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PubMed2000

Yeast nucleoporins involved in passive nuclear envelope permeability.

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

Biological Transport↗
PubMed1999

Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation.

Transcriptional activation of nuclear factor kappaB (NF-kappaB) is mediated by signal-induced phosphorylation and degradation of its inhibitor, IkappaBalpha. However, NF-kappaB activation induces rapid resynthesis of IkappaBalpha, which is responsible for post-induction repression of transcription. Newly synthesized IkappaBalpha translocates to the nucleus, where it dissociates NF-kappaB from DNA and transports NF-kappaB from the nucleus to the cytoplasm in a nuclear export sequence-dependent process that is sensitive to leptomycin B (LMB). In the present study, LMB was used as a tool to inhibit nuclear export sequence-mediated nuclear protein export and evaluate the consequences for regulation of NF-kappaB-dependent transcriptional activity. Pretreatment of cells with LMB inhibits NF-kappaB-dependent transcriptional activation mediated by interleukin 1beta or tumor necrosis factor alpha. This is a consequence of the inhibition of signal-induced degradation of IkappaBalpha. Although LMB treatment does not affect the signal transduction pathway leading to IkappaBalpha degradation, it blocks IkappaBalpha nuclear export. IkappaBalpha is thus accumulated in the nucleus, and in this compartment it is resistant to signal-induced degradation. These results indicate that the signal-induced degradation of IkappaBalpha is mainly, if not exclusively, a cytoplasmic process. An efficient nuclear export of IkappaBalpha is therefore essential for maintaining a low level of IkappaBalpha in the nucleus and allowing NF-kappaB to be transcriptionally active upon cell stimulation.

Biological Transport↗
PubMed2005

Yeast poly(A)-binding protein Pab1 shuttles between the nucleus and the cytoplasm and functions in mRNA export.

Pab1 is the major poly(A)-binding protein in yeast. It is a multifunctional protein that mediates many cellular functions associated with the 3'-poly(A)-tail of messenger RNAs. Here, we characterize Pab1 as an export cargo of the protein export factor Xpo1/Crm1. Pab1 is a major Xpo1/Crm1-interacting protein in yeast extracts and binds directly to Xpo1/Crm1 in a RanGTP-dependent manner. Pab1 shuttles rapidly between the nucleus and the cytoplasm and partially accumulates in the nucleus when the function of Xpo1/Crm1 is inhibited. However, Pab1 can also be exported by an alternative pathway, which is dependent on the MEX67-mRNA export pathway. Import of Pab1 is mediated by the import receptor Kap108/Sxm1 through a nuclear localization signal in its fourth RNA-binding domain. Interestingly, inhibition of Pab1's nuclear import causes a kinetic delay in the export of mRNA. Furthermore, the inviability of a pab1 deletion strain is suppressed by a mutation in the 5'-3' exoribonuclease RRP6, a component of the nuclear exosome. Therefore, nuclear Pab1 may be required for efficient mRNA export and may function in the quality control of mRNA in the nucleus.

Active Transport, Cell Nucleus↗
PubMed1994

Efflux of RNA from resealed nuclear envelope ghosts.

mRNA translocation across the nuclear envelope and the appropriate signal-receptor interactions have been studied using resealed rat liver nuclear envelope ghosts (RNEG). We compared export kinetics of nonadenylated (tRNAs, histone-2 poly(A)- mRNA), and adenylated RNAs (poly(A)+ tRNAs, synthetic histone-2 poly(A) +mRNA, albumin mRNA, beta-globin poly(A) +mRNA and a total poly(A) + mRNA extract from rat liver cells). ATP-dependent export of mRNAs and of total poly(A)+ RNA was prevented by inhibitors of a nuclear envelope NTPase. All adenylated RNA species competed with each other for export, but nonadenylated RNAs did not. This indicates the existence of different translocation mechanisms for different RNA species with their appropriate nuclear envelope associated RNA receptors involved in export. The attachment of a poly(A)250 sequence at the 3'-end of tRNA or histone messenger masks the intrinsic RNA export signal of nonadenylated RNAs and results in efflux comparable to that of beta-globin poly(A)+ mRNA. The attachment on oligo(A)5 does not have any comparable effect of nonadenylated RNA translocation. Export of all polyadenylated RNAs from RNEGs is blocked by a monoclonal antibody, which is directed against an intranuclear envelope poly(A) binding protein. The results suggest that the pore complexes do not select RNAs for export to the cytoplasm and are therefore not responsible for nuclear restriction of mRNA precursors.

