SUMO is a small ubiquitin-like modifier. The fluorescent fused SUMO (active for sumoylation) localized in the nucleus, while C-terminal truncated SUMO (inactive for sumoylation) diffused in the cytoplasm. Daxx is a SUMO target protein, locates predominantly in the nucleus. It has been identified as a component of the PODs. During extracellular stimulation, Daxx could be recruited to the cytoplasm with the existence of Ask1. Therefore, it is a shuttle protein. Daxx should contain nuclear localization signal (NLS) and nuclear export signal (NES) motifs. To identify the NES and NLS motifs on Daxx, Daxx were truncated into four segments. Several amino acids on the predicted NES and NLS motifs were mutated. Our results showed that the truncated Daxx fragments D1 (containing NES) and D4 (containing NLS2) could be translocated into nucleus independently. However, either NES or NLS2 mutants disrupted their translocation into nucleus. It indicated that both NES and NLS2 motif of Daxx were involved in the nuclear transport. Nevertheless the co-transfection of SUMOs and Daxx showed that the interactions between SUMO active form and Daxx mutants and between inactive SUMO and Daxx wild type rescued the nuclear transport function of Daxx mutants and inactive SUMO. Therefore, SUMO may play a role in the nuclear transport of Daxx by either sumoylation or interaction with Daxx in cytoplasm, and Daxx may recruit inactive SUMOs into nucleus by interaction.

Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions exhibit a substantial export of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. In the current report, we have defined nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1 dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

Potato yellow dwarf virus (PYDV) is the type species of the genus nucleorhabdovirus which is typified by its nucleotropic characters of the members. The virus accomplishes its replication and morphogenesis in the nuclei of infected cells. Two strains, Constricta strain (CYDV) and Sanguinolenta strain (SYDV) have been described at the level of vector-specificity. CYDV is vectored by Agallia constricta and SYDV is transmitted by Aceratagllia sanguinolenta. The full-length genome of CYDV was sequenced. The 12,792 nt antisense genome encodes seven open reading frames in the order of, nucleocapsid protein (N), unknown protein (X), phosphoprotein (P), movement protein (Y), matrix protein (M), glycoprotein (G), and large polymerase protein (L). The features of each protein including a nuclear localization signal, isoelectric point, and transmembrane domain, were determined by predictive algorithms. The gene coding region was flanked by leader and trailer, and each ORF was separated by a conserved intergenic junction. In the intergenic junctions, the highly conserved cis-regulatory elements, polyadenylation signal, gene spacer, and transcription start site, were identified. The similarities of amino acid sequences between each cognate protein of SYDV and CYDV were higher than 80% except for X and P proteins. The protein localization and interaction assays of each CYDV protein identified strain-specific associations in comparison with those of SYDV and generated unique protein interaction and localization map compared to SYDV. Phylogenetic analysis using L protein identified that CYDV forms a clade with other leafhopper-transmitted rhabdoviruses. Protein sequence comparisons revealed that CYDV X has greater similarity to the cognate protein of Eggplant mottle disease virus than to SYDV X. The localization patterns of CYDV-N and -Y were different compared the cognate proteins of SYDV. The functional nuclear export domain of SYDV M was identified using c-terminal fragments of the Mwt(aa 211-243), MLL223AA(aa 211-243), and MKR225AA(aa 211-243). Based on the data, the functional domains M mediating membrane association, nuclear import and export were mapped for both strains and suggested a model whereby M mediates intra- and intercellular movement of PYDV nucleocapsid.

In Tabak interagieren das Ankyrin-repeat Protein NtANK1 und der basische Leucin Zipper (bZIP)-Transkriptionsfaktor NtBZI-1. Diese Proteine sind in Auxin-vermittelter Genaktivierung und in Pathogenabwehr involviert. Ziel dieser Arbeit war es, die Funktion der homologen Ankyrin-repeat Proteine AKR2A und AKR2B aus Arabidopsis thaliana zu untersuchen. Dazu wurde die Interaktion zwischen AKR2A/B, und den homologen bZIP-Transkriptionsfaktoren der Gruppe C getestet. Mit verschiedenen Methoden, wie Hefe- und Protoplasten-two-hybrid und BiFC ( bimolecular fluorescence complementation ) konnte eine Interaktion der Arabidopsis Proteine nicht bestätigt werden. Lokalisationsstudien von YFP-AKR2A/B-Fusionsproteinen bestätigten, dass die Proteine im Cytoplasma lokalisiert sind. Sie besitzen ein funktionsfähiges Kernexportsignal und akkumulieren nach Inhibierung des Kernexports im Kern. Zur Funktionsaufklärung wurden AKR2-RNAi Pflanzen erzeugt, die sich phänotypisch vom Wildtyp unterscheiden: sie zeigen ein verringertes Wachstum und einen reduzierten Chlorophyllgehalt, abhängig von der Ausprägung des RNAi-Effektes. In elektronenmikroskopischen Untersuchungen ist zu erkennen, dass sich die Blattchloroplasten der AKR2-RNAi Pflanzen von denen des Wildtyps morphologisch unterscheiden und in ihrer Entwicklung unspezifisch beeinträchtigt sind. Eine Transkriptomanalyse der AKR2-RNAi Pflanzen zeigte, dass Gene des Endomembransystems herunterreguliert sind und viele Stress-induzierte Gene hochreguliert sind. Deshalb wurden die Pflanzen verschiedenen Stressbedingungen unterzogen und übereinstimmend stellte sich heraus, dass sie anfälliger gegenüber oxidativem Stress, Infektion mit dem biotrophen Bakterium Pseudomonas syringae und Infektion mit dem nekrotrophen Pilz Botrytis cinerea waren. Diese erhöhte Anfälligkeit kann als sekundärer Effekt aufgrund der beeinträchtigten Chloroplasten-Biogenese oder als spezifische Reaktion auf die reduzierte AKR2A/B-Proteinmenge interpretiert werden. Da AKR2A/B bereits als Importver mittler für chloroplastidäre Membranproteine beschrieben wurden (Bae et al., 2008), werden zusammenfassend mit den hier erhaltenen Daten multiple Funktionen für AKR2A und AKR2B diskutiert: Transport von Proteinen zu verschiedenen Endomembransystemen, eine Funktion im Signalaustausch zwischen Chloroplast und Kern, und eine Regulation der Transkriptionskontrolle im Kern.

