22 research outputs found
The Nup358-RanGAP complex is required for efficient importin alpha/beta-dependent nuclear import
No full textIn vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin alpha/beta-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin beta, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin beta strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore-associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin beta recycling and reformation of novel import complexes.Deutsche Forschungsgemeinschaft [KE 660/5-1, SFB523, TP18
Charakterisierung des RanGAP1-RanBP2 Komplexes in Mitose
No full textDer RanGAP1-RanBP2 Komplex stellt ein faszinierendes makromolekulares Gebilde dar, welches mindestens zwei enzymatische Aktivitäten umfasst. Zum einen beinhaltet der Komplex die GTP-Hydrolyse aktivierende Funktion, die RanGAP1 zusammen mit RanBP2 ausübt, zum anderen besitzt RanBP2 zusammen mit Ubc9 Sumo-konjugierende Aktivität. Gemeinsam sind diese Proteine essentielle Regulatoren des nukleo-zytoplasmatischen Transportes in Interphasezellen, und spielen des Weiteren eine wichtige, bislang jedoch kaum verstandene Rolle für die Funktion der Kinetochore in Mitose.Um den RanGAP1-RanBP2 Komplex spezifisch in mitotischen Zellen näher zu untersuchen, habe ich spezifisch in Mitose nach Interaktionspartnern gesucht. Dies führte zur Identifikation des Kernexportrezeptors Crm1 und der GTPase Ran als stabile Komponenten in einem Komplex mit RanGAP1, RanBP2 und Ubc9 in mitotischen Zellen. Zusätzlich schien dieser Komplex zahlreiche weitere Proteine in substöchiometrischen Mengen zu enthalten. Diese könnten beispielsweise NES-enthaltende Interaktionspartner von Crm1 und/oder Substrate für RanBP2-abhängige Sumoylierung sein. Da bisher RanBP2-abhängige Sumo-Substrate weitgehend unbekannt sind, habe ich eine Strategie entwickelt, um sumoylierte Proteine aus immungereinigtem RanGAP1-RanBP2 Komplex anzureichern. Dies ermöglichte die massenspektrometrische Identifizierung von ungefähr 90 potentiellen Sumo-Substraten, die spezifisch in mitotischen RanGAP1 Komplexen angereichert waren; 6 dieser Substrate wurden zur weiteren Charakterisierung ausgewä! hlt. Alle Kandidaten assoziierten mit mitotischen RanGAP1 Komplexen (Topo II alpha, TACC2, CKAP-5, Plk1, USP7, PIAS1). Die meisten davon konnten entweder in vitro mit rekombinanten Faktoren (TACC2, Plk1) oder als endogene Proteine, die mit mitotischem RanGAP1-RanBP2 Komplex als Quelle der Sumo E3 Ligase-Aktivität assoziiert waren (TopoII alpha, Plk1, USP7), sumoyliert werden. Auffällig war die Co-Reinigung der Sumo E3 Ligase PIAS1 mit RanGAP1 aus mitotischen Zellen; diese wurde ebenfalls effizient sumoyliert. Weitere Analysen deuteten darauf hin, dass RanGAP1 in einem Komplex mit PIAS1 enthalten ist, der sich vom RanGAP1-RanBP2 Komplex unterscheidet.In einem Nebenprojekt konnte ich zeigen, dass das Sumo-konjugierende Enzym Ubc9 in Zellen an Lysin 14 sumoyliert werden kann. Dieser Befund war wichtig, um eine biochemische Studie von Knipscheer et al. zu ergänzen, die einen neuen Mechanismus der Selektion von Sumo-Substraten identifizierte; diese Ergebnisse flossen in die Veröffentlichung Knipscheer, Flotho, Klug et al. (2008) Mol Cell ein.The RanGAP1-RanBP2 complex represents a fascinating macromolecular assembly comprising at least two enzymatic activities. On one hand, it harbors the GTP hydrolysis activating function of RanGAP1 together with RanBP2, and on the other hand, RanBP2 in concert with Ubc9 contains Sumo conjugating activity. Together, these proteins are not only crucial regulators of nucleocytoplasmic transport in interphase cells but they also play an important yet ill-defined role in kinetochore function during mitosis.To gain insight into the RanGAP1-RanBP2 complex specifically in mitotic cells, I searched for mitosis-specific interaction partners. This led to the identification of the nuclear export receptor Crm1 and the