title: The GAL genetic switch: visualisation of the interacting proteins by split-EGFP bimolecular fluorescence complementation.
abstract: A split-EGFP bimolecular fluorescence complementation assay was used to visualise and locate three interacting pairs of proteins from the GAL genetic switch of the budding yeast, Saccharomyces cerevisiae. Both the Gal4p-Gal80p and Gal80p-Gal3p pairs were found to be located in the nucleus under inducing conditions. However, the Gal80p-Gal1p complex was located throughout the cell. These results support recent work establishing an initial interaction between Gal3p and Gal80p occurring in the nucleus. Labelling of all three protein pairs impaired the growth of the yeast strains and resulted in reduced galactokinase activity in cell extracts. The most likely cause of this impairment is decreased dissociation rates of the complexes, caused by the essentially irreversible reassembly of the EGFP fragments. This suggests that a fully functional GAL genetic switch requires dynamic interactions between the protein components. These results also highlight the need for caution in the interpretation of in vivo split-EGFP experiments.
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, Belfast, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Barnard
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D005453:Fluorescence; D005686:Galactokinase / Q000235:genetics / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods
chemical_list: D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D005686:Galactokinase