Reverse Two-Hybrid Techniques in the Yeast Saccharomyces cerevisiae

Abstract

Use of the yeast two-hybrid system has provided definition to many previously uncharacterized pathways through the identification and characterization of novel protein-protein interactions. The two-hybrid system uses the bifunctional nature of transcription factors, such as the yeast enhancer Gal4, to allow protein-protein interactions to be monitored through changes in transcription of reporter genes. Once a positive interaction has been identified, either of the interacting proteins can be mutated by site-specific or randomly introduced changes, to produce proteins with a decreased ability to interact. Mutants generated using this strategy are very powerful reagents in tests of the biological significance of the interaction and in defining the residues involved in the interaction. Such techniques are termed reverse two-hybrid methods. We describe a reverse two-hybrid method that generates loss-of-interaction mutations of the catalytic subunit of the Escherichia coli heat-labile toxin (LTA1) with decreased binding to the active (GTP-bound) form of human ARF3, its protein cofactor. While newer methods are emerging for performing interaction screens in mammalian cells, instead of yeast, the use of reverse two-hybrid in yeast remains a robust and powerful means of identifying loss-of-interaction point mutants and compensating changes that remain among the most powerful tools of testing the biological significance of a protein-protein interaction.