title: Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method.
abstract: An alternative method for monitoring protein-protein interactions in Saccharomyces cerevisiae has been developed. It relies on the ability of two fragments of enhanced green fluorescent protein (EGFP) to reassemble and fluoresce when fused to interacting proteins. Since this fluorescence can be detected in living cells, simultaneous detection and localisation of interacting pairs is possible. DNA sequences encoding N- and C-terminal EGFP fragments flanked by sequences from the genes of interest were transformed into S. cerevisiae JPY5 cells and homologous recombination into the genome verified by PCR. The system was evaluated by testing known interacting proteins: labelling of the phosphofructokinase subunits, Pfk1p and Pfk2p, with N- and C-terminal EGFP fragments, respectively, resulted in green fluorescence in the cytoplasm. The system works in other cellular compartments: labelling of Idh1p and Idh2p (mitochondrial matrix), Sdh3p and Sdh4p (mitochondrial membrane) and Pap2p and Mtr4p (nucleus) all resulted in fluorescence in the appropriate cellular compartment.
authors.affiliation: School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Barnard
authors.forename: Neil V
authors.initials: NV
authors.lastname: McFerran
authors.forename: Alan
authors.initials: A
authors.lastname: Trudgett
authors.forename: John
authors.initials: J
authors.lastname: Nelson
authors.forename: David J
authors.initials: DJ
authors.lastname: Timson
mesh_terms: D053487:DEAD-box RNA Helicases / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D025221:Phosphofructokinases / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007770:L-Lactate Dehydrogenase; D013385:Succinate Dehydrogenase; D025221:Phosphofructokinases; C101004:MTR4 protein, S cerevisiae; D053487:DEAD-box RNA Helicases