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.
Highlights
small ubiquitin-like modifier (SUMO) chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes
NFATC2IP SLD2 crystals belonged to the P1211 space group, having cell dimensions of 32.5 Å, 32.4 Å, and 32.9 Å and 90°, 96.7°, and 90°; Ubc9:SLD2 crystals belonged to the P6 space group, having cell dimensions of 115.2 Å, 115.2 Å, and 34.8 Å and 90.0°, 90.0°, and 120.0°
Little conformational change is induced in SLD2 upon binding to Ubc9, with structural superimposition of complex and apo forms of this protein domain having a root mean square deviation (RMSD) of 0.63 Å
Summary
SUMO chain formation, and genome stabilization are all outputs generated by a limited repertoire of enzymes. Ubc9:SLD2 acts in the Nse SUMO E3 ligase-dependent pathway for DNA repair, whereas Ubc9:SUMO instead promotes global sumoylation and chain formation, via the Pli E3 SUMO ligase. This Pli1-dependent SUMO chain formation causes the genome instability phenotypes of SUMO-targeted ubiquitin ligase (STUbL) mutants. In the fission yeast Schizosaccharomyces pombe the SUMO pathway includes a single E2 called Ubc (Hus5) and two known SUMO E3 ligases, Pli and Nse2 [51]. These two E3 ligases are responsible for sumoylating largely distinct targets, how substrate specificity is generated is poorly characterized. The Rad C-terminal SLD interacts directly with Ubc9 [39], raising the possibility that Rad could selectively facilitate Nse2-dependent sumoylation via recruitment of Ubc
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