A fluorescent two-hybrid assay for direct visualization of protein interactions in living cells.
Genetic high throughput screens have yielded large sets of potential protein-protein interactions now to be verified and further investigated. Here we present a simple assay to directly visualize protein-protein interactions in single living cells. Using a modified lac repressor system, we tethered a fluorescent bait at a chromosomal lac operator array and assayed for co-localization of fluorescent prey fusion proteins. With this fluorescent two-hybrid assay we successfully investigated the interaction of proteins from different subcellular compartments including nucleus, cytoplasm, and mitochondria. In combination with an S phase marker we also studied the cell cycle dependence of protein-protein interactions. These results indicate that the fluorescent two-hybrid assay is a powerful tool to investigate protein-protein interactions within their cellular environment and to monitor the response to external stimuli in real time.
Zolghadr
Kourosh
K
Munich Center for Integrated Protein Science (CiPSM) and Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany.
Mortusewicz
Oliver
O
Rothbauer
Ulrich
U
Kleinhans
Regina
R
Goehler
Heike
H
Wanker
Erich E
EE
Cardoso
M Cristina
MC
Leonhardt
Heinrich
H
D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006816:Huntington Disease / Q000235:genetics / Q000378:metabolism; D007763:Lac Operon; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D040901:Proteomics / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques
D017931:DNA Primers; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D049452:Green Fluorescent Proteins; D045643:DNA Repair Enzymes