Adenosine Triphosphate↗
PubMed2005

The tRNA aminoacylation co-factor Arc1p is excluded from the nucleus by an Xpo1p-dependent mechanism.

Arc1p, a yeast tRNA-binding protein, forms a complex with the aminoacyl-tRNA synthetases, methionyl tRNA synthetase (MetRS) and glutamyl tRNA synthetase (GluRS). Although this complex localizes normally in the cytoplasm, in the absence of Arc1p the two free synthetases are also found inside the nucleus. In this work, in order to localize free Arc1 we abolished complex assembly by deleting the appended domains from both MetRS and GluRS. Surprisingly, free Arc1p remained cytoplasmic even when fitted with a strong nuclear localization signal (NLS). However, NLS-Arc1p accumulated in the nucleus when Xpo1/Crm1, the export receptor for NES-containing cargo proteins, was mutated. Thus, the cytoplasmic location of Arc1p is maintained by Xpo1p-dependent nuclear export and Arc1p could act as an adapter in the nucleocytoplasmic trafficking of tRNA and/or the tRNA-aminoacylation machinery.

Cell Nucleus↗
PubMed2006

Controlling protein compartmentalization to overcome disease.

Over the past decade, considerable progress has been made to improve our understanding of the intracellular transport of proteins. Mechanisms of nuclear import and export involving classical receptors have been studied. Signal sequences required for directing a protein molecule to a specific cellular compartment have been defined. Knowledge of subcellular trafficking of proteins has also increased our understanding of diseases caused due to mislocalization of proteins. A specific protein on deviating from its native cellular compartment may result in disease due to loss of its normal functioning and aberrant activity in the "wrong" compartment. Mislocalization of proteins results in diseases that range from metabolic disorders to cancer. In this review we discuss some of the diseases caused due to mislocalization. We further focus on application of nucleocytoplasmic transport to drug delivery. Various rationales to treat diseases by exploiting intracellular transport machinery have been proposed. Although the pathways for intracellular movement of proteins have been defined, these have not been adequately utilized for management of diseases involving mislocalized proteins. This review stresses the need for designing drug delivery systems utilizing these mechanisms as this area is least exploited but offers great potential.

Animals↗
PubMed1999

The yeast nucleoporin Nup2p is involved in nuclear export of importin alpha/Srp1p.

The importin alpha.beta heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin alpha subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in Deltanup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in Deltanup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an N-terminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export. Formation of the Srp1p.Cse1p. RanGTP export complex releases Srp1p from its binding site in Nup2p. We propose that Nup2p may act as a scaffold that facilitates formation of the Srp1p export complex.

Animals↗
PubMed2003

Impacts of altered RNA metabolism on abscisic acid signaling.

The plant hormone abscisic acid (ABA) regulates many essential processes in growth and development. The recent characterization of ABA-sensitivity mutations in RNA-binding proteins has led to the recognition of a functional link between post-transcriptional mRNA processing and the ABA signal transduction machinery. By influencing transcript abundance, these RNA-binding proteins may modulate ABA signaling through the alteration of mRNA processing events such as splicing, 3' processing, nuclear export, transcript stability and RNA degradation.

Abscisic Acid↗
PubMed1999

Nucleocytoplasmic traffic of MAP kinases.

MAPK pathways represent a unique extracellular signal response system. An important feature of such a multicomponent system appears to be the spatial intracellular organization of individual components. Recent studies demonstrate that the MAP kinases of such pathways are the molecular link between the plasma membrane sensors and the nuclear transcription factors. Stimulation of several MAPK pathways induces rapid and transient nuclear accumulation of MAP kinases. Investigations on the mode of regulation of this process using higher eukaryotes Erk2 and lower eukaryotes Hog1 and Sty1/Spc1 have revealed that at least three events contribute to signal-induced nuclear localization of these MAP kinases: activation by phosphorylation, regulated nuclear import and export, and nuclear retention.

