Fusions of ubiquitin and a target protein are recognized and cleaved by ubiquitin-specific proteases (UBPs) after a double glycine motif (Gly-Gly-X) located at the C-terminus of ubiquitin. As opposed to many other proteases, UBPs do not recognize a specific sequence of a polypeptide chain but instead recognize the folded ubiquitin.

 

 

 

Ubiquitin can be expressed in yeast as an N-terminal half (Nub) as well as a C-terminal half (Cub). The two halves retain their affinity for each other and spontaneously reassemble to form the so-called split-ubiquitin.

 

 

 

If the Nub and Cub moieties are co-expressed within a single cell, the reporter protein that is fused to the C-terminus of ubiquitin will be cleaved off upon re-assembly of the Nub and Cub moieties into split-ubiquitin.

 

 

 

A point mutation in the N-terminal half of ubiquitin (NubG) completely abolishes the affinity of the two halves for each other. As the separate NubG and Cub parts are not recognized by ubiquitin-specific proteases (UBPs), no cleavage of the reporter protein takes place.

 

 

 

The bait protein is fused to the Cub domain followed by a reporter protein, and a prey protein is fused to a mutated NubG domain. If bait and prey interact, their interaction brings the NubG and Cub domains close enough together to reconstitute split-ubiquitin, resulting in the release of the reporter protein (red) by the action of the UBPs.