immunohistochemistry and Western blot. Statistical analysis demonstrated that patients with high CRM1 levels indicated shorter survival period. We further found that silencing CRM1 caused apoptosis in ESCC cell lines. Moreover, knockdown of CRM1 disturbed the expression of tumor suppressor proteins and inhibited NF-κB activity in ESCC cell lines, especially if the cell line was treated with 5-fluorouracil. In consequence, our results for the first time indicated that CRM1 was dysregulated in ESCC, and suppression of CRM1 expression which resulted in inhibiting of NF-κB signaling might be developed into a new strategy in ESCC therapy.

Expression of proteins involved in epigenetic regulation in human cutaneous melanoma and peritumoral skin.

Epigenetic processes play a critical role in melanoma development. However, little is known about proteins responsible for epigenetic transformations in melanoma cells. The processes in the peritumoral skin within the excision margin are almost unstudied. We studied the changes in expression of 112 proteins involved in epigenetic regulation of gene expression in the human cutaneous melanoma and its peritumoral zone using "The Proteomic Antibody Microarrays" (GRAA2, Sigma-Aldrich). Dimethylated histone H3 at lysines 4 and 9 as well as proteins involved in the regulation of transcription (histone deacetylases HDAC-1 and HDAC-11, DNA methyl-binding protein Kaiso), cell cycle control (protein kinases Aurora-В and PKR, chromosome protein CENP-E , and phosphorylated and acetylated histone H3), DNA repair (phosphorylated histone H2AX), and nuclear protein import (importins α3 and α5/7) were over-expressed in the melanoma tissue as compared with normal skin. At the same time, HDAC-10 and proliferating cell nuclear antigen PCNA were downregulated. In the peritumoral skin, at the excision margin (1-2 cm from the melanoma edge), we observed similar changes in expression of these proteins and, additionally, over-expression of arginine methyltransferases PRMT5 and NAD-dependent histone deacetylase SIR2. Histone methyltransferase G9a and metastasis-associated protein 2 were downregulated. Therefore, epigenetic regulation that requires histone modifications and expression of some regulatory proteins is of importance for melanoma development and propagation. The observed changes in the peritumoral skin may indicate the epigenetic pre-tuning in this zone possibly involved in malignant transformation. These results can be potentially useful for melanoma diagnostics and targeted therapy.

Rtp1p is a karyopherin-like protein required for RNA polymerase II biogenesis.

The assembly and nuclear transport of RNA polymerase II (RNA pol II) are processes that require the participation of many auxiliary factors. In a yeast genetic screen, we identified a previously uncharacterized gene, YMR185w (renamed RTP1), which encodes a protein required for the nuclear import of RNA pol II. Using protein affinity purification coupled to mass spectrometry, we identified interactions between Rtp1p and members of the R2TP complex. Rtp1p also interacts, to a different extent, with several RNA pol II subunits. The pattern of interactions is compatible with a role for Rtp1p as an assembly factor that participates in the formation of the Rpb2/Rpb3 subassembly complex and its binding to the Rpb1p-containing subcomplex. Besides, Rtp1p has a molecular architecture characteristic of karyopherins, composed of HEAT repeats, and is able to interact with phenylalanine-glycine-containing nucleoporins. Our results define Rtp1p as a new component of the RNA pol II biogenesis machinery that plays roles in subunit assembly and likely in transport through the nuclear pore complex.

An intracellular targeted antibody detects EGFR as an independent prognostic factor in ovarian carcinomas.

BACKGROUND: In ovarian cancer, the reported rate of EGFR expression varies between 4-70% depending on assessment method and data on patient outcome are conflicting. METHODS: In this study we investigated EGFR expression and its prognostic value in a cohort of 121 invasive ovarian carcinomas, using a novel antibody against the intracellular domain of the receptor. We further evaluated an association between EGFR, the nuclear transporter CRM1 as well as COX-2. Furthermore, we evaluated EGFR expression in ten ovarian cancer cell lines and incubated cancer cells with Leptomycin B, a CRM1 specific inhibitor. RESULTS: We observed a membranous and cytoplasmic EGFR expression in 36.4% and 64% of ovarian carcinomas, respectively. Membranous EGFR was an independent prognostic factor for poor overall survival in ovarian cancer patients (HR 2.7, CI 1.1-6.4, p = 0.02) which was also found in the serous subtype (HR 4.6, CI 1.6-13.4, p = 0.004). We further observed a significant association of EGFR with COX-2 and nuclear CRM1 expression (chi-square test for trends, p = 0.006 and p = 0.013, respectively). In addition, combined membranous EGFR/COX-2 expression was significantly related to unfavorable overall survival (HR 7.2, CI 2.3-22.1, p = 0.001).In cell culture, we observed a suppression of EGFR protein levels after exposure to Leptomycin B in OVCAR-3 and SKOV-3 cells. CONCLUSIONS: Our results suggest that the EGFR/COX-2/CRM1 interaction might be involved in progression of ovarian cancer and patient prognosis. Hence, it is an interesting anti-cancer target for a combination therapy. Further studies will also be needed to investigate whether EGFR is also predictive for benefit from EGFR targeted therapies.

