Topic Introduction The Yeast Two-Hybrid System: A Tool for Mapping Protein–Protein Interactions Jitender Mehla, J. Harry Caufield, and Peter Uetz 1 Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284 Virtually all processes in living cells are dependent on protein–protein interactions (PPIs). Under- standing PPI networks is thus essential for molecular biology and disease research. One powerful genetic system for mapping PPIs both at a small scale and in a high-throughput manner is the yeast two hybrid (Y2H) screen. In Y2H screening, PPIs are detected through the activation of reporter genes responding to a reconstituted transcription factor. In this introduction, we describe library- and array- based Y2H methods and explain their basic theory. We also include the rationale behind different Y2H approaches and strategies for optimizing results. INTRODUCTION Proteins are responsible for all major biological processes inside cells, either individually or in close coordination with other macromolecules. Outside cells, proteins and their interactions help to mediate cell–cell communication. Millions of micro- and macromolecular interactions are required to maintain the normal functional and structural architecture of a cell at any given time. Comprehensive analysis of specific protein–protein interactions (PPIs) on a genome-wide scale is a challenging task of proteomics and has been best explored in organisms such as budding yeast, Escher- ichia coli, and human. However, major portions of most genomes remain uncharacterized, including 20–50% of the predicted human genes (Pawlowski 2008) and 745 predicted genes of Saccharomyces cerevisiae (http://www.yeastgenome.org/cache/genomeSnapshot.html). High-throughput approaches have been developed to characterize unknown proteins and determine inter- and intramolecular interactions and networks in many organisms. Affinity purification and subsequent analysis by mass spectrometry (AP/MS) and the yeast two hybrid (Y2H) system are the preferred methods for mapping protein–protein networks on a large scale. The Y2H assay is a genetic method that employs in vivo screening for binary PPIs (Fields and Song 1989). A two hybrid screen may make use of random libraries (from genomic DNA or cDNA) or defined clone sets called ORFeomes. The latter may be used in array-based Y2H assays and are the preferred choice if such clones are available, because they are easily scalable from a few proteins to whole genomes. Most importantly, arrays are more efficient at avoiding false positives, although the number of false negatives may be also higher than in random library screens. RATIONALE FOR THE YEAST TWO HYBRID SYSTEM The original concept of the Y2H system is based on detection of an interaction between two proteins via the reconstitution of a transcription factor that activates one or more reporter genes (Fields and 1 Correspondence: phu878@gmail.com © 2015 Cold Spring Harbor Laboratory Press Cite this introduction as Cold Spring Harb Protoc; doi:10.1101/pdb.top083345 1