Development and implementation of split-GFP-based bimolecular fluorescence complementation (BiFC) assays in yeast.
BiFC (bimolecular fluorescence complementation) is a tool for investigating interactions between proteins. Non-fluorescent fragments of, for example, GFP (green fluorescent protein) are fused to the interacting partners. The interaction brings the fragments together, which then fold, reassemble and fluoresce. This process can be carried out in living cells and provides information both on the interaction and its subcellular location. We have developed a split-GFP-based BiFC assay for use in the budding yeast Saccharomyces cerevisiae in which the modifications are carried out at the genomic level, thus resulting in the tagged yeast proteins being expressed at wild-type levels. The system is capable of detecting interactions in all subcellular compartments tested (the cytoplasm, mitochondria and nucleus) and makes a valuable addition to techniques for the investigation of protein-protein interactions in this model organism.
Barnard
Emma
E
School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, Northern Ireland, UK.
McFerran
Neil V
NV
Trudgett
Alan
A
Nelson
John
J
Timson
David J
DJ
D001681:Biological Assay / Q000379:methods*; D002451:Cell Compartmentation; D005453:Fluorescence; D005800:Genes, Fungal; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D011995:Recombination, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*
D049452:Green Fluorescent Proteins