The RanBP2/RanGAP1*SUMO1/Ubc9 complex is a multisubunit SUMO E3 ligase

Mol Cell. 2012 May 11;46(3):287-98. doi: 10.1016/j.molcel.2012.02.017. Epub 2012 Mar 29.

Abstract

RanBP2/Nup358 is an essential protein with roles in nuclear transport and mitosis, and is one of the few known SUMO E3 ligases. However, why RanBP2 functions in vivo has been unclear: throughout the cell cycle it stably interacts with RanGAP1*SUMO1 and Ubc9, whose binding sites overlap with the E3 ligase region. Here we show that cellular RanBP2 is quantitatively associated with RanGAP1, indicating that complexed rather than free RanBP2 is the relevant E3 ligase. Biochemical reconstitution of the RanBP2/RanGAP1*SUMO1/Ubc9 complex enabled us to characterize its activity on the endogenous substrate Borealin. We find that the complex is a composite E3 ligase rather than an E2-E3 complex, and demonstrate that complex formation induces activation of a catalytic site that shows no activity in free RanBP2. Our findings provide insights into the mechanism of an important E3 ligase, and extend the concept of multisubunit E3 ligases from ubiquitin to the SUMO field.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Basic Helix-Loop-Helix Transcription Factors / metabolism
  • Basic Helix-Loop-Helix Transcription Factors / physiology
  • Cell Cycle Proteins / metabolism
  • Crystallography, X-Ray
  • GTPase-Activating Proteins / chemistry
  • GTPase-Activating Proteins / metabolism
  • GTPase-Activating Proteins / physiology*
  • Humans
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / metabolism
  • Molecular Chaperones / physiology*
  • Nuclear Pore Complex Proteins / chemistry
  • Nuclear Pore Complex Proteins / metabolism
  • Nuclear Pore Complex Proteins / physiology*
  • Repressor Proteins / metabolism
  • Repressor Proteins / physiology
  • SUMO-1 Protein / chemistry
  • SUMO-1 Protein / metabolism
  • SUMO-1 Protein / physiology*
  • Sumoylation
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Conjugating Enzymes / physiology*
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Protein Ligases / physiology

Substances

  • Basic Helix-Loop-Helix Transcription Factors
  • CDCA8 protein, human
  • Cell Cycle Proteins
  • GTPase-Activating Proteins
  • Molecular Chaperones
  • Nuclear Pore Complex Proteins
  • RANGAP1 protein, human
  • Repressor Proteins
  • SIM1 protein, human
  • SUMO-1 Protein
  • SUMO1 protein, human
  • ran-binding protein 2
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases
  • ubiquitin-conjugating enzyme UBC9