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

MacPherson, Lisa Frances (2018) Split-DHFR as a protein complementation assay for localisation and interactions of yeast mitochondrial proteins. MSc(R) thesis, University of Glasgow.

Abstract

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.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Keywords: pca,dhfr,ims, mitochondria, ppi, yeast, intermembrane space ,protein complementation assay, dihydrofolate reductase, protein protein interaction.
• Subjects: Q Science > QH Natural history; Q Science > QH Natural history > QH301 Biology; Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
• Supervisor's Name: Tokatlidis, Professor Kostas and Christie, Professor John
• Date of Award: 2018
• Depositing User: Miss Lisa Frances MacPherson
• Unique ID: glathesis:2018-8948
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 17 Apr 2018 12:34
• Last Modified: 27 Apr 2018 08:18
• URI: https://theses.gla.ac.uk/id/eprint/8948