Abstract
Protein ubiquitination is implicated in several critical cellular processes. Ubiquitination is mediated by the sequential action of at least three enzymes, the E1, E2 and E3 proteins. Although Arabidopsis genome research estimates over 1,300 proteins involved in ubiquitination, little is known about the biochemical functions of these proteins. Here we demonstrated a novel method for high-throughput in vitro ubiquitination analysis based on wheat cell-free protein synthesis and luminescent detection. The recombinant proteins without purification were used for the assay, and luminescent analysis showed the ubiquitin-conjugation of 29 E2s and a HECT-type E3. Furthermore, this analysis also detected the polyubiquitination of a HECT- and RING-type E3s. 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. Based on this study, we are developing the convenient and versatile method to elucidate plant ubiquitination pathway.