JAB1 expression is associated with inverse expression of p27(kip1) in hepatocellular carcinoma.

BACKGROUND/AIMS: Recent studies have shown that overexpression of c-jun activation domain binding protein 1 (JAB1) and reduced expression of p27(kip1) are associated with advanced tumor stage and poor prognosis in several human cancers. Here, We investigated the functional role and correlation of JAB1 and p27(kip1) in hepatocellular carcinoma (HCC). METHODOLOGY: Immunohistochemical study for JAB1, p27(kip1) was performed on 76 cases of HCC and adjacent nontumorous tissues. 6 Fresh specimens of HCC and the adjacent liver tissue were collected for Western blot analysis. The influence of As2O3 on HCC SMMC-7721 cells, was detected by flow cytometry and Hochest staining. The expression and subcellular localization of p27(kip1) and JAB1 were investigated by Western blot and immunofluorescence. RESULTS: The expression of JAB1 was higher but p27(kip1) was lower in HCC than that in adjacent liver tissue. As2O3 treatment inhibited the growth of SMMC-7721 cells. In As2O3-treated cells, p27(kip1) expression was increased while JAB1 was decreased. The location of p27(kip1) and JAB1 were transferred from cytoplasm to nucleus. CONCLUSIONS: In HCC, JAB1 was inversely correlated with p27(kip1). As2O3 attenuated the expression of JAB1, disturbed the location and expression of p27(kip1), which may participate in regulating the growth of human hepatoma cells.

Enhanced beetle luciferase for high-resolution bioluminescence imaging.

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin alpha by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.

Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae.

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, (7)KKAKAKK(13), in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, (7)AAAKAAA(13), were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.

Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancer.

BACKGROUND: The human nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) mediates the nuclear export of proteins and messenger RNAs and, thus, is an important regulator of subcellular distribution of key molecules. Whereas cell-biologic studies have suggested a fundamental role for CRM1 in the regulation of mitosis, the expression of this protein in human tumor tissue has not been investigated to date. METHODS: In this study, the expression of CRM1 was analyzed in a cohort of 88 ovarian tumors and 12 ovarian cell lines for the first time to the authors' knowledge. RESULTS: Immunohistochemistry revealed increased nuclear (52.7%) and cytoplasmic (56.8%) expression of CRM1 in 74 carcinomas compared with the expression revealed in borderline tumors and benign lesions. Similarly, CRM1 expression was increased in ovarian cancer cell lines compared with human ovarian surface epithelial cells. Cytoplasmic CRM1 expression was related significantly to advanced tumor stage (P= .043), poorly differentiated carcinomas (P= .011), and higher mitotic rate (P= .008). Nuclear CRM1 was associated significantly with cyclooxygenase-2 (COX-2) expression (P= .002) and poor overall survival (P= .01). Because it was demonstrated previously that blocking of CRM1 by leptomycin B (LMB) contributes to the inhibition of nuclear export, the authors used a set of mechanistic assays to study the effects of CRM1 inhibition in cancer cells. Treatment of OVCAR-3 cells with LMB revealed a significant reduction of cell proliferation and increased apoptosis as well as suppressed interleukin-1beta-induced COX-2 expression. CONCLUSIONS: The current results indicated that CRM1 is expressed in a subpopulation of ovarian carcinomas with aggressive behavior and is related to poor patient outcome. A correlation also was demonstrated between CRM1 and COX-2 expression in ovarian cancer tissue. Furthermore, the treatment of ovarian cancer cells with LMB revealed a reduction in COX-2 expression. Therefore, the authors suggest that CRM1 may be an interesting biomarker for the assessment of patient prognosis and a molecular target for anticancer treatment.

Subcellular distribution of importins correlates with germ cell maturation.

