Epitope-tagged ubiquitin. A new probe for analyzing ubiquitin function.
In the present work, a method based on an epitope-tagged ubiquitin derivative is described that allows for the unambiguous detection of ubiquitin-protein conjugates formed in vivo or in vitro. Expression in the yeast Saccharomyces cerevisiae of ubiquitin that has been tagged at its amino terminus with a peptide epitope results in the formation of tagged ubiquitin-protein conjugates that are detectable by immunoblotting with a monoclonal antibody that recognizes the tag. The expression of tagged ubiquitin has no adverse effect on vegetative growth and, moreover, can suppress the stress-hypersensitive phenotype of yeast lacking the polyubiquitin gene UBI4. We also show that tagged ubiquitin is correctly conjugated in vivo and in vitro to a short-lived test protein and can be covalently extended into the multimeric ubiquitin chain that is normally required for the degradation of this protein. Surprisingly, however, conjugation of tagged ubiquitin inhibits proteolysis. These and related results suggest that the amino-terminal region of ubiquitin is important in protease-substrate recognition and that the multiubiquitin chain is a dynamic transient structure. The potential of tagged ubiquitin for the identification and isolation of ubiquitin-protein conjugates and ubiquitin-related enzymes, and as a tool in mechanistic studies is discussed.
Ellison
M J
MJ
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Hochstrasser
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D015153:Blotting, Western; D000939:Epitopes; D016259:Genes, myc; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000276:immunology; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013312:Stress, Physiological / Q000378:metabolism; D014452:Ubiquitins / Q000276:immunology* / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D009841:Oligonucleotides; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D001616:beta-Galactosidase