Constructing new yeast strains and testing interactions between protomers of yeast potassium translocation proteins by Bimolecular Fluorescence Complementation.
TRK1 encodes the main potassium translocation systems in Saccharomyces cerevisiae. TRK1 encodes on one polypeptide chain four domains that are homologous to (inwardly rectifying) potassium channel subunits. Even if Trk1 could be functional as monomer, it is thought that the system is made up out of more than one protomer in the plasma membrane.
To determine possible interactions between protomers and the number of protomers within the “physiological” assembly in the membrane, Bimolecular Fluorescence complementation (BiFC) will be used. BiFC is based on the ability of some proteins to form functional complexes when their polypeptide chain is split into two parts and if these two parts are in close proximity. In this project split “Venus” (a variant of the green fluorescent protein, GFP) will be used to analyze interactions between Trk1 protomers. The amino-terminal and carboxy-terminal parts of Venus will be inserted at different positions of the Trk1 polypeptide chain. Yeast strains co-“expressing” these proteins will be generated and analyzed by fluorescence microscopy. Fluorescence should only occur if these “half-proteins” are close enough to allow constitution of full Venus. The results should help answering the questions: (i) How many protomers are present in functional Trk1? And (ii) how are these protomers arranged?