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.
Highlights
The GTPase Ran is the key regulator of nucleocytoplasmic transport
Sumoylation of Ran by the RanBP2 Complex Depends on Ran-binding Domains in the E3 Ligase—Most Small ubiquitin-related modifier (SUMO) targets, with the notable exception of RanGAP1, cannot be sumoylated efficiently by physiologically relevant concentrations of the SUMO E1 and E2 enzymes; they require E3 ligases
The physical proximity between RanBP2’s E3 ligase region, its Ran binding domains and the Ran GTPase-activating enzyme RanGAP1 in the RanBP2 SUMO E3 ligase complex has long suggested that the GTPase Ran could be a target for RanBP2-dependent sumoylation
Summary
The GTPase Ran is the key regulator of nucleocytoplasmic transport. Results: RanGDP is modified with SUMO1 by the E3 ligase RanBP2 and deSUMOylated by the isopeptidase SENP1. Conclusion: Ran is subject to reversible sumoylation at nuclear pore complexes. Significance: SUMOylation of Ran might be a novel way of regulating the directionality of nucleocytoplasmic transport for certain cargoes. The SUMO E3 ligase complex RanBP2/RanGAP1*SUMO1/ Ubc localizes at cytoplasmic nuclear pore complex (NPC) filaments and is a docking site in nucleocytoplasmic transport. 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. Mapping Ran sumoylation sites revealed that transport receptors may block access of the E2-conjugating enzyme Ubc, the acceptor lysines are perfectly accessible in Ran/NTF2 complexes. Recognition requires interaction of Ran with RanBP2’s RBDs, which is prevented by the transport factor NTF2
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