Importin proteins regulate access to the nucleus by recognizing and transporting distinct cargo proteins. Building on studies in Drosophila and Caenorhabditis elegans, we hypothesized that regulated expression and subcellular localization of specific importins may be linked to mammalian gonadal differentiation. We identified distinct developmental and cellular localization patterns for importins beta1, alpha3, alpha4 and RanBP5 (importin beta3) in fetal and postnatal murine testes using Western blotting and immunohistochemistry. Importin beta1 protein is detected in selected germ and somatic cells in fetal gonads, with a striking perinuclear staining evident from embryonic day (E) 14.5 within testicular gonocytes. RanBP5 exhibits age- and gender-specific subcellular localization within fetal gonads. At E12.5, RanBP5 protein is cytoplasmic in gonocytes but predominantly nuclear in oogonia, but by E14.5 RanBP5 appears nuclear in gonocytes and cytoplasmic in oogonia. In postnatal testes, importin alpha3 and alpha4 in spermatocytes, spermatids, and Sertoli cells display cytoplasmic and nuclear localization, respectively.

Identification of gene expression indicators for thyroid axis disruption in a Xenopus laevis metamorphosis screening assay. Part 2. Effects on the tail and hindlimb.

Thyroid hormones (TH), thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)), play crucial roles in regulation of growth, development and metabolism in vertebrates and are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the US Environmental Protection Agency is developing a high throughput screen using TH-dependent metamorphosis of the Xenopus laevis tadpole as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. Using a combination of cDNA array and real time quantitative polymerase chain reaction (QPCR) analyses, this study identifies molecular markers in tissues peripheral to the central thyroid axis. We examine the hindlimb and tail of tadpoles up to 96 h of continuous exposure to T(3), T(4), methimazole, propylthiouracil, or perchlorate. Several novel biomarker candidates are indicated that include transcripts encoding importin, RNA helicase II/Gu, and defender against death protein, DAD1. In combination with previously-identified biomarker candidates, these transcripts will greatly augment the predictive and diagnostic power of the Xenopus metamorphosis assay for perturbation of TH action.

Dynamic intracellular survivin in oral squamous cell carcinoma: underlying molecular mechanism and potential as an early prognostic marker.

Survivin functions as an apoptosis inhibitor and a regulator of cell division in many tumours. The intracellular localization of survivin in tumours has been suggested as a prognostic marker. However, current reports are inconsistent and the underlying molecular mechanisms are not understood. The present study has examined the localization and prognostic value of nuclear and cytoplasmic survivin in the pre-therapeutic biopsies from 71 oral and oropharyngeal squamous carcinoma (OSCC) patients. Statistical analysis indicated that preferential nuclear versus cytoplasmic survivin correlated with favourable versus unfavourable disease outcome. Uni- and multi-variate analysis showed that in contrast to total survivin expression, the difference between nuclear and cytoplasmic survivin was a strong predictor for relapse-free survival (p=0.0003). As a potential underlying molecular mechanism, it is shown in OSCC cell lines that predominantly cytoplasmic survivin mediates protection against chemo- and radio-therapy-induced apoptosis. Importantly, the cytoplasmic localization of survivin is regulated by its nuclear export signal (NES), and export-deficient nuclear survivin is not cytoprotective. This study suggests that the difference between cytoplasmic and nuclear survivin is an indicator for survivin activity in tumour cells. Thus, this difference may serve as a predictive marker of outcome in OSCC patients undergoing multi-modality therapy. The pharmacogenetic interference with survivin's cytoplasmic localization is also to be pursued as a potential therapeutic strategy.

Intramolecular masking of nuclear localization signals: analysis of importin binding using a novel AlphaScreen-based method.

Active nuclear import of proteins requires the recognition of a nuclear localization sequence (NLS) by members of the importin (IMP) family of proteins. We have developed a modified AlphaScreen-based assay able to estimate the solution binding affinities of such interactions using biotinylated IMPs and His6-tagged NLS-containing proteins. We describe this assay in detail as well as its application in documenting the phenomenon of intramolecular masking of NLSs using recombinant green fluorescent protein (GFP) fusion proteins containing sequences from the SV40 large tumor T antigen (T-ag). We also use it to examine, for the first time, IMP binding to the cancer cell-specific proapoptotic factor viral protein 3 (VP3) from the chicken anemia virus (CAV). High-affinity binding of the IMPalpha/beta heterodimer to the T-ag NLS was observed when the GFP tag was fused to its N terminus but not to its C terminus. Effects of flanking residues were also observed in GFP-T-ag fusion derivatives containing the Thr128 NLS-inactivating mutation, whereby the absence of flanking sequences N terminal to the T-ag NLS appeared to decrease the specificity of the mutation in terms of oblating IMPalpha/beta binding. IMPbeta, but not IMPalpha or the IMPalpha/beta heterodimer, was found to bind to CAV VP3 with high affinity. Interestingly, GFP-VP3(74-121) bound to IMPbeta with threefold higher affinity than the full-length protein, GFP-VP3(1-121), implying that the NLS is masked to a significant extent in the context of full-length protein. This may represent a regulatory mechanism to control nuclear import in a tumor cell-specific fashion.

