Split-DHFR as a protein complementation assay for localisation and interactions of yeast mitochondrial proteins

QH Natural history;QH301 Biology;QH345 Biochemistry

MacPherson, Lisa Frances ; Tokatlidis, Kostas

University:University of Glasgow

Department:Institute of Molecular Cell and Systems Biology

关键词: pca,dhfr,ims, mitochondria, ppi, yeast, intermembrane space ,protein complementation assay,dihydrofolate reductase, protein protein interaction.;   

Others  :  http://theses.gla.ac.uk/8948/1/2018macphersonmsc%28r%29.pdf

来源: University of Glasgow

【 摘 要 】

Protein complementation assays (PCAs) utilising two fragments of a reporter protein – fused to two potentially interacting proteins of interest – are a common method of analysing protein-protein interactions (PPIs). This approach, using split dihydrofolate reductase (DHFR) as a reporter protein, has been previously carried out for cytosolic Saccharomyces cerevisiae proteins. The focus of this study was to establish a split-DHFR assay specifically for use in analysing yeast mitochondrial PPIs in the intermembrane space (IMS), which has not been done before. A strategy to overcome the problem endogenous DHFR activity had to be developed using a modified strain of S. cerevisiae for the specific application here. Further, plasmids containing two positive control proteins, Tim9 and Tim10 (two well-known interacting proteins of the IMS) were cloned for transformation into yeast strain BY4741. Several other plasmids bearing various control proteins were designed and some of them cloned, although we required more time to have the full set of tools to establish the assay.