RT Journal Article
T1 IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells
A1 Brandariz Núñez, Alberto
A1 Menaya Vargas, Rebeca
A1 Benavente Martínez, Francisco Javier
A1 Martínez Costas, José Manuel
K1 Cytoplasmic inclusions
K1 Immunostaining
K1 Protein expression
K1 Protein interactions
K1 Birds
K1 Plasmid construction
K1 SV40
K1 Protein-protein interactions
AB Background: Characterization of protein-protein interactions is essential for understanding cellular functions. Althoughthere are many published methods to analyze protein-protein interactions, most of them present serious limitations. In adifferent 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 eukaryoticcells (tested in primate and avian cells). When p53 was tagged with Intercoil (IC; muNS residues 477–542), it not only gotintegrated into muNS cytoplasmic inclusions, but also attracted its known ligand SV40 large T antigen (TAg) to thesestructures. We have also adapted this system to work within the cell nucleus, by creating muNS-related protein chimerasthat form nuclear inclusions. We show that nuclear muNS-derived inclusions are as efficient as cytoplasmic ones in capturingIC-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 reconstructing a transcription factor as any of the two-hybrid systems do. The method is simple and requires only celltransfection and a fluorescence microscope. Our tagging method can be used either in the cytoplasm or the nucleus ofliving cells to test protein-protein interactions or to perform functional studies by protein ligand sequestration.
PB PLOS
YR 2010
FD 2010
LK http://hdl.handle.net/10347/22843
UL http://hdl.handle.net/10347/22843
LA eng
NO Brandariz-Nuñez A, Menaya-Vargas R, Benavente J, Martinez-Costas J (2010) IC-Tagging and Protein Relocation to ARV muNS Inclusions: A Method to Study Protein-Protein Interactions in the Cytoplasm or Nucleus of Living Cells. PLoS ONE 5(11): e13785. https://doi.org/10.1371/journal.pone.0013785
NO This work was supported by grants from the European Commission under contracts ERAS-CT-2003-980409 (as part of the European ScienceFoundation EUROCORES Programme EuroSCOPE, web: http://www.esf.org/euroscope); the Spanish Ministerio de Ciencia y Tecnología (BFU2007-61330, BFU 205-24982-E, web: http://www.mityc.es/) and Xunta de Galicia (08CSA009203PR, web: http://www.conselleriaiei.org/ga/dxidi/index.php). ABN was the recipient of apredoctoral FPI fellowship from the Spanish Ministerio de Ciencia y Tecnología
DS Minerva
RD 24 abr 2026