The proliferation marker pKi-67 organizes the nucleolus during the cell cycle depending on Ran and cyclin B.

The proliferation marker pKi-67 ('Ki-67 antigen') is commonly used in clinical and research pathology to detect proliferating cells, as it is only expressed during cell-cycle progression. Despite the fact that this antigen has been known for nearly two decades, there is still no adequate understanding of its function. This study has therefore identified proteins that interact with pKi-67, using a yeast two-hybrid system. A mammalian two-hybrid system and immunoprecipitation studies were used to verify these interactions. Among other cell-cycle regulatory proteins, two binding partners associated with the small GTPase Ran were identified. In addition, DNA-structural and nucleolus-associated proteins binding to pKi-67 were found. Moreover, it was demonstrated that the N-terminal domain of pKi-67 is capable of self-binding to its own repeat region encoded by exon 13. Since RanBP, a protein involved in the transport of macromolecules over the nuclear lamina, was found to be a binding partner, a possible effect of pKi-67 on the localization of cell-cycle regulatory proteins was proposed. To test this hypothesis, a tetracycline-responsive gene expression system was used to induce the pKi-67 fragments previously used for the two-hybrid screens in HeLa cells. Subsequent immunostaining revealed the translocation of cyclin B1 from cytoplasm to nucleoli in response to this expression. It is suggested that pKi-67 is a Ran-associated protein with a role in the disintegration and reformation of the nucleolus and thereby in entry into and exit from the M-phase.

Subcellular localization of the hypusine-containing eukaryotic initiation factor 5A by immunofluorescent staining and green fluorescent protein tagging.

Eukaryotic initiation factor 5A (eIF-5A) is the only protein in nature that contains hypusine, an unusual amino acid residue formed posttranslationally by deoxyhypusine synthase and deoxyhypusine hydroxylase. Although the eIF-5A gene is essential for cell survival and proliferation, the precise function and localization of eIF-5A remain unclear. In this study, we have determined the subcellular distribution of eIF-5A by indirect immunofluorescent staining and by direct visualization of green fluorescent protein tagged eIF-5A (GFP-eIF5A). Immunofluorescent staining of the formaldehyde-fixed cells showed that eIF-5A was present in both the nucleus and cytoplasm. Only the nuclear eIF-5A was resistant to Triton extraction. Direct visualization of GFP tagged eIF-5A in living cells revealed the same whole-cell distribution pattern. However, a fusion of an additional pyruvate kinase (PK) moiety into GFP-eIF-5A precluded the nuclear localization of GFP-PK-eIF-5A fusion protein. Fusion of the GFP-PK tag with three different domains of eIF-5A also failed to reveal any nuclear localization of the fusion proteins, suggesting the absence of receptor-mediated nuclear import. Using interspecies heterokaryon fusion assay, we could detect the nuclear export of GFP-Rev, but not of GFP-eIF-5A. The whole-cell distribution pattern of eIF-5A was recalcitrant to the treatments that included energy depletion, heat shock, and inhibition of transcription, translation, polyamine synthesis, or CRM1-dependent nuclear export. Collectively, our data indicate that eIF-5A gains nuclear entry via passive diffusion, but it does not undergo active nucleocytoplasmic shuttling.

CRM1-mediated nuclear export is present during porcine embryogenesis, but is not required for early cleavage.

Regulated movement of cellular factors between the cytoplasm and nucleus is required for fundamental cellular processes ranging from cell cycle control to transcriptional regulation. CRM1 is a nuclear export factor whose function is to actively transport nuclear cargos that bear nuclear export sequences to the cytoplasm. Because CRM1 likely plays a role in the intracellular regulation of many cellular processes, we set out to characterize CRM1 function during early mammalian embryogenesis. A series of embryo culture experiments that employed a specific inhibitor of CRM1, leptomycin B, indicated that CRM1 function is not required for development until after the 4-cell stage of porcine embryo development. Immunolocalization of CRM1 in fixed embryos revealed that CRM1 is localized in a unique pattern during the period of time when the embryo does not have a developmental requirement for CRM1. Despite these findings, a microinjection assay showed that CRM1 function persists during this period of development. This demonstrates that although CRM1 is present in a functional form throughout mammalian embryo development, its function is not required for early cleavage.