Using the yeast interaction trap and other two-hybrid-based approaches to study protein-protein interactions

The detection of physical interaction between two or more molecules of inte rest can be facilitated if the act of association between the interactive p artners leads to the production of a readily observed biological or physica l readout. Many interacting molecule pairs (X, Y) can be made to induce suc h a readout if X and Y are each fused to defined protein elements with desi red properties. For example, in the yeast forward two-hybrid system, X is s ynthesized as a translational fusion to a DNA-binding domain (DBD), Y is sy nthesized as a fusion to a transcriptional activation domain (AD), and coex pression of DBD-X and AD-Y induces transcription of easily scored responsiv e reporters. Other approaches use paradigms based on the artifical producti on of two, hybrid, molecules, but substitute a variety of readouts includin g the repression of transcription, activation of signal transduction pathwa ys, or reconstitution of a disrupted enzymatic activity. In this article, w e summarize a number of two-hybrid-based approaches, and detail the use of the forward yeast two-hybrid system in a screen to identify novel interacti ng partners for a protein of interest. (C) 2001 Academic Press.