The RanBP2/RanGAP1∗SUMO1/Ubc9 Complex Is a Multisubunit SUMO E3 Ligase Andreas Werner, Annette Flotho, Frauke Melchior  Molecular Cell  Volume 46, Issue 3, Pages 287-298 (May 2012) DOI: 10.1016/j.molcel.2012.02.017 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 RanBP2 Associates with RanGAP1∗SUMO1 and Is Required for SUMO E3 Activity at Nuclear Pore Complexes RanBP2 forms a major part of the cytoplasmic filaments of the NPC and stably associates with sumoylated RanGAP1 and Ubc9. It comprises a Leu-rich domain anchoring the protein to the NPC, several zinc finger motifs, four Ran binding domains (1–4), several FG repeats (indicated by dashes), and a cyclophilin-like domain (Cy). The E3 ligase region is situated between RB3 and RB4. Numbers indicate amino acid positions. RanBP2 complexes used in this study were reconstituted from His-RanBP2RB3-4, full-length sumoylated RanGAP1, and untagged Ubc9. Both IR1 and IR2 of RanBP2 have been described as binding sites for Ubc9. N terminal of IR1 is a functional SIM; in an equivalent position of IR2 is a short sequence motif resembling a SIM. See also Figure S1. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 RanBP2 Is Stably Bound to Sumoylated RanGAP1 and Ubc9 (A) All RanBP2 colocalizes with RanGAP1. RanBP2 and RanGAP1 were immunostained in fixed HeLa cells and analyzed by confocal microscopy. Shown are the single channels and the merged image of a confocal section (top panel; scale bar, 5 μm). White lines in the merge correspond to the line profile plots shown in the lower right panel. Pearson's correlation coefficient and thresholded Manders' overlap coefficient 1+2 were calculated in comparison to a RanBP2/RanBP2 stained sample; a randomized RanGAP1 image served as negative control (lower left panel; mean ±SD, n = 3). (B) RanBP2 codepletes with RanGAP1 from mitotic HeLa lysates. RanGAP1 was immunodepleted in two subsequent steps from cytosol of nocodazole-arrested cells; empty beads served as control. Samples including the starting material and the proteins recovered on αRanGAP1 beads were analyzed by SDS-PAGE and immunoblotting. (C) Stable interaction between RanGAP1∗SUMO1 and RanBP2 requires Ubc9. RanBP2RB3-4 (BP2), RanGAP1∗SUMO1 (GAP1∗S), and Ubc9 (each 2.7 μM) were incubated for 20 min on ice in the indicated combinations. Mixtures were analyzed by gel filtration. Peak fractions of the trimeric complex were visualized on a Coomassie-stained SDS gel. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Stable Complex Formation of RanBP2, RanGAP1∗SUMO1, and Ubc9 Requires RanBP2's IR1 Region and Its SUMO-Interacting Motif, SIM1 (A) RanBP2 variants used in this study included the following: RanBP2RB3-4 WT, RanBP2RB3-4-L2651A/L2653A/F2658A (IR1mut), RanBP2RB3-4-L2729A/L2731A/F2736A (IR2mut), RanBP2RB3-4-I2634A/V2635A (SIM1mut), RanBP2RB3-4-I2711A/I2712A (SIM2mut), and RanBP2ΔFG, RanBP2ΔFG-ΔIR2. (B) SIM1 and IR1 are required for the formation of isopeptidase-resistant RanBP2 complexes in vitro. Purified RanBP2/RanGAP1∗SUMO1/Ubc9 complexes were incubated with GST-Senp1 and analyzed for desumoylation of RanGAP1 by western blot. Free RanGAP1∗SUMO1 was used as control. (C) SIM1 and IR1 are required for stable complex formation in vivo. Nuclear pore-associated RanGAP1 was analyzed in untransfected HeLa cells and in cells transfected with HA-RanBP2ΔFG or its variants (mutations identical to mutations in RanBP2RB3-4 described in 3A) by immunofluorescence using αRanGAP1 and αHA antibodies; DNA was stained with Hoechst. Cells transfected with RanBP2ΔFG WT, SIM1mut, or IR1mut are shown as an example representing typical phenotypes. Based on these categories, >150 cells from three independent experiments were counted for quantification (mean ±SEM). (D) Interactions mapped in this study resemble those described by Reverter and Lima (2005). See also Figure S2. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Two Functionally Distinct Molecules of Ubc9 Are Needed for Sumoylation by the RanBP2/RanGAP1∗SUMO1/Ubc9 E3 Ligase Complex (A) RanBP2/RanGAP1∗SUMO1/Ubc9 complex as the only source for E2 and E3 is not sufficient for sumoylation. A time course of YFP-Sp100 sumoylation was performed with 24 nM Ubc9 and 24 nM free RanBP2 or 24 nM RanBP2/RanGAP1∗SUMO1/Ubc9 complex as the only source for E2–E3. The lower panel shows the amount of RanBP2 and Ubc9 in the reaction. (B) The trimeric complex is an active E3 ligase. Sumoylation of YFP-Sp100 was performed with 24 nM RanBP2/RanGAP1∗SUMO1/Ubc9 complex in the presence of 55 nM (+) or 110 nM (++) additional Ubc9. (C) The catalytic cysteine of Ubc9 has no contribution to the stability of the trimeric RanBP2/RanGAP1∗SUMO1/Ubc9 complex. Trimeric complexes were reconstituted using either Ubc9 WT or its inactive variant C93S and tested for isopeptidase protection of RanGAP1∗SUMO1. Free RanGAP1∗SUMO1 was used as control. (D) Two functionally distinct Ubc9 molecules are required for E2–E3 function. A trimeric RanBP2/RanGAP1∗SUMO1/Ubc9 complex containing either Ubc9 WT or its inactive variant C93S was added to a YFP-Sp100 sumoylation reaction containing additional Ubc9 (WT or C93S). The lower panel shows the protein levels of both trimeric complexes. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 The RanBP2/RanGAP1∗SUMO1/Ubc9 Complex Is an E3 Ligase for Its Physiological Substrate Borealin (A) A complex of His-Borealin, Survivin, and the N terminus of Incenp (aa 1–58) was reconstituted from bacterially expressed proteins. A Coomassie gel after the final purification step is shown (∗, contaminating protein). (B) Borealin is sumoylated by the IR1 region of free RanBP2. Borealin was sumoylated over time with free RanBP2 WT, IR1mut, or IR2mut variant. The lower panel shows the quantification of immunoblots (mean ±SD, n = 3) and the amount of RanBP2 and Ubc9 in the reaction. (C) The RanBP2/RanGAP1∗SUMO1/Ubc9 complex is an efficient E3 ligase for Borealin in vitro. Borealin was sumoylated over time with the RanBP2/RanGAP1∗SUMO1/Ubc9 complex. The right panel shows the quantification of immunoblots (mean ±SD, n = 3). See also Figure S3. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Complex Formation Turns RanBP's IR2 Region into the Catalytic Center (A) The E3 ligase activity of the complex requires an IR2–Ubc9 interaction. Time courses of Borealin sumoylation were performed with trimeric complexes containing RanBP2 WT or IR2mut variant deficient in Ubc9 binding. As E2 enzyme in the reaction, Ubc9 WT or a variant defective in binding to RanBP2 (Ubc9-L25A/V57A) was used. The right panel shows the quantification of immunoblots (mean ±SD, n = 3) and the amount of trimeric complexes and Ubc9 in the reaction. (B and C) Time courses of Borealin (B) or YFP-Sp100 (C) sumoylation with free RanBP2 WT, free IR1mut variant, or the trimeric RanBP2/RanGAP1∗SUMO1/Ubc9 complex as E3 ligase. Right panels show the quantification of immunoblots (mean ±SD, n = 3) and the amounts of RanBP2 and Ubc9 in the reaction. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 E3 Ligase Activity of the Complex Requires Specific Binding of SUMO (A) E3 ligase activity does not require the putative SIM N-terminal of IR2. Time courses of Borealin sumoylation were performed with trimeric complexes that contained either RanBP2 WT or the SIM2mut variant. The right panel shows the quantification of immunoblots (mean ±SD, n = 3) and the amount of complexes in the reaction. (B) The thioester-bound SUMO interacts with the complex via its residues important for SIM binding. Time courses of Borealin sumoylation were performed with RanBP2 complex using WT SUMO1 or SUMO1 variants defective in binding to SIMs (SUMO1-V38A/K39A, SUMO1-K37A/V38A, SUMO1-F36L). The right panel shows the quantification of immunoblots (mean ±SD, n = 3). (C) The RanBP2/RanGAP1∗SUMO1/Ubc9 complex as multisubunit SUMO E3 ligase. See text for details and also Figure S4. Molecular Cell 2012 46, 287-298DOI: (10.1016/j.molcel.2012.02.017) Copyright © 2012 Elsevier Inc. Terms and Conditions