N. Johnsson et A. Varshavsky, SPLIT UBIQUITIN AS A SENSOR OF PROTEIN INTERACTIONS IN-VIVO, Proceedings of the National Academy of Sciences of the United Statesof America, 91(22), 1994, pp. 10340-10344
We describe an assay for in vivo protein interactions. Protein fusions
containing ubiquitin, a 76-residue, single-domain protein, are rapidl
y cleaved in vivo by ubiquitin-specific proteases, which recognize the
folded conformation of ubiquitin. When a C-terminal fragment of ubiqu
itin (C-ub) is expressed as a fusion to a reporter protein, the fusion
is cleaved only if an N-terminal fragment of ubiquitin (N-ub) is also
expressed in the same cell. This reconstitution of native ubiquitin f
rom its fragments, detectable by the in vivo cleavage assay, is not ob
served with a mutationally altered N-ub. However, if C-ub and the alte
red N-ub are each linked to polypeptides that interact in vivo, the cl
eavage of the fusion containing C-ub is restored, yielding a generally
applicable assay for kinetic and equilibrium aspects of in vivo prote
in interactions. This method, termed USPS (ubiquitin-based split-prote
in 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.