title: Sumoylation of the GTPase Ran by the RanBP2 SUMO E3 Ligase Complex.
abstract: The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/Ubc9 localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. RanBP2 has four Ran binding domains (RBDs), two of which flank RanBP2's E3 ligase region. We thus wondered whether the small GTPase Ran is a target for RanBP2-dependent sumoylation. Indeed, Ran is sumoylated both by a reconstituted and the endogenous RanBP2 complex in semi-permeabilized cells. Generic inhibition of SUMO isopeptidases or depletion of the SUMO isopeptidase SENP1 enhances sumoylation of Ran in semi-permeabilized cells. As Ran is typically associated with transport receptors, we tested the influence of Crm1, Imp β, Transportin, and NTF2 on Ran sumoylation. Surprisingly, all inhibited Ran sumoylation. Mapping Ran sumoylation sites revealed that transport receptors may simply block access of the E2-conjugating enzyme Ubc9, however the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Isothermal titration calorimetry revealed that NTF2 prevents sumoylation by reducing RanGDP's affinity to RanBP2's RBDs to undetectable levels. Taken together, our findings indicate that RanGDP and not RanGTP is the physiological target for the RanBP2 SUMO E3 ligase complex. Recognition requires interaction of Ran with RanBP2's RBDs, which is prevented by the transport factor NTF2.
authors.affiliation: From the Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ, ZMBH Alliance, Heidelberg, Germany.
authors.forename: Volkan
authors.initials: V
authors.lastname: Sakin
authors.affiliation: From the Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ, ZMBH Alliance, Heidelberg, Germany.
authors.forename: Sebastian M
authors.initials: SM
authors.lastname: Richter
authors.affiliation: Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany, and.
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
authors.affiliation: Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany, and Department of Clinical Chemistry, University Medical Center, 37075 Göttingen, Germany.
authors.forename: Henning
authors.initials: H
authors.lastname: Urlaub
authors.affiliation: From the Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ, ZMBH Alliance, Heidelberg, Germany, f.melchior@zmbh.uni-heidelberg.de.
authors.forename: Frauke
authors.initials: F
authors.lastname: Melchior
mesh_terms: D003546:Cysteine Endopeptidases; D010450:Endopeptidases / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018832:Molecular Chaperones / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism*; D058207:Sumoylation; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D020931:ran GTP-Binding Protein / Q000378:metabolism*
chemical_list: D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C094493:ran-binding protein 2; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C404512:SENP1 protein, human; D003546:Cysteine Endopeptidases; D020931:ran GTP-Binding Protein
keywords: E3 ligase; nuclear pore; nuclear transport; small GTPase; small GTPase Ran; small ubiquitin-like modifier (SUMO); sumoylation