Split ubiquitin as a sensor of protein interactions in vivo.
We describe an assay for in vivo protein interactions. Protein fusions containing ubiquitin, a 76-residue, single-domain protein, are rapidly cleaved in vivo by ubiquitin-specific proteases, which recognize the folded conformation of ubiquitin. When a C-terminal fragment of ubiquitin (C(ub)) is expressed as a fusion to a reporter protein, the fusion is cleaved only if an N-terminal fragment of ubiquitin (Nub) is also expressed in the same cell. This reconstitution of native ubiquitin from its fragments, detectable by the in vivo cleavage assay, is not observed with a mutationally altered Nub. However, if C(ub) and the altered Nub are each linked to polypeptides that interact in vivo, the cleavage of the fusion containing C(ub) is restored, yielding a generally applicable assay for kinetic and equilibrium aspects of in vivo protein interactions. This method, termed USPS (ubiquitin-based split-protein sensor), makes it possible to monitor a protein-protein interaction as a function of time, at the natural sites of this interaction in a living cell.
Johnsson
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Division of Biology, California Institute of Technology, Pasadena 91125.
Varshavsky
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A
D000818:Animals; D015415:Biomarkers; D051379:Mice; D008961:Models, Structural; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism*
D015415:Biomarkers; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D013762:Tetrahydrofolate Dehydrogenase