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Molecular & Cellular Proteomics 5:2175-2184, 2006.
© 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

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Technology

A Differential Cytolocalization Assay for Analysis of Macromolecular Assemblies in the Eukaryotic Cytoplasm^*,S

Daniel Blanchard^,,¶, Harald Hutter^||, Jamie Fleenor^¶ and Andrew Fire^,¶,**

From the Departments of Pathology and Genetics, Stanford University School of Medicine, Stanford, California 94305-5324, Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, ^¶ Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland 21210, and ^|| Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

We have developed a differential cytolocalization assay (DCLA) that allows the observation of cytoplasmic protein/protein interactions in vivo. In the DCLA, interactions are visualized as a relocalization of a green fluorescent protein-tagged "prey" by a membrane-bound "bait." This assay was tested and utilized in Caenorhabditis elegans to probe interactions among proteins involved in RNA interference (RNAi) and nonsense-mediated decay (NMD) pathways. Several previously documented interactions were confirmed with DCLA, whereas uniformly negative results were obtained in several controls in which no interaction was expected. Novel interactions were also observed, including the association of SMG-5, a protein required for NMD, to several components of the RNAi pathway. The DCLA can be readily carried out under diverse conditions, allowing a dynamic assessment of protein interactions in vivo. We used this property to test a subset of the RNAi and NMD interactions in animals in which proteins central to each mechanism were mutated; several key associations in each machinery that can occur in vivo in the absence of a functional process were identified.

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