A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation

Joshua J. Peter, Helge M. Magnussen, Paul A. DaRosa, David Millrine, Stephen P. Matthews, Frederic Lamoliatte, Ramasubramanian Sundaramoorthy, Ron R. Kopito, Yogesh Kulathu (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

21 Citations (Scopus)
152 Downloads (Pure)


Protein UFMylation, i.e., post-translational modification with ubiquitin-fold modifier 1 (UFM1), is essential for cellular and endoplasmic reticulum homeostasis. Despite its biological importance, we have a poor understanding of how UFM1 is conjugated onto substrates. Here, we use a rebuilding approach to define the minimal requirements of protein UFMylation. We find that the reported cognate E3 ligase UFL1 is inactive on its own and instead requires the adaptor protein UFBP1 to form an active E3 ligase complex. Structure predictions suggest the UFL1/UFBP1 complex to be made up of winged helix (WH) domain repeats. We show that UFL1/UFBP1 utilizes a scaffold-type E3 ligase mechanism that activates the UFM1-conjugating E2 enzyme, UFC1, for aminolysis. Further, we characterize a second adaptor protein CDK5RAP3 that binds to and forms an integral part of the ligase complex. Unexpectedly, we find that CDK5RAP3 inhibits UFL1/UFBP1 ligase activity in vitro. Results from reconstituting ribosome UFMylation suggest that CDK5RAP3 functions as a substrate adaptor that directs UFMylation to the ribosomal protein RPL26. In summary, our reconstitution approach reveals the biochemical basis of UFMylation and regulatory principles of this atypical E3 ligase complex.

Original languageEnglish
Article numbere111015
Number of pages19
JournalEMBO Journal
Issue number21
Early online date19 Sept 2022
Publication statusPublished - 2 Nov 2022


  • Ubiquitin-like modifier
  • E3 ligase
  • ribosome
  • posttranslational modification
  • enzyme substrate
  • post-translational modification
  • ubiquitin-like modifier

ASJC Scopus subject areas

  • General Biochemistry,Genetics and Molecular Biology
  • General Immunology and Microbiology
  • Molecular Biology
  • General Neuroscience


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