title: Genome-wide protein-protein interaction screening by protein-fragment complementation assay (PCA) in living cells.
abstract: Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval.
authors: Samuel
authors: S
authors: Rochette
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval.
authors: Guillaume
authors: G
authors: Diss
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval.
authors: Marie
authors: M
authors: Filteau
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval.
authors: Jean-Baptiste
authors: JB
authors: Leducq
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval.
authors: Alexandre K
authors: AK
authors: Dubé
authors: Département de Biologie, Institut de biologie intégrative et des systémes & PROTEO, Université Laval; Christian.Landry@bio.ulaval.ca.
authors: Christian R
authors: CR
authors: Landry
mesh_terms: D057166:High-Throughput Screening Assays / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase