Abstract
Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.
Highlights
The ubiquitin pathway is conserved throughout eukaryotic evolution and is implicated in numerous cellular processes [1]
Development of the Luminescent Ubiquitination Assay—The new luminescent assay was designed to monitor the ubiquitination of GST fusion proteins in reactions containing purified E1, E2, and E3 (Rsp5) enzymes, biotinylated ubiquitin (b-Ub), and ATP (Fig. 1B)
The ubiquitination of four other in vitro GSTtagged substrates of Rsp5 (Ybr196c, Ypr084w, Ynl136w, and Ydr203c, which were discovered in the course of the study) and five proteins not ubiquitinated by Rsp5 in vitro
Summary
Purification of Yeast E2 Enzymes—Yeast E2 genes UBC1 and UBC4 were cloned into pET15b plasmids as described [24]. To prepare the ubiquitination reactions for analysis using AlphaScreen, the samples were diluted 270-fold with AlphaScreen assay buffer (unless otherwise indicated) and transferred to 384-well OptiPlateNEW microplates containing 10 l of AlphaScreen anti-GST acceptor and streptavidin donor beads (both at a final concentration of 20 g/ml). This dilution step brings the concentration of b-Ub and GST proteins into the nanomolar range, which corresponds to the binding capacities of AlphaScreen beads. The proteins extracted from the mutant and wild-type strains were analyzed using SDS-PAGE and probed with ␣-GST and ␣-ubiquitin (Covance) antibodies
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