Animals↗
PubMed1998

A protein required for nuclear-protein import, Mog1p, directly interacts with GTP-Gsp1p, the Saccharomyces cerevisiae ran homologue.

We previously isolated 25 temperature-sensitive gsp1 alleles of Saccharomyces cerevisiae Ran homologue, each of which possesses amino acid changes that differ from each other. We report here isolation of three multicopy suppressors-PDE2, NTF2, and a gene designated MOG1-all of which rescued a growth defect of these gsp1 strains. The gsp1 suppression occurred even in the absence of GSP2, another S. cerevisiae GSP1-like gene. Previously, NTF2 was reported to suppress gsp1 but not PDE2. Mog1p, with a calculated molecular mass of 24 kDa, was found to be encoded by the yeast ORF YJR074W. Both MOG1 and NTF2 suppressed a series of gsp1 alleles with similar efficiency, and both suppressed gsp1 even with a single gene dose. Consistent with the high efficiency of gsp1 suppression, Mog1p directly bound to GTP, but not to GDP-Gsp1p. The disruption of MOG1 made yeast temperature-sensitive for growth. Deltamog1, which was suppressed by overexpression of NTF2, was found to have a defect in both classic and nonclassic nuclear localization signal-dependent nuclear-protein imports, but not in mRNA export. Thus, Mog1p, which was localized in the nucleus, is a Gsp1p-binding protein involved in nuclear-protein import and that functionally interacts with Ntf2p. Furthermore, the finding that PDE2 suppressed both gsp1 and rna1-1 indicates that the Ran GTPase cycle is regulated by the Ras-cAMP pathway.

Amino Acid Sequence↗
PubMed2004

Regulation of heme oxygenase-1 gene transcription: recent advances and highlights from the International Conference (Uppsala, 2003) on Heme Oxygenase.

Recent investigations into the regulation of heme oxygenase-1 gene (hmox-1) transcription have exposed mechanisms of increasing diversity and complexity worthy of a gene whose expression is modulated by a seemingly endless array of physiological, pathophysiological, and nonphysiological agents and conditions. For instance, contrary to initial and prevalent assumptions that inducer-dependent gene stimulation is mediated principally by the positive action of transcription activators, it now appears that such induction may occur secondarily to deactivation of the repressor protein, Bach1. As a further complication, heme and cadmium, two potent inducers of the hmox-1 gene, inhibit Bach1 function by different mechanisms-by inhibition of DNA binding or promotion of nuclear export, respectively. Bach1 also plays a role in signal-dependent hmox-1 gene repression, an increasingly appreciated phenomenon that is manifested in a species- and cell-specific manner. Although extreme concentrations of the heme oxygenase-1 protein resulting from the opposing phenomena of gene activation and repression have physiological consequences, even minor modulation in the level of this enzyme, as elicited by variations in the length of a dinucleotide repeat region within the human hmox-1 promoter, may be of clinical relevance. Finally, mechanistic diversity is also apparent in the type and combination of protein kinase-dependent, signal transduction pathways used during hmox-1 gene activation.

Animals↗
PubMed1998

The plurifunctional nucleolus.

The nucleolus of eukaryotic cells was first described in the early 19th century and was discovered in the 1960s to be the seat of ribosome synthesis. Although rRNA transcription, rRNA processing and ribosome assembly have been clearly established as major functions of the nucleolus, recent studies suggest that the nucleolus participates in many other aspects of gene expression as well. Thus, the nucleolus has been implicated in the processing or nuclear export of certain mRNAs. In addition, new results indicate that biosyntheses of signal recognition particle RNA and telomerase RNA involve a nucleolar stage and that the nucleolus is also involved in processing of U6 RNA, one of the spliceosomal small nuclear RNAs. Interestingly, these three nucleolus-associated small nuclear RNAs (signal recognition particle RNA, telomerase RNA and U6 RNA) are components of catalytic ribonucleoprotein machines. Finally, recent work has also suggested that some transfer RNA precursors are processed in the nucleolus. The nucleolus may have evolutionarily descended from a proto-eukaryotic minimal genome that was spatially linked to vicinal RNA processing and ribonucleoprotein assembly events involved in gene read-out. The nucleolus of today's eukaryotes, now surrounded by the chromatin of over 2 billion years of genome expansion, may still perform these ancient functions, in addition to ribosome biosynthesis. The plurifunctional nucleolus concept has a strong footing in contemporary data and adds a new perspective to our current picture of the spatial-functional design of the cell nucleus.

Cell Nucleolus↗
PubMed1997

Nuclear localization of I kappa B alpha promotes active transport of NF-kappa B from the nucleus to the cytoplasm.

I kappa B alpha tightly regulates the transcriptional activity of NF-kappa B by retaining it in the cytoplasm in an inactive form. In the present work, we report that I kappa B alpha, when expressed in the nuclear compartment, not only abrogates NF-kappa B/DNA interactions and NF-kappa B-dependent transcription, but also transports NF-kappa B back to the cytoplasm. This function of I kappa B alpha is insured by a nuclear export sequence located in the C-terminal domain of I kappa B alpha and homologous to the previously described export signal found in HIV-1 Rev protein as well as in PKI (the inhibitor of the catalytic subunit of protein kinase A). Thus, inhibition of NF-kappa B/DNA binding and the consecutive efficient nuclear export of the transcription factor of I kappa B alpha could represent an important mechanism for the control of the expression of NF-kappa B-dependent genes.

Amino Acid Sequence↗
PubMed2005

RanBP3 enhances nuclear export of active (beta)-catenin independently of CRM1.

beta-Catenin is the nuclear effector of the Wnt signaling cascade. The mechanism by which nuclear activity of beta-catenin is regulated is not well defined. Therefore, we used the nuclear marker RanGTP to screen for novel nuclear beta-catenin binding proteins. We identified a cofactor of chromosome region maintenance 1 (CRM1)-mediated nuclear export, Ran binding protein 3 (RanBP3), as a novel beta-catenin-interacting protein that binds directly to beta-catenin in a RanGTP-stimulated manner. RanBP3 inhibits beta-catenin-mediated transcriptional activation in both Wnt1- and beta-catenin-stimulated human cells. In Xenopus laevis embryos, RanBP3 interferes with beta-catenin-induced dorsoventral axis formation. Furthermore, RanBP3 depletion stimulates the Wnt pathway in both human cells and Drosophila melanogaster embryos. In human cells, this is accompanied by an increase of dephosphorylated beta-catenin in the nucleus. Conversely, overexpression of RanBP3 leads to a shift of active beta-catenin toward the cytoplasm. Modulation of beta-catenin activity and localization by RanBP3 is independent of adenomatous polyposis coli protein and CRM1. We conclude that RanBP3 is a direct export enhancer for beta-catenin, independent of its role as a CRM1-associated nuclear export cofactor.

Active Transport, Cell Nucleus↗
PubMed2000

Poly(ADP-ribose) binds to specific domains in DNA damage checkpoint proteins.

Poly(ADP-ribose) is formed in possibly all multicellular organisms by a familiy of poly(ADP-ribose) polymerases (PARPs). PARP-1, the best understood and until recently the only known member of this family, is a DNA damage signal protein catalyzing its automodification with multiple, variably sized ADP-ribose polymers that may contain up to 200 residues and several branching points. Through these polymers, PARP-1 can interact noncovalently with other proteins and alter their functions. Here we report the discovery of a poly(ADP-ribose)-binding sequence motif in several important DNA damage checkpoint proteins. The 20-amino acid motif contains two conserved regions: (i) a cluster rich in basic amino acids and (ii) a pattern of hydrophobic amino acids interspersed with basic residues. Using a combination of alanine scanning, polymer blot analysis, and photoaffinity labeling, we have identified poly(ADP-ribose)-binding sites in the following proteins: p53, p21(CIP1/WAF1), xeroderma pigmentosum group A complementing protein, MSH6, DNA ligase III, XRCC1, DNA polymerase epsilon, DNA-PK(CS), Ku70, NF-kappaB, inducible nitric-oxide synthase, caspase-activated DNase, and telomerase. The poly(ADP-ribose)-binding motif was found to overlap with five important functional domains responsible for (i) protein-protein interactions, (ii) DNA binding, (iii) nuclear localization, (iv) nuclear export, and (v) protein degradation. Thus, PARPs may target specific signal network proteins via poly(ADP-ribose) and regulate their domain functions.

