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Detection of Protein Ubiquitination
Published on: August 19, 2009
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Sensitive detection of protein ubiquitylation using a protein fragment complementation assay
Marie Le Boulch1, Audrey Brossard1, Gaëlle Le Dez1
1Univ Rennes, CNRS, IGDR (Institute of Genetics and Development of Rennes) - UMR 6290, F-35000 Rennes, France.
Journal of Cell Science
|May 16, 2020
Summary
We developed NUbiCA, a sensitive assay to detect ubiquitylation, a key protein modification. This new tool accurately monitors ubiquitylation in cells and model organisms, aiding the study of challenging cellular events.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Ubiquitylation is a crucial, reversible post-translational modification regulating diverse cellular functions.
- Detecting ubiquitylated proteins is difficult due to their low endogenous abundance.
- Existing methods often struggle with low-abundance or transient ubiquitylation events.
Purpose of the Study:
- To introduce NUbiCA, a novel and sensitive protein-fragment complementation assay.
- To enable robust monitoring of ubiquitylation in cultured cells and model organisms.
- To provide a resource for studying challenging ubiquitylation dynamics.
Main Methods:
- Development and application of the NUbiCA assay.
- Utilizing a yeast model system for validation.
- Creation of a genome-wide yeast strain collection for NUbiCA screening.
Main Results:
- NUbiCA accurately detects mono- and polyubiquitylation of endogenous proteins in yeast.
- The assay can determine the topology of ubiquitin conjugates.
- A comprehensive yeast strain collection for ubiquitylation studies was established.
Conclusions:
- NUbiCA offers a sensitive and versatile method for studying ubiquitylation.
- This assay facilitates the analysis of low-abundance and transient ubiquitylation events.
- The NUbiCA yeast resource will accelerate research into ubiquitylation's cellular roles.
