The yeast three-hybrid system as an experimental platform to identify proteins interacting with small signaling molecules in plant cells: Potential and limitations

Abstract

Chemical genetics is a powerful scientific strategy that utilizes small bioactive molecules as experimental tools to unravel biological processes. Bioactive compounds occurring in nature represent an enormous diversity of structures that can be used to dissect functions of biological systems. Once the bioactivity of a natural or synthetic compound has been critically evaluated the challenge remains to identify its molecular target and mode of action, which usually is a time-consuming and labor-intensive process. To facilitate this task, we decided to implement the yeast three-hybrid (Y3H) technology as a general experimental platform to scan the whole Arabidopsis proteome for targets of small signaling molecules. The Y3H technology is based on the yeast two-hybrid system and allows direct cloning of proteins that interact in vivo with a synthetic hybrid ligand, which comprises the biologically active molecule of interest covalently linked to methotrexate (Mtx). In yeast nucleus the hybrid ligand connects two fusion proteins: the Mtx part binding to dihydrofolate reductase fused to a DNA-binding domain (encoded in the yeast strain), and the bioactive molecule part binding to its potential protein target fused to a DNA-activating domain (encoded on a cDNA expression vector). During cDNA library screening, the formation of this ternary, transcriptional activator complex leads to reporter gene activation in yeast cells, and thereby allows selection of the putative targets of small bioactive molecules of interest. Here we present the strategy and experimental details for construction and application of a Y3H platform, including chemical synthesis of different hybrid ligands, construction of suitable cDNA libraries, the choice of yeast strains, and appropriate screening conditions. Based on the results obtained and the current literature we discuss the perspectives and limitations of the Y3H approach for identifying targets of small bioactive molecules.

Utilizing Yeast Surface Human Proteome Display Libraries to Identify Small Molecule-Protein Interactions

“…Since its development, the three-hybrid method has been successfully used to identify many protein-small molecule interactions [33, 34]. Very recently, the three-hybrid approach has been used to identify proteins that bind to the drugs erlotinib, atorvastatin, and sulfasalazine [35], the glaucoma therapeutic anecortave acetate [36], and a panel of anti-tuberculosis drugs [37].…”

Two-hybrid-based systems: Powerful tools for investigation of membrane traffic machineries

“…There are several types of three-hybrid assays. The RNA-based three-hybrid system allows rapid detection of RNA-binding proteins, while the ligand-based three-hybrid system searches for proteins that interact with different ligands (Becker et al, 2004;Cottier et al, 2011;Putz et al, 1996;SenGupta et al, 1996). Additionally, three-hybrid assays can be used to identify factors interacting with heteromeric proteins, and in fact have been used to identify physiological caspase-1 substrates and inhibitors (Van Criekinge et al, 1998).…”

“…Identification of different kind of ligands as interactors of heteromeric proteins and candidate drug targets (Becker et al, 2004;Cottier et al, 2011;de Felipe et al, 2004).…”

“…Membrane proteins are not identified (Jaeger et al, 2004); yeasts have the capacity to efficiently extrude some kind of drugs (Cottier et al, 2011). (Baker et al, 2003) Reverse two-hybrid assay (yeast, bacteria and mammalian cells)…”

“…The ligand-based three-hybrid system has been successfully used to detect protein-small molecule interactions and is thus generally useful in the discovery of candidate drug targets having the necessary sensitivity (Becker et al, 2004;Cottier et al, 2011;de Felipe et al, 2004).…”