The long term goal of this research is to take advantage of the powerful genetic capabilities available in the yeast, Saccharomyces cerevisiae to understand 1) The kinds of potassium transporters present in this eukaryote and 2) the structure and function of the transporters. The questions to be addressed in the near term are the following: What are the structural and functional similarities, if any, between the high and low affinity potassium transporters already identified? What is the topological organization of these potassium transporters in the plasma membrane? Which amino acids in the transporter polypeptides are involved in the mechanism of potassium transport? A gene, TRK1, likely to encode the high-affinity potassium transporter, has been cloned, as has a gene, TRK2, required for low-affinity potassium uptake. Goals for the next three years are: 1) to define topological features of the high-affinity transporter (TRK1); 2) to determine the relationship between TRK1 structure and function by combining site-directed mutagenesis with in vivo functional assays; 3) to determine the molecular basis of potassium affinity and identify amino acids likely to interact directly with potassium ions by isolation and analysis of mutations in TRK1 that increase discrimination between potassium and rubidium; 4) to pursue a molecular analysis of the TRK2 gene; 5) to determine the molecular basis of mutations in the TRK2 gene and determine their physiological effects, 6) to determine the molecular changes in TRK2D mutants (alleles of TRK2 that enhance low-affinity potassium uptake); and 7) to assess the functional roles of the high and low- affinity potassium transporters. Future goals include genetically identifying potassium channel genes in yeast. The proposed research is a continuation of the molecular and genetic analysis of potassium transport in the yeast, Saccharomyces cerevisiae. This small eukaryote is useful as a model system in which to examine the molecular basis of potassium transport which is a highly conserved and fundamental physiological function of all cells. Through the isolation of mutants that exhibit altered potassium transport, proteins involved in the movement of this essential ion across cell membranes can be identified genetically, and the relationship between the structure and function of potassium transporter molecules can be investigated.
