A differential cytolocalization assay for analysis of macromolecular assemblies in the eukaryotic cytoplasm.
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.
Blanchard
Daniel
D
Departments of Pathology and Genetics, Stanford University School of Medicine, Stanford, California 94305-5324, USA.
Hutter
Harald
H
Fleenor
Jamie
J
Fire
Andrew
A
D000595:Amino Acid Sequence; D000818:Animals; D001681:Biological Assay; D017173:Caenorhabditis elegans / Q000737:chemistry / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000032:analysis / Q000378:metabolism*; D002352:Carrier Proteins / Q000032:analysis / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D025941:Protein Interaction Mapping / Q000379:methods*; D034622:RNA Interference; D020871:RNA Stability
D029742:Caenorhabditis elegans Proteins; D002352:Carrier Proteins; C472410:SMG-5 protein, C elegans