A co-localization assay for the analysis of protein-protein interactions.
The understanding and analysis of protein associations in living cells is a major goal of molecular biology. Here, we describe an assay for the analysis of protein-protein interactions based on the co-localization of a fused site-specific protease with a cleavable reporter in close proximity to the interaction partner under examination. We exemplified this scheme in the temperature-sensitive Saccharomyces cerevisiae cdc25-2 mutant strain using the nuclear inclusion protease of tobacco etch virus fused to the adaptor protein growth factor receptor binding protein 2 (Grb2). The growth-defective phenotype of cdc25-2 was complemented by expression of a membrane-targeted constitutively active Ras protein, which contained a TEV protease substrate sequence allowing for release from the membrane upon proteolysis. Interaction of Grb2 with the membrane-targeted intracellular domain of the oncogene vErbB resulted in co-localization of the TEV protease with its substrate, release of Ras from the membrane and restoration of the temperature-sensitive phenotype of cdc25-2. The flexibility of the general scheme of this approach may allow for its application in many different assay scenarios and may represent a suitable alternative in cases where other approaches fail.
Köhler
Fabian
F
Institute for Biology III, Albert-Ludwigs-University of Freiburg and Max-Planck-Institute for Immunobiology, Stuebeweg 51, 79108 Freiburg, Germany. koehler@immunbio.mpg.de
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D051380:GRB2 Adaptor Protein / Q000235:genetics / Q000378:metabolism; D005816:Genetic Complementation Test; D009154:Mutation; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism; D010942:Plant Viruses / Q000201:enzymology; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014026:Nicotiana / Q000821:virology; D014170:Transformation, Genetic; D020666:ras-GRF1 / Q000235:genetics / Q000378:metabolism
D018797:Cell Cycle Proteins; D051380:GRB2 Adaptor Protein; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D010447:Peptide Hydrolases