9406577 Gaber The PI, Dr. Richard Gaber, proposes to conduct genetic and molecular analyses of K+ transporters in the yeast Saccharomyces cerevisiae. The proposal has four aims. These are to investigate by molecular and genetic methods: 1) the potassium transporting glucose transporters. A detailed analysis of potassium uptake by one of these transporters, HXT3, will be carried out. An extensive collection of spontaneous suppressor mutations in HXT3 will be obtained and the nature of each suppressor mutation will be determined. Their effects on potassium/glucose stoichiometry, glucose transport kinetics and substrate specificity will be determined. 2) Genetic and molecular analysis of potassium transporting amino acid transporters will be carried out. Some trk1d trk2d suppressors do not show dependency on glucose. DNA sequence analysis of one of these, RPD4-1, revealed that it encodes a new member of a family of genes that encode amino acid transporters. Further experiments will determine whether this mutant transporter mediates potassium uptake, and if so, whether potassium uptake is dependent on co-transport of the permeant amino acid. 3) Genetic and molecular analysis of additional trk1d and trk2d suppressors will be carried out. Genetic analysis of the collection of these suppressors will be completed and a representative of each genetically distinct locus will be cloned and sequenced. The collection contains at least two more suppressors of interest, RPD7, represented by 10 independent dominant alleles, and rpd6-1, which is allelic to VPS16, a gene encoding a vacuolar sorting protein. 4) Suppressor mutations will be targeted to specific transporters. %%% Simple mutations in sugar transporters can confer upon these proteins the ability to transport potassium ions thus suppress the potassium-uptake-defective phenotype of Saccharomyces cerevisiae cells deleted for their normal potassium transporters. These suppressor mutations alter single amino acids within putative membrane spanning domains to confer potassium/sugar co-transport. A similar suppressor mutation in a putative amino acid transporter has also been found. These novel transporters offer a unique opportunity to study the molecular basis of solute transport. ***
