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Evaluation of Substrate Ubiquitylation by E3 Ubiquitin-ligase in Mammalian Cell Lysates
Published on: May 10, 2022
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A non-canonical scaffold-type E3 ligase complex mediates protein UFMylation
Joshua J Peter1, Helge M Magnussen1, Paul A DaRosa2
1Medical Research Council Protein Phosphorylation & Ubiquitylation Unit (MRC-PPU), School of Life Sciences, University of Dundee, Dundee, UK.
The EMBO Journal
|September 19, 2022
Summary
Protein UFMylation, a vital modification, requires the UFL1 E3 ligase complex with adaptors UFBP1 and CDK5RAP3. This study reveals UFM1 conjugation mechanisms and CDK5RAP3
Area of Science:
- Biochemistry
- Molecular Biology
- Cellular Homeostasis
Background:
- Protein UFMylation, a post-translational modification involving ubiquitin-fold modifier 1 (UFM1), is crucial for cellular and endoplasmic reticulum homeostasis.
- The precise mechanisms of UFM1 conjugation onto substrates remain poorly understood.
Purpose of the Study:
- To define the minimal requirements for protein UFMylation using a rebuilding approach.
- To elucidate the biochemical basis and regulatory principles of the UFM1 E3 ligase complex.
Main Methods:
- Reconstitution of the UFMylation machinery in vitro.
- Structural prediction of the UFL1/UFBP1 complex.
- Biochemical assays to characterize E3 ligase activity and substrate recognition.
Main Results:
- The E3 ligase UFL1 requires the adaptor protein UFBP1 to form an active complex.
- The UFL1/UFBP1 complex functions as a scaffold-type E3 ligase, activating the E2 enzyme UFC1.
- CDK5RAP3 acts as a substrate adaptor, directing UFMylation to RPL26, but inhibits ligase activity in vitro.
Conclusions:
- This study reveals the essential components and mechanism of the UFM1 E3 ligase complex.
- CDK5RAP3 plays a dual role as a substrate adaptor and negative regulator in UFMylation.
- The findings provide critical insights into the regulation of this atypical post-translational modification.
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