GTPase Ran as stable components in complex with RanGAP1, RanBP2 and Ubc9 in mitotic cells. In addition, the complex seemed to contain many different proteins at substochiometric levels. These could, for example, be NES containing Crm1 interactors and/or targets for RanBP2 dependent sumoylation. As RanBP2 dependent Sumo targets are largely unknown, I devised a strategy to enrich sumoylated proteins from immunoprecipitated RanGAP1-RanBP2 complexes. This allowed mass-spectrometric identification of 90 putative Sumo substrates specifically enriched in mitotic RanGAP1 complexes; 6 of these were selected for further validation. All candidates associated with mitotic RanGAP1 complexes (Topo II alpha, TACC2, CKAP-5, Plk1, USP7, PIAS1), and most of these could be sumoylated in vitro with recombinant factors (TACC2, Plk1) or as proteins associated with mitotic RanGAP1-RanBP2 complexes as source of Sumo E3 ligase activity (TopoII alpha, Plk1, USP7). Strikingly, the Sumo E3 ligase PIAS1 also co-purified with RanGAP1 from mitotic cells and was efficiently sumoylated in these experiments. Further analysis suggested that mitotic RanGAP1 is present in a complex with PIAS1 distinct from the RanGAP1-RanBP2 complex.In a side project, I could show that the Sumo conjugating enzyme Ubc9 is sumoylated on lysine 14 in cells. This finding was crucial to supplement a biochemical study by Knipscheer et al. that identified a novel mechanism for Sumo substrate selection and contributed to the publication Knipscheer, Flotho, Klug et al. (2008) Mol Cell
Transport nucléaire et mitose : pourquoi partager des acteurs communs ?
No full textColloque organisé par Valérie Doye et Richard Wozniak du 2 au 7 juillet 2007 Liens entre les acteurs du transport noyau-cytoplasme (protéines des pores nucléaires) et la machinerie de division cellulaire (fuseau mitotique et kinétochores). Participants Ariane Abrieu, Yves Barral, Valérie Doye, Annette Flotho, Vincent Galy, Helder Maiato, Michael Matunis, Andrea Musacchio, Jonathon Pines, Bernd Pulverer, Robert Scott, Jason Swedlow, Jan van Deursen, Karsten Weis, Richard Wozniak, Timothy Yen, ..
Ubc9 sumoylation regulates SUMO target discrimination
No full textPosttranslational modification with small ubiquitin-related modifier, SUMO, is a widespread mechanism for rapid and reversible changes in protein function. Considering the large number of known targets, the number of enzymes involved in modification seems surprisingly low: a single El, a single E2, and a few distinct E3 ligases. Here we show that autosumoylation of the mammalian E2-conjugating enzyme Ubc9 at Lys14 regulates target discrimination. While not altering its activity toward HDAC4, E2-25K, PML, or TDG, sumoylation of Ubc9 impairs its activity on RanGAP1 and strongly activates sumoylation of the transcriptional regulator Sp100. Enhancement depends on a SUMO-interacting motif (SIM) in Sp100 that creates an additional interface with the SUMO conjugated to the E2, a mechanism distinct from Ubc9 similar to SUMO thioester recruitment. The crystal structure of sumoylated Ubc9 demonstrates how the newly created binding interface can provide a gain in affinity otherwise provided by E3 ligases
The Sumo proteome of proliferating and neuronal-differentiating cells reveals Utf1 among key Sumo targets involved in neurogenesis
No full textPost-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.J.F.C.-V. and F.J.-V. were the recipients of FPI (MICIU, BES-2016-076500) and JAE Ph.D. (CSIC) fellowships, respectively. We acknowledge the ZMBH Core Facility for Mass Spectrometry and Proteomics. We thank Dr. Annette Flotho for help with data analysis. We also acknowledge L.M. Buch and A. Romero-Franco for preliminary results on Kctd15 and Sall4, respectively
Identification and analysis of endogenous SUMO1 and SUMO2/3 targets in mammalian cells and tissues using monoclonal antibodies
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