Detecting ProteinProtein Interactions with the Split-Ubiquitin Sensor

A detailed understanding of a cellular process requires the knowledge about the interactions between its protein constituents. The Split-Ubiquitin technique allows to monitor and detect interactions of very diverse proteins, including transcription factors and membrane-associated proteins. The technique is based on unique features of ubiquitin, the enzymes of the ubiquitin pathway, and the reconstitution of a native-like ubiquitin from its N- and C-terminal fragments. Using Ura3p as a reporter for the reconstitution of the ubiquitin fragments, methods are presented that enable to screen in yeast for interaction partners of a given protein with either a randomly generated expression library or a defined but more limited array of protein fusions.