Abstract
BackgroundUbiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is also implicated in several critical cellular processes. Although Arabidopsis genome research has estimated more than 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrate a novel, simple and high-sensitive method for in vitro analysis of ubiquitination and polyubiquitination based on wheat cell-free protein synthesis and luminescent detection.ResultsUsing wheat cell-free synthesis, 11 E3 proteins from Arabidopsis full-length cDNA templates were produced. These proteins were analyzed either in the translation mixture or purified recombinant protein from the translation mixture. In our luminescent method using FLAG- or His-tagged and biotinylated ubiquitins, the polyubiquitin chain on AtUBC22, UPL5 and UPL7 (HECT) and CIP8 (RING) was detected. Also, binding of ubiquitin to these proteins was detected using biotinylated ubiquitin and FLAG-tagged recombinant protein. Furthermore, screening of the RING 6 subgroup demonstrated that At1g55530 was capable of polyubiquitin chain formation like CIP8. Interestingly, these ubiquitinations were carried out without the addition of exogenous E1 and/or E2 proteins, indicating that these enzymes were endogenous to the wheat cell-free system. The amount of polyubiquitinated proteins in the crude translation reaction mixture was unaffected by treatment with MG132, suggesting that our system does not contain 26S proteasome-dependent protein degradation activity.ConclusionIn this study, we developed a simple wheat cell-free based luminescence method that could be a powerful tool for comprehensive ubiquitination analysis.
Highlights
Ubiquitination is mediated by the sequential action of at least three enzymes: the E1, E2 and E3 proteins
Since E3 binds to both E2 and the target protein, and acts as scaffold between E2 and the substrate protein, the E3 ligase is the major determinant for selecting target proteins for ubiquitination
The HECT-type E3 ligase is distinct from the other two ligases in that it forms a thioesterbond with ubiquitin prior to the transfer of ubiquitin to target proteins
Summary
Ubiquitination is mediated by the sequential action of at least three enzymes: the E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) proteins. Polyubiquitination of target proteins is implicated in several critical cellular processes. Together with an E3 ligase enzyme, ubiquitin is attached via its carboxyl-terminus to an e-amino group of a lysine residue in the target protein. There is large number of genes encoding E3 ligases in all eukaryotes, and the diversity of E3s is thought to contribute to the substrate specificity of numerous target proteins. The HECT-type E3 ligase is distinct from the other two ligases in that it forms a thioesterbond with ubiquitin prior to the transfer of ubiquitin to target proteins. The RING-type E3 ligase contains a unique domain similar to the zinc finger motif that mediates protein-protein interactions [5] and is further divided into two classes: one that can function alone and another that forms a complex with other E3 components [4]
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