Deciphering Protein Interaction Dynamics Using the Yeast Three-Hybrid System.
Proteins often act together with other proteins to regulate crucial biological processes. Beyond binary interactions, they often operate within multi-protein assemblies, where alterations in binding affinity or structural conformation of one or more components can significantly impact the overall function of the complex. The yeast three-hybrid (Y3H) system extends the conventional yeast two-hybrid (Y2H) by adding a bridge protein to test ternary interactions ex planta. Here, we present a Y3H protocol utilizing the pBridge and pACT2 AD vectors in the Saccharomyces cerevisiae Y2HGold strain and examine the role of SUPPRESSOR OF PHYA-105 1 (SPA1) in mediating the interaction between CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) and CRYPTOCHROME 1 (CRY1) in Arabidopsis thaliana. COP1 and SPA proteins form a functional E3 ubiquitin ligase complex that prevents photomorphogenesis in darkness, while CRY1 antagonizes this activity under blue light conditions. Using the Y3H system, we show that co-expression of SPA1 enhances the interaction between COP1 and CRY1, as detected via MEL1 reporter gene activation in a quantitative assay using p-nitrophenyl alpha-D-galactopyranoside (PNPG). This protocol offers a practical framework for dissecting ternary protein interactions using the Y3H assay.