Biochem. J. (2010) 429, 593–602 (Printed in Great Britain) doi:10.1042/BJ20100240 593 The role of transmembrane domain 9 in substrate recognition by the fungal high-affinity glutathione transporters Anil THAKUR and Anand K. BACHHAWAT 1 Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India Hgt1p, a high-affinity glutathione transporter from Saccharo- myces 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. Phe 523 was found to be critical for glutathione recognition, since F523A mutants showed a 4-fold increase in K m without affecting expression or localization. Phe 523 and the previously identified polar residue Gln 526 were on the same face of the helix suggesting a joint participation in glutathione recognition, whereas two other polar residues, Ser 519 and Asn 522 , of TMD9, although also orientated on the same face, did not appear to be involved. The size and hydrophobicity of Phe 523 were both key features of its functionality, as seen from mutational analysis. Sequence alignments revealed that Phe 523 and Gln 526 were conserved in a cluster of OPT homologues from different fungi. A second cluster contained isoleucine and glutamate resi- dues 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. Key words: alanine scanning, glutathione, glutathione transporter, high-affinity glutathione transporter 1 (HGT1), oligopeptide transporter family (OPT), Saccharomyces cerevisiae oligo- peptide transporter family 1 (ScOPT1/OPT1). INTRODUCTION Hgt1p (or ScOpt1p), a high-affinity glutathione transporter of the yeast Saccharomyces cerevisiae [1,2], is a member of the relatively novel, and poorly characterized OPT (oligopeptide transporter) family [3]. The members of the OPT family [Transporter Classification (TC) # 2.A.67; http://www.tcdb.org/] are found in plants, fungi, bacteria and archaea, but are absent from metazoans [4]. The OPT family has been divided into two distinct clades: the PT (peptide transport) clade and the YS (yellow stripe) clade [3,5]. Despite the discovery of the family a decade ago, only a few members of the OPT family have been functionally characterized for their substrate specificity and physiological role. Among the fungi, the functionally characterized members of the PT clade include Hgt1p from S. cerevisiae, and Pgt1 from Schizosaccharomyces pombe, both of which have been shown to function as high-affinity glutathione transporters, although Hgt1p has also been shown to transport oligopeptides, albeit with lower affinity [1,2,6]. A second PT homologue in S. cerevisiae, OPT2, in contrast does not transport glutathione and has not been assigned any function. In S. pombe also, in addition to Pgt1, there are two other OPT members, and, of these, whereas Isp4 was shown to be able to transport oligopeptides, neither Isp4 nor the uncharacterized ORF (open reading frame) SPCC1840.12 was able to transport glutathione [1,3,6]. Candida albicans has eight OPT members: CaOPT1–CaOPT8. These have been shown to be involved in oligopeptide uptake, but the role in glutathione uptake, if any, has not been evaluated [7,8]. Among the PT clade members in plants, Arabidopsis thaliana has eight members, but their substrate specificities have not been clearly defined. AtOPT1 and AtOPT4 appear to be OPTs, AtOPT3 is a transporter of a still undefined Fe–chelator complex, whereas AtOPT6 has also been shown to be able to transport glutathione, albeit at a very low affinity [9–13]. A very weak glutathione transporter activity has been shown for the rice OPT, OsOPT6, and Brassica juncea, BjOPT6 [14,15]. In contrast, few of the plant YS members from Arabidopsis and Zea mays have been implicated in the transport of metal-chelating secondary amino acids, such as iron–deoxymugineic acid complexes [16–18]. However, no information is available on the substrate specificity or functions of the fungal or bacterial counterparts in the YS clade. The ability to assign functions to the members of the OPT family has been hampered by the very limited information available on mechanistic or structural aspects of members of the OPT family, in both the PT and YS clades. Although it is known that members of this family are proton-coupled transporters [11], the structural features that confer such distinct substrate specificity between the two clades and among the individual members within each clade need to be elucidated. The most extensively studied OPT member is Hgt1p. Investigations into the role of the cysteine residues, as well as the polar and charged amino acids in the TMDs (transmembrane domains) of Hgt1p have revealed that the TMD helices 1, 4 and 9 and the intracellular loop region 537–568 are important for substrate translocation [19,20]. Furthermore, two residues, Gln 222 in TMD4 and Gln 526 in TMD9, were found to be required for substrate recognition [19]. Although Gln 222 in TMD4 was widely conserved in the OPT family, Gln 526 of TMD9 appeared to be present only in two known glutathione Abbreviations used: ECL, enhanced chemiluminescence; HA, haemagglutinin; HGT, high-affinity glutathione transporter; OPT, oligopeptide transporter; ORF, open reading frame; PT, peptide transport; SD, synthetic-defined; TEF, transcriptional enhancer factor; TMD, transmembrane domain; YS, yellow stripe. 1 To whom correspondence should be addressed (email anand@imtech.res.in). c  The Authors Journal compilation c  2010 Biochemical Society