In S. cerevisiae the AGT1 permease is responsible for the active H⁺-symport of a series of a-glucosides used in industrial applications of yeasts, such as baking, brewing and production of fuel ethanol. This permease has a high affinity for sucrose and trehalose, and a lower affinity for maltose and maltotriose. Transport of these sugars into the cells is not only the first step in their metabolism, but it is also the rate-limiting step for fermentation. In order to improve the catalytic properties of this permease, it is necessary to identify which amino acids are involved in the active H⁺ co-transport and/or substrate specificity. Previous studies with the E. coli lactose-H⁺ symporter encoded by LacY, has revealed that proton binding and translocation is mediated by four (Glu-126, Arg-144, Glu-269, and His-322) of the >10 charged amino acids present in the transmembrane helices of the permease. Analyzing the predicted structure of the AGT1 transporter we identify only 4 charged amino acids in its transmembrane segments (Glu-120, Asp-123, Glu-167 e Arg-504), residues that are significantly conserved at the same positions in several other known and/or putative a-glucoside transporters from Saccharomyces, Torulaspora, Kluyveromyces, and Candida yeasts. We have thus performed site-directed mutagenesis of these residues using PCR and an over-expressing plasmid containing the AGT1 gene. The functionality of the permease was assayed through transformation and maltotriose growth and fermentation tests by an agt1-deleted S. cerevisiae strain, and compared with the same strain carrying the wild-type permease. The results obtained so far indicate that when the Asp-123 residue was substituted by a glycine, the yeast strain carrying this mutated permease could still grow on maltotriose, but the rates of maltotriose consumption and fermentation where significantly lower that those obtained with the wild-type AGT1 transporter. Although our results suggest that Asp-123 is probably involved in the sugar-H⁺ co-transport, we are currently analyzing the involvement of the other three charged amino acid residues in the active sugar-H⁺ symport mediated by the AGT1 permease.