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

Stéphanie Cottier

doi:10.3389/fpls.2011.00101

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.

Highlights

FUNCTIONAL IMPORTANCE OF SMALL MOLECULES Organic small molecules participate in many biological processes, such as metabolic pathways, signal transduction mechanisms, and developmental programs, in which they often play important, sometimes decisive roles
The number of candidate targets we identified in our screens is too low to draw general conclusions and yet, intriguingly, we uncovered three enzymes interacting with jasmonic acid (JA) or salicylic acid (SA) derivatives and three members of a transcription factor family interacting with compound 8 (Cpd8)
EVALUATION OF THE METHODOLOGY: PROS AND CONS OF THE yeast three-hybrid (Y3H) SYSTEM We have demonstrated that the Y3H technology is a valid approach to isolate binding protein candidates of small bioactive molecules from plant sources in extension of previous successful applications to yeast and mammalian systems, which were nurtured from drug research aiming at drug target identification (Licitra and Liu, 1996; Becker et al, 2004; Dirnberger et al, 2006; Chidley et al, 2011)

Summary

Introduction

FUNCTIONAL IMPORTANCE OF SMALL MOLECULES Organic small molecules participate in many biological processes, such as metabolic pathways, signal transduction mechanisms, and developmental programs, in which they often play important, sometimes decisive roles. Once a fish is hooked, i.e., a target protein has bound to its small molecule ligand contained in the bait, the assembled transcriptional activator complex initiates expression of the reporter gene (Figure 1).

Results

Conclusion