Abstract
The process of protein ubiquitination typically consists of three sequential steps to add an ubiquitin (Ub) or Ub chain to a substrate protein, requiring three different enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and ubiquitin protein ligase (E3). Most E2s possess the classical E2 activity in forming E2-Ub complex through a thioester linkage, in presence of an E1 and Ub. Additionally, some E2s have the ability of catalyzing the formation of free Ub dimer. Such activity indicates an important role of these E2s in ubiquitination pathway. Thus, we developed an in vitro Ub dimer formation assay to determine the activity of certain E2s. Moreover, by using Ub mutants, in which different lysine residues are mutated, the specific linkage of dimer can also be determined.
Keywords: Ubiquitination, Ubiquitin dimer formation, E2, Arabidopsis UBC22, K11 linkage
Background
The existing protocols for E2 conjugation initiation assay (without adding E3 and substrate) aim to detect the thioester linkage (E2-S-Ub). Our method focuses on the E2 activity of catalyzing free Ub dimer formation (Ub-Ub). It provides a convenient way to detect an important biochemical feature of E2 in different species. Further, the specific linkage of dimer can be determined by using different Ub mutants.
Materials and Reagents
Equipment
Procedure
Data analysis
Purified Arabidopsis recombinant E2 UBC22 fused with 6x His tag (His-UBC22) was tested in the in vitro Ub dimer formation assay. As shown in Figure 1, Ub dimers could be detected when His-UBC22 was added into the reaction (lane 2 compared to the lane 1), indicating the biochemical activity of UBC22 catalyzing free dimer formation. In addition, similar dimer formation was observed when the Ub-K48R mutant or Ub-K63R mutant was used, which lacks K48 residue or K63 residue (lane 6 compared to lane 5, or lane 8 compared to lane 7). Interestingly, when the Ub-K11R which lacks K11 residue was used, little dimer was produced (lane 4 compared to lane 3). These results indicate that UBC22 could catalyze Ub dimer formation in vitro specifically through K11 residue of Ub. Three independent experiments were performed and produced similar results.
Notes
Recipes
Acknowledgments
We gratefully acknowledge the financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC) (Discovery grant) to HW.
References
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