title: Detection and characterization of protein interactions in vivo by a simple live-cell imaging method.
abstract: Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
authors: Institute for Research in Biomedicine-IRB, Barcelona, Spain.
authors: Oriol
authors: O
authors: Gallego
authors: Tanja
authors: T
authors: Specht
authors: Thorsten
authors: T
authors: Brach
authors: Arun
authors: A
authors: Kumar
authors: Anne-Claude
authors: AC
authors: Gavin
authors: Marko
authors: M
authors: Kaksonen
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D004705:Endocytosis; D005089:Exocytosis; D036681:Fluorescence Recovery After Photobleaching; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008856:Microscopy, Fluorescence; D046912:Multiprotein Complexes / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D060066:Protein Interaction Maps; D055503:Protein Multimerization; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D059010:Single-Cell Analysis; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; C510360:Ede1 protein, S cerevisiae; D046912:Multiprotein Complexes; C080321:STE5 protein, S cerevisiae; C079506:STE50 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C546997:Syp1 protein, S cerevisiae; D033921:Vesicular Transport Proteins; D020930:MAP Kinase Kinase Kinases; C067018:Ste11 protein, S cerevisiae
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