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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Pages (from-to)597-604
Number of pages8
JournalFungal Genetics and Biology
Volume45
Issue number5
DOIs
Publication statusPublished - 1 May 2008

Keywords

  • Biomolecular fluorescence complementation assay
  • Enhanced green fluorescent protein
  • Hapto-GFP
  • Isocitrate dehydrogenase
  • Phosphofructokinase
  • Protein-fragment complementation assay
  • Succinate dehydrogenase
  • TRAMP complex

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