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Originally published In Press as doi:10.1074/mcp.M700548-MCP200 on July 12, 2008.
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Molecular & Cellular Proteomics 7:2279-2287, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

Research

A Fluorescent Two-hybrid Assay for Direct Visualization of Protein Interactions in Living Cells*,S

Kourosh Zolghadr{ddagger}, Oliver Mortusewicz{ddagger}, Ulrich Rothbauer{ddagger}, Regina Kleinhans{ddagger}, Heike Goehler§,¶, Erich E. Wanker§, M. Cristina Cardoso§ and Heinrich Leonhardt{ddagger},||

From the {ddagger} Munich Center for Integrated Protein Science (CiPSM) and Department of Biology, Ludwig Maximilians University Munich, 82152 Planegg-Martinsried, Germany and § Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany

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.

|| To whom correspondence should be addressed. E-mail: h.leonhardt{at}lmu.de


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