Identification and Characterization of Physiological Pairing of E2 Ubiquitin-Conjugating Enzymes and E3 Ubiquitin Ligases.
Autoubiquitination
BiFC
E2-E3 pairing
E3 ubiquitin ligase
Ubiquitin
Ubiquitin-conjugating enzyme (E2)
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
22
11
2022
pubmed:
23
11
2022
medline:
25
11
2022
Statut:
ppublish
Résumé
The posttranslational attachment of the small protein modifier ubiquitin (Ub) is best known for its function in targeting proteins for degradation by the proteasome. However, ubiquitination also serves as a signal determining protein localization, activity, and interaction. Ubiquitination requires the sequential activity of E1 ubiquitin-activating enzyme (UBA), E2 ubiquitin-conjugating enzyme (UBC), and E3 ubiquitin ligase. Recognition of a target protein by an Ub-E2-E3 complex can result in its mono-ubiquitination (attachment of a single Ub moiety) or poly-ubiquitination, i.e., attachment of Ub chains. While the E3 ligase is important for the reaction specificity, the E2s catalyze the attachment of Ub to the target and to Ub itself to generate chains. In Arabidopsis thaliana, there are two E1s, 37 UBCs (and two ubiquitin-like conjugating enzymes) and more than 1400 E3 ligases, working in a combinatorial way. Therefore, in order to understand E3 ligase function, it is important to frame it within its possible E2s interactors. In this chapter, we propose a two-step identification and characterization of physiological E2-E3 pairs. In a first step, in vivo interacting E2s are identified through bimolecular fluorescence complementation (BiFC) using transient expression in Arabidopsis protoplast. In the second step, the activity of E2-E3 pairs is analyzed by a synthetic biology approach in which autoubiquitination is reconstituted in bacteria.
Identifiants
pubmed: 36413307
doi: 10.1007/978-1-0716-2784-6_2
doi:
Substances chimiques
Ubiquitin-Protein Ligases
EC 2.3.2.27
Ubiquitin-Conjugating Enzymes
EC 2.3.2.23
Ubiquitin
0
Ubiquitin-Activating Enzymes
EC 6.2.1.45
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
13-29Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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