Constructing new yeast strains and testing interactions between monomers of yeast cation translocation proteins by Bi-and Trimolecular Fluorescence Complementation.
TRK1 and TRK2 encode structurally similar potassium translocation systems in Saccharomyces cerevisiae. Both consist of a single polypeptide chain containing four domains that are homologous to (inwardly rectifying) potassium channel subunits. Therefore, Trk1 could be functional as monomer. However, it is thought that the system is made up out of more than one monomer in the plasma membrane and it seems possible that Trk1 and Trk2 can form heteromers.
To determine possible interactions between monomers and the number of monomers within the “physiological” assembly in the membrane, Bi- and Tri-molecular Fluorescence complementation (BiFC/TriFC) will be used. These methods are based on the ability of some proteins to form functional complexes when their polypeptide chain is split into several parts if these parts are in close proximity. In this project BiFC or TriFC will be used to analyze interactions between Trk1 and/or Trk2 monomers. Yeast strains co-“expressing” these proteins will be generated and analyzed by fluorescence microscopy. The results should help answering the questions: (i) How many monomers are present in functional Trk1? And (ii), can Trk1 and Trk2 form heteromers in the plasma membrane?