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In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
N-end rule specificity within the ubiquitin/proteasome pathway is not an affinity effect
O V Baboshina1, R Crinelli, T J Siepmann
1Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
The Journal of Biological Chemistry
|August 9, 2001
Summary
The N-end rule pathway targets proteins for degradation based on their amino-terminal residue. This study identifies the mammalian E3alpha/Ubr1 ligase and reveals specificity arises from catalytic rate, not binding affinity, explaining protein degradation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- The N-end rule pathway regulates protein degradation via the ubiquitin/26 S proteasome system.
- Protein degradation is linked to the amino-terminal residue, with basic or hydrophobic termini (type 1 and 2) predicted to degrade faster than others (type 3).
- A significant portion of eukaryotic protein degradation follows the N-end rule, despite most proteins being type 3 substrates.
Purpose of the Study:
- To purify the mammalian N-end rule ligase E3alpha/Ubr1.
- To elucidate the biochemical basis of N-end rule specificity.
- To reconcile conflicting observations regarding N-end rule targeting and inhibition.
Main Methods:
- Purification of mammalian N-end rule ligase E3alpha/Ubr1.
- In vitro ubiquitination assays using model substrates.
- Kinetic analysis (kcat, Km) of E3alpha activity.
- Inhibition studies with N-end rule dipeptides.
Main Results:
- E3alpha/Ubr1 was purified to near homogeneity.
- E3alpha forms lysine 48-linked polyubiquitin chains on all substrate types, dependent on Ubc2/Rad6.
- Specificity is determined by catalytic rate (kcat), not binding affinity (Km), with type 1 and 2 substrates processed significantly faster.
- Dipeptides act as general noncompetitive inhibitors, contradicting previous predictions.
Conclusions:
- The N-end rule specificity is primarily a catalytic rate (kcat) phenomenon.
- A model is proposed where substrate binding induces conformational changes in E3alpha, affecting catalytic efficiency.
- This model explains N-end rule targeting and the inhibitory effects of specific dipeptides.
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These groups modify specific amino acids in a protein.
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