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The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. <p>Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.<p>The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues 101 to 113 in dTIS11 sequence. We also identify a cryptic Transportin-dependent PY nuclear localization signal (PY-NLS) in the tandem zinc finger region of dTIS11 and show that it is conserved across the TIS11 protein family. This PY-NLS partially overlaps the second zinc finger (ZnF2) of dTIS11. Importantly, mutations disrupting the capacity of the ZnF2 to coordinate a Zn2+ ion unmask dTIS11 and TTP PY-NLS and promote nuclear import. Taken together, our results indicate that the nuclear export of Drosophila and mammalian TIS11 proteins is mediated by CRM1 through diverging NESs, while their nuclear import mechanism might rely on a conserved PY-NLS whose activity is negatively regulated by ZnF2 folding.<p>In the second part, we present preliminary results which implicate the nucleo-cytoplasmic transport machinery in the assembly of stress granules (SGs) in mammalian cells. SGs contain silenced mRNPs which resemble stalled initiation complexes, and they form transiently in response to acute stress, concomitantly with a global arrest of translation. While their exact role remains undefined, it seems clear that SGs are able to exchange mRNPs with polysomes and with PBs, and that they are connected to post-transcriptional and translational regulations of gene expression during stress. Here, we show that inhibition of Transportin-1 expression or function does not affect the translational status of cells but impairs the assembly of stress granules. Finally, we show that Transportin-1 and -2B, but not -2A, localize into stress granules in response to several stresses. <p>In conclusion, we suggest two potential new roles for Transportins, in the nucleo-cytoplasmic traffic of TIS11 proteins on the one hand and in the assembly of stress granules on the other hand.<p>/<p>Le compartimentage nucléo-cytoplasmique permet aux cellules eucaryotes de réguler l’expression génétique par des mécanismes post-transcriptionnels élaborés. Les ARN messagers subissent plusieurs étapes de maturation dans le noyau avant d’être exportés vers le cytoplasme où ils sont traduits et dégradés. Ces processus sont effectués via des protéines de liaison à l’ARN, ou RBPs. Beaucoup de RBPs exercent des fonctions différentes dans le noyau et dans le cytoplasme, et leur activité peut dès lors être rapidement modulée par une modification de leur localisation.<p>Le transport nucléo-cytoplasmique actif des protéines s’effectue à travers les pores nucléaires et fait majoritairement appel à des transporteurs solubles de la famille des karyophérines. Ceux-ci reconnaissent au sein des protéines à transporter une séquence-passeport appelée NLS (nuclear localization signal) ou NES (nuclear export signal) selon la direction nécessitée. <p>Le présent travail comporte deux parties. La première porte sur la localisation subcellulaire et le transport nucléo-cytoplasmique des protéines de la famille TIS11, et plus particulièrement de dTIS11 qui est le seul représentant de cette famille chez la Drosophile. Comme ses homologues dans d’autres espèces, dTIS11 est une RBP qui favorise la déadénylation et la dégradation de ses ARN messagers cibles. Nos résultats démontrent que dTIS11 fait la navette entre le noyau et le cytoplasme. L’export de dTIS11 hors du noyau est réalisé par la karyophérine CRM1 et fait appel à un NES différent de celui présent chez les protéines TIS11 mammaliennes. Nous identifions également un NLS cryptique au sein du domaine à deux doigts de zinc avec lequel dTIS11 lie l’ARN. Ce NLS correspond partiellement au signal consensus reconnu par la Transportine. Il est démasqué par la mutation du second doigt de zinc ;dans ces conditions, il permet l’import de dTIS11 par la Transportine. Enfin, nous montrons qu’il est conservé dans d’autres protéines de la famille TIS11. <p>Dans la seconde partie, nous nous intéressons aux granules de stress, qui sont des microdomaines cytoplasmiques dans lesquels se concentrent des RBPs et des ARN messagers non traduits en réponse à un stress cellulaire. Nous montrons que les karyophérines appartenant à la sous-famille des Transportines sont présentes dans ces granules et que l’inhibition de l’expression ou de la fonction des Transportines réduit la formation de ces granules en réponse à divers stress cellulaires. Nous écartons la possibilité que ce résultat soit un effet indirect d’un ralentissement du métabolisme traductionnel. Nos résultats suggèrent donc une implication des Transportines dans la formation des granules de stress. <p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished