Histidine-Tagged Ubiquitin Substitutes for Wild-Type Ubiquitin in Saccharomyces cerevisiae and Facilitates Isolation and Identification of in Vivo Substrates of the Ubiquitin Pathway

摘要: A general method for purification of any substrate the ubiquitin pathway, major eukaryotic proteolytic should utilize common characteristic covalent linkage to lysyl residues. The utility a N-terminal histidine-tagged (HisUb) in vivo conjugation and isolation ubiquitinated proteins by metal chelation chromatography is conditioned requirement that HisUb conjugate same set as wild-type ubiquitin. Stringent tests with Saccharomyces cerevisiae strains expressing ubiquitins only from plasmids were performed show could substitute was demonstrated yeast extracts immunoblotting Rpb1, largest subunit RNA polymerase II. fraction Rpb1 present form vivo. ability use expression transgenic organisms retain their endogenous genes through Arabidopsis thaliana or its variant HisUbK48R. UbK48R version capable proteins, but cannot serve an attachment site via interubiquitin linkage. Whereas plants showed insignificant enrichment lines HisUbK48R gave much better yield.