The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters

Abstract

Hgt1p, a high-affinity glutathione transporter from Saccharomyces cerevisiae belongs to the recently described family of OPTs (oligopeptide transporters), the majority of whose members still have unknown substrate specificity. To obtain insights into substrate recognition and translocation, we have subjected all 21 residues of TMD9 (transmembrane domain 9) to alanine-scanning mutagenesis. Phe⁵²³ was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in K_m without affecting expression or localization. Phe⁵²³ and the previously identified polar residue Gln⁵²⁶ were on the same face of the helix suggesting a joint participation in glutathione recognition, whereas two other polar residues, Ser⁵¹⁹ and Asn⁵²², of TMD9, although also orientated on the same face, did not appear to be involved. The size and hydrophobicity of Phe⁵²³ were both key features of its functionality, as seen from mutational analysis. Sequence alignments revealed that Phe⁵²³ and Gln⁵²⁶ were conserved in a cluster of OPT homologues from different fungi. A second cluster contained isoleucine and glutamate residues in place of phenylalanine and glutamine residues, residues that are best tolerated in Hgt1p for glutathione transporter activity, when introduced together. The critical nature of the residues at these positions in TMD9 for substrate recognition was exploited to assign substrate specificities of several putative fungal orthologues present in these and other clusters. The presence of either phenylalanine and glutamine or isoleucine and glutamate residues at these positions correlated with their function as high-affinity glutathione transporters based on genetic assays and the K_m of these transporters towards glutathione.