Three critical aromatic sites have been identified in the yeast galactose transporter Gal2: Tyr352 at the extracellular boundary of putative transmembrane segment (TM) 7, Tyr446 in the middle of TM10 and Phe504 in the middle of TM12. The relationship between these sites was investigated by random mutagenesis of each combination of two of the three residues. Galactose transport-positive clones selected by plate assays encoded Tyr446 and specific combinations of aromatic residues at sites 352 and 504. Double-site mutants containing aromatic residues at these latter two positions showed either essentially full galactose transport activity (Phe352Trp504 and Trp352Trp504) or no significant activity (Phe352Tyr504 and Trp352Tyr504), whereas single-site mutants showed markedly reduced activity. These results are indicative of a specific interaction between sites 352 and 504 of Gal2.