Alanine↗
PubMed2001

Regulated nucleocytoplasmic transport of protein kinase D in response to G protein-coupled receptor activation.

Protein kinase D (PKD)/protein kinase C mu is a serine/threonine protein kinase activated by growth factors, antigen-receptor engagement, and G protein-coupled receptor (GPCR) agonists via a phosphorylation-dependent mechanism that requires protein kinase C (PKC) activity. In order to investigate the dynamic mechanisms associated with GPCR signaling, the intracellular distribution of PKD was analyzed in live cells by imaging fluorescent protein-tagged PKD and in fixed cells by immunocytochemistry. We found that PKD shuttled between the cytoplasm and the nucleus in both fibroblasts and epithelial cells. Cell stimulation with mitogenic GPCR agonists that activate PKD induced a transient nuclear accumulation of PKD that was prevented by inhibiting PKC activity. The nuclear import of PKD requires its cys2 domain in conjunction with a nuclear import receptor, while its nuclear export requires its pleckstrin homology domain and a competent Crm1-dependent nuclear export pathway. This study thus characterizes the regulated nuclear transport of a signaling molecule in response to mitogenic GPCR agonists and positions PKD as a serine kinase whose kinase activity and intracellular localization is coordinated by PKC.

3T3 Cells↗
PubMed2000

Early steps in termination of the immortalization state in Burkitt lymphoma: induction of genes involved in signal transduction, transcription, and trafficking by the ganglioside IV(3)NeuAc-nLcOse(4)Cer.

Stimulation by the ganglioside IV(3)NeuAc-nLcOse(4)Cer leads to growth arrest in the Burkitt lymphoma cell line Raji. In order to analyze the primary response of Raji cells to that stimulus, a cDNA array screen and a suppression subtractive hybridization-PCR approach were performed. Twenty-four genes with assigned functions were confirmed to be induced by the ganglioside in reverse Northern blot experiments covering e.g. protein kinase B, phospholipase C, the MAP-kinase ERK3, the transcription factors YY1, DR1 and NSEP, the membrane traffic protein TAP, and the nuclear export protein CRM1. Most of the genes identified are involved in signal transduction, transcription, and cell trafficking. For selected genes, the induction of expression was quantified by semiquantitative RT-PCR.

Burkitt Lymphoma↗
PubMed2000

Nucleocytoplasmic translocation of Stat1 is regulated by a leucine-rich export signal in the coiled-coil domain.

Signal transducer and activator of transcription (Stat) proteins are latent transcription factors that reside in the cytoplasm before activation. On cytokine-induced tyrosine phosphorylation, these molecules dimerize and accumulate transiently in the nucleus. No specific signals mediating these processes have been identified to date. In this report, we examine the nuclear export of Stat1. We find that treatment of cells with the export inhibitor leptomycin B does not affect steady-state localization of Stat1 but impedes nuclear export after IFNgamma-induced nuclear accumulation. We identify a conserved leucine-rich helical segment in the coiled-coil domain of Stat1, which is responsible for the efficient nuclear export of this protein. Mutation of two hallmark leucines within this segment greatly attenuate the back transport of Stat1 in the cytoplasm. When fused to a carrier protein, the Stat1 export sequence can mediate nuclear export after intranuclear microinjection. We show that prolonging the nuclear presence of Stat1 by inhibiting nuclear export reduces the transcriptional response to stimulation with IFNgamma. These data suggest that Stats are actively exported from the nucleus via several separate pathways and link this activity to transcriptional activation.

Amino Acid Sequence↗