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Sumoylation of Nuclear Transport Receptors and the small GTPase Ran

dc.contributor.advisorMelchior, Frauke Prof.
dc.contributor.authorSakin, Volkande
dc.titleSumoylation of Nuclear Transport Receptors and the small GTPase Rande
dc.contributor.refereeBrose, Nils Prof.
dc.description.abstractengSumoylation has been linked to nucleocytoplasmic transport since the discovery of SUMO as a modifier of vertebrate RanGAP1, which is targeted to the nuclear pore complex (NPC) after sumoylation. The link between sumoylation and nucleocytoplasmic transport has been strengthened even more with the discovery that the major component of NPC cytoplasmic filaments, RanBP2, acts as a SUMO E3 ligase. RanBP2 is in stable complex with Ubc9 and sumoylated RanGAP1, and it was recently discovered that it is the RanBP2 complex which acts as a multisubunit E3 ligase in cells. One fascinating feature of the RanBP2 complex is the very close proximity of the E3 ligase region and the binding site for sumoylated RanGAP1, both of which are embedded between binding sites for nuclear transport receptors and RanGTP. Consequently, nuclear transport receptors and the small GTPase Ran are candidates for RanBP2 dependent sumoylation. In line with this idea, in recent mass spectrometry based screens several nuclear transport receptors and Ran were found to be potential candidates for sumoylation. In this work, I aimed to test whether nuclear transport receptors and/or Ran are subjected to RanBP2-dependent sumoylation and to further investigate the mechanistics and the functional consequences of this modification. Indeed, upon enrichment of endogenously sumoylated proteins from HeLa cells by IP/peptide elution, in vivo sumoylation of Crm1, Impβ, and Imp5 could be confirmed. Although Ran sumoylation could not be detected by IPs, I could demonstrate that it is sumoylated in the context of semi-permeabilized cells. Subsequent in vitro sumoylation experiments revealed that an 80kDa RanBP2 fragment as well as the reconstituted RanBP2/Ubc9/RanGAP1-SUMO1 complex could stimulate the sumoylation of Ran, whereas PIAS E3 ligases could not. Large scale in vitro sumoylation of Ran by RanBP2 fragment followed by mass spectrometric analysis identified K130, 132, and 134 as SUMO acceptor sites. Mutation of these three lysines into arginines allowed to investigate functional consequences. While Ran triple mutant was predominantly nuclear and behaved identical to wild type in the canonical Imp α/β-dependent import, it showed a striking stimulation in Imp13-dependent import. Intriguingly, the sumoylation deficient Ran mutant lost dependency on its import receptor NTF2, suggesting that it may enrich in the nucleus by other means. In a side project, I could show that the import of the SUMO E1 enzyme subunit, Aos1, is not supported by transportin or Imp13 and demonstrate that Imp α/β interacts with the E1 heterodimer mainly via the Uba2 NLS. These findings complemented the study published as “Imp α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme” (Moutty M.C., Sakin V., Melchior F., 2011 MBoC).de
dc.contributor.coRefereeBraus, Gerhard Prof.
dc.subject.engNuclear Transport Receptorsde
dc.subject.engNucleocytoplasmic Transportde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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