Polyubiquitin-Photoactivatable Crosslinking Reagents for Mapping Ubiquitin Interactome Identify Rpn1 as a Proteasome Ubiquitin-Associating Subunit

Michal Chojnacki, Wissam Mansour, Dharjath S Hameed, Rajesh K Singh, Farid El Oualid, Rina Rosenzweig, Mark A Nakasone, Zanlin Yu, Fabian Glaser, Lewis E Kay, David Fushman, Huib Ovaa (Lead / Corresponding author), Michael H Glickman (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

34 Citations (Scopus)

Abstract

Ubiquitin (Ub) signaling is a diverse group of processes controlled by covalent attachment of small protein Ub and polyUb chains to a range of cellular protein targets. The best documented Ub signaling pathway is the one that delivers polyUb proteins to the 26S proteasome for degradation. However, studies of molecular interactions involved in this process have been hampered by the transient and hydrophobic nature of these interactions and the lack of tools to study them. Here, we develop Ub-phototrap (UbPT), a synthetic Ub variant containing a photoactivatable crosslinking side chain. Enzymatic polymerization into chains of defined lengths and linkage types provided a set of reagents that led to identification of Rpn1 as a third proteasome ubiquitin-associating subunit that coordinates docking of substrate shuttles, unloading of substrates, and anchoring of polyUb conjugates. Our work demonstrates the value of UbPT, and we expect that its future uses will help define and investigate the ubiquitin interactome.

Original languageEnglish
Pages (from-to)443-457.e6
Number of pages22
JournalCell Chemical Biology
Volume24
Issue number4
DOIs
Publication statusPublished - 20 Apr 2017

Keywords

  • Binding Sites
  • Cross-Linking Reagents/chemistry
  • Molecular Docking Simulation
  • Nuclear Magnetic Resonance, Biomolecular
  • Polyubiquitin/chemistry
  • Proteasome Endopeptidase Complex/chemistry
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Subunits/chemistry
  • Saccharomyces cerevisiae/metabolism
  • Saccharomyces cerevisiae Proteins/chemistry
  • Ubiquitin/chemistry
  • Ubiquitination/radiation effects
  • Ultraviolet Rays

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