Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method

Emma Barnard; Neil V McFerran; Alan Trudgett; John Nelson; David J Timson

doi:10.1016/j.fgb.2008.01.003

Detection and localisation of protein-protein interactions in Saccharomyces cerevisiae using a split-GFP method

Fungal Genet Biol. 2008 May;45(5):597-604. doi: 10.1016/j.fgb.2008.01.003. Epub 2008 Jan 26.

Authors

Emma Barnard¹, Neil V McFerran, Alan Trudgett, John Nelson, David J Timson

Affiliation

¹ School of Biological Sciences, Queen's University Belfast, Medical Biology Centre, 97 Lisburn Road, Belfast BT9 7BL, UK.

PMID: 18313953
DOI: 10.1016/j.fgb.2008.01.003

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.

Publication types

Research Support, Non-U.S. Gov't
Validation Study

MeSH terms

DEAD-box RNA Helicases / genetics
DEAD-box RNA Helicases / metabolism
Green Fluorescent Proteins / genetics
Green Fluorescent Proteins / metabolism
L-Lactate Dehydrogenase / genetics
L-Lactate Dehydrogenase / metabolism
Microscopy, Fluorescence
Phosphofructokinases / genetics
Phosphofructokinases / metabolism
Protein Interaction Mapping / methods*
Recombinant Fusion Proteins / analysis
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism
Saccharomyces cerevisiae / metabolism*
Saccharomyces cerevisiae Proteins / analysis*
Saccharomyces cerevisiae Proteins / genetics
Saccharomyces cerevisiae Proteins / metabolism*
Succinate Dehydrogenase / genetics
Succinate Dehydrogenase / metabolism
Transcription Factors / genetics
Transcription Factors / metabolism

Substances

Recombinant Fusion Proteins
Saccharomyces cerevisiae Proteins
Transcription Factors
enhanced green fluorescent protein
Green Fluorescent Proteins
L-Lactate Dehydrogenase
Succinate Dehydrogenase
Phosphofructokinases
MTR4 protein, S cerevisiae
DEAD-box RNA Helicases