Figure 4.

Monitoring small molecule–protein interactions using the yeast three-hybrid system. (A) Validation of the Mtx-based system: expression of the hook gene (DBD::DHFR) and the empty fish vector as control (AD) conferred cell growth on galactose-containing synthetic complete (SC −His −Ura −Trp +Leu) medium but not on SC −His −Ura −Trp −Leu medium. On selective medium (−Leu) only yeast expressing the Dex-binding fusion protein, AD::GR, grew in the presences of Mtx–Dex, and when X-Gal was simultaneously added, also the expression of the second reporter gene (lacZ) was detectable (galactosidase activity). Yeast suspensions were plated at two different densities, 4 × 10⁴ cells/dot (left) and 4 × 10² cells/dot (right). Competition assays indicated that all hybrid ligand were readily taken up by yeast cells: Addition of increasing concentrations of other baits to Mtx–Dex-containing medium, here shown for Mtx–CAMe, resulted in suppression of reporter gene expression and cell growth, indicating that formation of the trimeric transcriptional activation complex was prevented. (B) Verification of new candidate targets identified from Y3H screens with the hybrid ligand Mtx–CAMe. Plasmid DNA (fish vector) of two candidates identified in the initial screen was isolated and transfected into new yeast cells harboring the hook, DBD::DHFR. All yeast cells grew on +Leu but not on −Leu medium, including cells containing only the empty fish vector (control AD). By contrast, when hook vector (DBD::DHFR) and the fish vector harboring either of two CAMe-binding proteins (AD::EH or AD::MTK) were spotted on different selective media (−Leu), reporter gene activation (growth and galactosidase activity) was observed in presence of the specific bait, Mtx–CAMe, but not in the presence of alternative baits such as Mtx–CA, indicating that the interactions are highly selective. Addition of increasing concentrations of Mtx out-competed Mtx–CAMe-dependent growth. EH, epoxide hydrolase; MTK, 5-methylthioribose kinase; for other abbreviations see text.