title: Multiplex assay for condition-dependent changes in protein-protein interactions.
abstract: Changes in protein-protein interactions that occur in response to environmental cues are difficult to uncover and have been poorly characterized to date. Here we describe a yeast-based assay that allows many binary protein interactions to be assessed in parallel and under various conditions. This method combines molecular bar-coding and tag array technology with the murine dihydrofolate reductase-based protein-fragment complementation assay. A total of 238 protein-fragment complementation assay strains, each representing a unique binary protein complex, were tagged with molecular barcodes, pooled, and then interrogated against a panel of 80 diverse small molecules. Our method successfully identified specific disruption of the Hom3:Fpr1 interaction by the immunosuppressant FK506, illustrating the assay's capacity to identify chemical inhibitors of protein-protein interactions. Among the additional findings was specific cellular depletion of the Dst1:Rbp9 complex by the anthracycline drug doxorubicin, but not by the related drug idarubicin. The assay also revealed chemical-induced accumulation of several binary multidrug transporter complexes that largely paralleled increases in transcript levels. Further assessment of two such interactions (Tpo1:Pdr5 and Snq2:Pdr5) in the presence of 1,246 unique chemical compounds revealed a positive correlation between drug lipophilicity and the drug response in yeast.
authors.affiliation: Stanford Genome Technology Center, Department of Biochemistry, Stanford University, Palo Alto, CA 94304, USA.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Schlecht
authors.forename: Molly
authors.initials: M
authors.lastname: Miranda
authors.forename: Sundari
authors.initials: S
authors.lastname: Suresh
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
mesh_terms: D000818:Animals; D019295:Computational Biology; D004268:DNA-Binding Proteins; D051379:Mice; D046228:Microarray Analysis; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D044042:Receptors, Formyl Peptide / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries / Q000378:metabolism*; D016559:Tacrolimus; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism*; D015003:Yeasts
chemical_list: D004268:DNA-Binding Proteins; C068394:DST1 protein, S cerevisiae; C469988:Fpr1 protein, mouse; D044042:Receptors, Formyl Peptide; D029701:Saccharomyces cerevisiae Proteins; D054852:Small Molecule Libraries; D013762:Tetrahydrofolate Dehydrogenase; D016559:Tacrolimus