DNA repair and global sumoylation are regulated by distinct Ubc9 noncovalent complexes

Abstract

Global sumoylation, SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Mechanisms driving selectivity for each of these processes are largely uncharacterized. Here, through crystallographic analyses we show that the SUMO E2 Ubc9 forms a noncovalent complex with a SUMO-like domain of Rad60 (SLD2). Ubc9:SLD2 and Ubc9:SUMO noncovalent complexes are structurally analogous, suggesting that differential recruitment of Ubc9 by SUMO or Rad60 provides a novel means for such selectivity. Indeed, deconvoluting Ubc9 function by disrupting either the Ubc9:SLD2 or Ubc9:SUMO noncovalent complex reveals distinct roles in facilitating sumoylation. Ubc9:SLD2 acts in the Nse2 SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli1 E3 SUMO ligase. Moreover, this Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. Overall, we determine that, unexpectedly, Ubc9 noncovalent partner choice dictates the role of sumoylation in distinct cellular pathways.

Fig. 1.

Ubc9:SLD2 complex and NFAT2CIP SLD2 structure. (A) The 1.9-Å resolution crystal structure of Ubc9:SLD2 complex reveals that SLD2 (green) binds to the α1-β1 noncovalent interaction site of Ubc9 (cyan), instead of the active site centered on reside Cys93. (B) SLD2 FEG motif Glu380, 3₁₀-helix-β5 residues Glu396, Asp399, and Gln400, and the Arg339 side chain form hydrogen bond interactions (dashed black lines) with Ubc9 residues. (C) The upper panels shows the Rad60 SLD2 sequence, containing the Ubc9-interacting FEG motif, aligned to budding yeast Smt3p, human SUMO-1, and human NFATC2IP SLD2. Secondary structure of Rad60 SLD2 is depicted above the alignment. Red highlights amino acid side chains that form hydrogen bonds or salt bridges to Ubc9, green highlights residues having significant hydrophobic interactions in the Ubc9 interface, and NFATC2IP residues marked in blue show conserved residues likely having direct interactions with Ubc9. The lower panel shows fission yeast, budding yeast, mouse, and human Ubc9 structure-based sequence alignment, with fission yeast Ubc9 secondary structure depicted above the alignment. Red highlights side chains forming hydrogen bonds or salt bridges to SUMO/SLD2 residues at the noncovalent interface, green highlights residues having hydrophobic interactions at this interface, and bold marks the Asp-His-Pro-Phe-Gly-Phe motif that forms a key component of Ubc9's interaction with SUMO/SLD2. H. sapiens, Homo sapiens; M. musculus, Mus musculus. (D) The 1.6-Å crystal structure of NFATC2IP SLD2 reveals a β-GRASP fold composed of five β-strands, an α-helix, and a 3₁₀-helix. The ellipse highlights the region in SUMO between β-strand 2 and α-helix 1 observed to bind SIM sequences. (E) Electrostatic surface diagram for NFATC2IP SLD2 in the same orientation as in panel D, with the ellipse marking the surface of the β-strand 2 α-helix 1 region that shows a net negative charge and lack of a clear binding cleft, distinct from the electropositive SIM-binding pocket in SUMO. (F) Structural superimposition of the Ubc9:SLD2 complex (cyan, Ubc9; green, SLD2) with NFATC2IP SLD2 (orange), showing close structural similarity, despite low sequence identities. (G) Close-up view of structural superimposition shown in panel F revealing a well-conserved, noncovalent Ubc9-binding face in NFATC2IP SLD2, with well-conserved residues depicted as sticks. These residues include Asp394 of β3–β4-loop FDG motif and the carboxylate residues in the 3₁₀-helix β-strand 5 region, including residues Glu410 and Asp413, in addition to Glu416 that is suitably orientated for interactions with Ubc9.

Fig. 2.

Phenotypes caused by Ubc9 mutations at the noncovalent interface. (A) Western analysis following in vitro GSH-Sepharose pulldowns of either bacterially expressed GST-Ubc9 or GST-Ubc9^H20D incubated with His₆-SLD2. Immunoblots are shown using antiserum for hexahistidine (His₆) or GST. (B) Serial dilutions of the ubc9-3 mutant, plated on medium lacking thiamine to induce protein expression from the indicated plasmids. Cells were either untreated (No Drug) at the indicated temperatures or treated with HU at 32°C. pVector denotes an empty vector. (C) Serial dilutions of the indicated strains, which were plated on nonselective medium and either untreated (No Drug) at the indicated temperatures or treated with HU at 32°C. α, anti.

Fig. 3.

In vivo effect of abrogating the noncovalent Ubc9:SUMO complex. (A) Western analysis following in vitro GSH-Sepharose pulldowns of either bacterially expressed GST-Ubc9 or GST-Ubc9^H20D incubated with His₆-SUMO. Immunoblots are shown using antiserum for His₆ or GST. (B) Western analysis following in vitro GSH-Sepharose pulldowns of bacterially expressed GST-Ubc9 incubated with either His₆-SUMO or His₆-SUMO^D81R. Immunoblots are shown using antiserum for His₆ or GST. (C) Western analysis of a bacteria-based sumoylation assay. Total lysates of BL21(DE3) strains expressing S. pombe E1, E2, and either conjugatable SUMO (SUMO-gg), nonconjugatable SUMO (SUMO-Δgg), or conjugatable SUMO^D81R (SUMO^D81R-gg) are shown. Immunoblotting was performed using antiserum for SUMO. Coomassie staining is shown as a loading control. (D) Serial dilutions of the indicated strains plated on medium and either untreated (No Drug), treated with the indicated concentrations of genotoxins, or UV irradiated. (E) Western analysis of total lysates of the indicated strains, immunoblotted using antiserum for SUMO or tubulin (loading control). (F) Western analysis of total lysates of the indicated strains immunoblotted using antisera for SUMO, Cdc2, and GST (loading controls). Strains containing pRad60-SLD2 and pRad60-SLD2^E380R were cultured in medium lacking thiamine to induce overexpression (OP) of these ectopic vectors. (G) In vitro competition assay of SLD2 and SUMO binding to Ubc9. Proteins were detected following SDS-PAGE separation by Coomassie staining and then quantified. Numbers shown below gels indicate the ratio of bound SLD2 to SUMO (SLD2/SUMO). (H) Western analysis of lysates from the indicated strains, immunoblotted with antisera to SUMO and tubulin (loading control). Cells were cultured either with or without hydroxyurea (15 mM) treatment for 4 h.

Fig. 4.

Ubc9 partner choice promotes distinct sumoylation pathways. (A) Serial dilutions of the indicated strains plated on medium and either untreated (No Drug) or treated with the indicated concentrations of drugs. (B) A representative tetrad dissection is shown from a cross between rad60^E380R and SUMO^D81R mutant cells. Key depicts the genotypes present, which are denoted by various shapes placed around each colony within the tetrads. (C) As described above for panel B but with nse2-SA crossed with SUMO^D81R mutant cells. (D) Serial dilutions of the indicated strains plated on medium lacking thiamine to induce expression from the indicated vectors. Plates were either drug free (No Drug) or treated with HU. wt, wild type.

Fig. 5.

SUMO^D81R suppresses STUbL mutant phenotypes due to a defect in SUMO chain formation. (A) Serial dilutions of the indicated strains plated on nonselective medium and grown at either permissive (25°C) or restrictive (35°C) temperature. (B) Western analysis of total lysates of the indicated strains immunoblotted with antiserum for SUMO or tubulin (loading control). Cells were cultured at the restrictive temperature (35°C) in medium lacking thiamine to induce expression from the indicated vectors. (C) As described in panel B, but the indicated strains were cultured at 32°C. (D) Serial dilutions of the indicated strains plated on medium lacking thiamine to induce expression from the vectors and either untreated at a permissive (25°C) or restrictive (35°C) temperature or HU treated at 25°C. (E) Western analysis of total lysates of the indicated strains immunoblotted with antiserum for SUMO (*, loading control). (F and G) The indicated strains were serially diluted onto plates containing no drug and either grown at either the permissive or nonpermissive temperature or plated at 32°C with the indicated concentrations of HU.

Fig. 6.

Critical SUMO chain-independent functions for Pli1 and Ubc9:SUMO. (A) A representative tetrad dissection is shown of an rhp51Δ pli1Δ top1Δ strain back-crossed against a wild-type strain. The key depicts the genotypes present, which are denoted by various shapes around each colony; wild-type colonies are not marked. (B) As described above, with the rhp51Δ top1Δ strain crossed against the SUMO^D81R mutant. (C) As described above, from a cross between the rhp51Δ and SUMO^K14/30R mutants. (D and E) Serial dilutions of the indicated strains plated on medium that was either drug free (No Drug) or treated with the indicated concentrations of HU or camptothecin (CPT). All cells were grown at 30°C.

Fig. 7.

Model for the roles of Ubc9:SLD2 and Ubc9:SUMO complexes in sumoylation. The Ubc9:SUMO complex supports both Pli1-mediated global sumoylation and Pli1-mediated SUMO chain formation. The Ubc9:Rad60 SLD2 complex, through the observed interaction of Rad60 with the Smc5/6 complex, facilitates sumoylation by the Nse2 E3 SUMO ligase. Rad60 also has Ubc9 complex-independent functions as indicated. Although not supported by our data, we do not fully exclude the possibility that Rad60, by competing for Ubc9 binding, can play a minor role in modulating the function of Ubc9:SUMO. Additionally, Nse2 could also play a role in SUMO chain formation, but such species were not detectable in our analyses. See Discussion for further details.