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

Oliver Mortusewicz,Erich E. Wanker,Kourosh Zolghadr,Heike Goehler,Regina Kleinhans,M. Cristina Cardoso,Ulrich Rothbauer,Heinrich Leonhardt

doi:10.1074/mcp.m700548-mcp200

Abstract

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.

Highlights

Genetic high throughput screens have yielded large sets of potential protein-protein interactions to be verified and further investigated
We generated an expression construct encoding a fluorescent bait protein consisting of a fluorescent protein (FP), the lac repressor (LacI), and the protein X to be tested for interactions resulting in the triple fusion protein FP-LacI-X (Fig. 1a) or X-LacI-FP
Interaction of red fluorescent protein (RFP)-PCNA with the PCNA binding domain (PBD) part of the bait protein resulted in co-localization of the fluorescent signals at the lac operator array (Fig. 3c, upper panel), whereas deletion of the PBD in the bait protein led to a dispersed distribution of RFP-PCNA in non-S phase cells (Fig. 3d, upper panel)

Summary

EXPERIMENTAL PROCEDURES

Expression Constructs—The LacI-encoding sequence was PCRamplified from the p3ЈSS EGFP-LacI expression vector [9] using the following primers: forward primer 5Ј-TCT AGA AAG CTT TCC ATG GTG AAA CCA GTA-3Ј and reverse primer 5Ј-CCA TGC CCG GGA CAG GCT GCT TCG GGA AAC-3Ј (restriction sites in italic). This PCR fragment was digested with HindIII and XmaI and cloned into the same sites of two Dnmt1-YFP expression vectors (MTNY. and PBHD-YFP) [10] generating PBD-LacI-YFP and ⌬PBD-LacI-YFP. The NLS-PCNA-LacI-RFP and XRCC1-LacI-RFP constructs were generated by PCR amplification of the PCNA and XRCC1 cDNA using the following primers (restriction sites in italic): PCNA forward, 5Ј-CCCCCTCGAGATGTTCGAGGCGCGC-3Ј; PCNA reverse, 5Ј-GGGGAAGCTTGGAGATCCTTCTTCATCCTC-3Ј; XRCC1 forward, 5Ј-CCCCAGATCTATGCCGGAGATCCGC-3Ј; and XRCC1 reverse, 5Ј-GGGGGAATTCGGGGCTTGCGGCACCAC-3Ј. The PCR fragments were cloned into a LacI-RFP expression vector using the XhoI/HindIII sites for the NLS-PCNA-LacI-RFP and the BglII/EcoRI sites for the XRCC1-LacIRFP expression vector

A Fluorescent Two-hybrid Assay to Study Protein Interactions

RESULTS

DISCUSSION