title: Reverse PCA, a systematic approach for identifying genes important for the physical interaction between protein pairs.
abstract: Protein-protein interactions (PPIs) are of central importance for many areas of biological research. Several complementary high-throughput technologies have been developed to study PPIs. The wealth of information that emerged from these technologies led to the first maps of the protein interactomes of several model organisms. Many changes can occur in protein complexes as a result of genetic and biochemical perturbations. In the absence of a suitable assay, such changes are difficult to identify, and thus have been poorly characterized. In this study, we present a novel genetic approach (termed "reverse PCA") that allows the identification of genes whose products are required for the physical interaction between two given proteins. Our assay starts with a yeast strain in which the interaction between two proteins of interest can be detected by resistance to the drug, methotrexate, in the context of the protein-fragment complementation assay (PCA). Using synthetic genetic array (SGA) technology, we can systematically screen mutant libraries of the yeast Saccharomyces cerevisiae to identify those mutations that disrupt the physical interaction of interest. We were able to successfully validate this novel approach by identifying mutants that dissociate the conserved interaction between Cia2 and Mms19, two proteins involved in Iron-Sulfur protein biogenesis and genome stability. This method will facilitate the study of protein structure-function relationships, and may help in elucidating the mechanisms that regulate PPIs.
authors.affiliation: Faculty of Life Sciences Bar-Ilan University, Ramat-Gan, Israel.
authors.forename: Ifat
authors.initials: I
authors.lastname: Lev
authors.forename: Marina
authors.initials: M
authors.lastname: Volpe
authors.forename: Liron
authors.initials: L
authors.lastname: Goor
authors.forename: Nelly
authors.initials: N
authors.lastname: Levinton
authors.forename: Liach
authors.initials: L
authors.lastname: Emuna
authors.forename: Shay
authors.initials: S
authors.lastname: Ben-Aroya
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D005813:Genes, Synthetic; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D007506:Iron-Sulfur Proteins; C103317:MET18 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors