Ubiquitin-Activated Interaction Traps (UBAITs): Tools for Capturing Protein-Protein Interactions
UBAITs (Ubiquitin-Activated Interaction Traps) are reagents that capitalize on the biochemistry of the ubiquitin system to covalently trap transient protein-protein interactions. UBAITs consist of an affinity-tagged protein of interest fused to a short linker followed by a C-terminal ubiquitin moiety. When charged in an E1- and E2-dependent manner, the C-terminal ubiquitin moiety of the UBAIT has the potential to form an amide linkage with lysine side chains of a protein that interacts transiently with the protein of interest, thereby covalently trapping the protein-protein interaction. The partner protein can then be identified by affinity-based purification of the UBAIT coupled with mass spectroscopy methods. While originally designed to identify substrates of ubiquitin ligases, UBAITs can, in principle, be used for identifying interaction partners of virtually any protein of interest. Here we describe methods for utilizing UBAITs in both cell-based and in vitro experiments.
Key wordsUBAIT Protein-protein interactions Ubiquitin ligases HECT E3 RING E3s
- 5.Ling R, Colón E, Dahmus ME, Callis J (2000) Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway. Anal Biochem 282:54–64. https://doi.org/10.1006/abio.2000.4586CrossRefPubMedGoogle Scholar