IC-tagging and protein relocation to ARV muNS inclusions: a method to study protein-protein interactions in the cytoplasm or nucleus of living cells.

Abstract

BACKGROUND: Characterization of protein-protein interactions is essential for understanding cellular functions. Although there are many published methods to analyze protein-protein interactions, most of them present serious limitations. In a different study we have characterized a novel avian reovirus muNS-based protein tagging and inclusion targeting method, and demonstrated its validity to purify free an immobilized protein. METHODOLOGY/PRINCIPAL FINDINGS: Here we present a method to identify protein-protein interactions inside living eukaryotic cells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477-542), it not only got integrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to these structures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimeras that form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturing IC-tagged proteins, and that the proteins targeted to nuclear inclusions are able to interact with their known ligands. CONCLUSIONS/SIGNIFICANCE: Our protein redistribution method does not present the architectural requirement of re-constructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only cell transfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus of living cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.