Membrane-based yeast two-hybrid system to detect protein interactions

Abstract

The classical yeast two-hybrid system and its modifications have been successfully used over the past decade to investigate interactions between most classes of proteins expressed in a given cell or tissue. However, some proteins (e.g., integral membrane proteins or nuclear proteins) are relatively difficult to investigate by standard yeast two-hybrid methods either because they are retained at cellular membranes or they activate the system in the absence of a true protein interaction. The membrane-based yeast two-hybrid (MbY2H) system presented in this unit overcomes some of these limitations. It is based on the split-ubiquitin protein complementation assay and detects protein interactions directly at the membrane, thereby allowing the use of full-length integral membrane proteins and membrane-associated proteins as baits to hunt for novel interaction partners. A simple modification also allows the use of proteins that are self-activating in a classical yeast two-hybrid system (e.g., acidic proteins and many transcription factors). Like the yeast two-hybrid system, the MbY2H system can also be used for interaction discovery by screening complex cDNA libraries for novel interaction partners.