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 Km 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 Km of these transporters towards glutathione.
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 . 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 . 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 , 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, Gln222 in TMD4 and Gln526 in TMD9, were found to be required for substrate recognition . Although Gln222 in TMD4 was widely conserved in the OPT family, Gln526 of TMD9 appeared to be present only in two known glutathione transporters, Hgt1p and Pgt1p, further suggesting that it might be specific for glutathione recognition.
In an effort to further investigate TMD9, which appeared to be important for substrate recognition among the family of OPT members, we have, in the present study, carried out an alanine-scanning mutagenesis of the 21 amino acid residues of TMD9 of Hgt1p followed by their functional characterization. Whereas several residues led to a moderate loss in activity, kinetic analysis of the most severely affected mutant, F523A, revealed that it plays a critical role in substrate recognition. Multiple sequence alignments of the TMD9 region of fungal OPTs revealed that the two key residues of TMD9 involved in glutathione recognition, Phe523 and Gln526, were conserved in some OPT members. Some members contained glutamate and isoleucine residues at these positions, two changes that we examined in Hgt1p, and found to be tolerated in relation to glutathione transport by Hgt1p. Expression and kinetic analysis of representive members of these putative orthologues indicated that they functioned as high-affinity glutathione transporters. In contrast, CaOPT1p of C. albicans, which has hydrophobic amino acids at both of these positions in TMD9 despite sharing a high overall similarity with Hgt1p, retained a very weak glutathione transport activity. In addition to defining the residues important for substrate recognition in Hgt1p, the present study thus also succeeds in identifying a glutathione transporter signature present in the fungal OPT family.
Chemicals and reagents
All of the chemicals used in the present study were analytical grade and obtained from commercial sources. Medium components were purchased from Difco, Sigma–Aldrich, HiMedia, Merck India and USB Corporation. Oligonucleotides were purchased from Sigma India. Restriction enzymes, Vent DNA polymerase and other DNA-modifying enzymes were obtained from New England Bio Labs. A DNA sequencing kit (ABI PRISM 310 XL with dye-termination cycle sequencing ready reaction kit) was obtained from PerkinElmer. Gel-extraction kits and plasmid miniprep columns were obtained from Qiagen or Sigma. [35S]GSH (specific activity 1000 Ci/mmol) was purchased from the Bhabha Atomic Research Centre. HA (haemagglutinin)-tagged (6E2) mouse monoclonal antibody and horse anti-mouse HRP (horseradish peroxidase)-linked antibody were bought from Cell Signaling Technology. Alexa Fluor® 488-conjugated goat anti-mouse antibody was obtained from Molecular Probes. Hybond ECL (enhanced chemiluminescence) nitrocellulose membrane and ECL plus Western blotting detection reagents were purchased from Amersham Biosciences.
Strains, medium and growth conditions
The Escherichia coli strain DH5α was used as a cloning host. The S. cerevisiae strain ABC 817 (MATa his3Δ1 leu2Δ0 met15Δ-0 ura3Δ0 hgt1Δ:: LEU2) and the S. pombe strain ABC 2187 (h-leu1-32 ura4-c190T cys1aΔ pgt1Δ::ura4) deficient in glutathione uptake ability were used as hosts in the complementation studies [1,6]. Kluyveromyces lactis (MTCC 223) and Pichia guilliermondii (MTCC 1311) were obtained from the Microbial Type Culture Collection (Institute of Microbial Technology, Chandigarh, India). C. albicans Sc5314 was obtained from Dr K. Ganesan (Institute of Microbial Technology, Chandigarh, India), and Schizosaccharomyces japonicus (NCYC 419) was a laboratory stock strain . S. cerevisiae was regularly maintained on YPD (yeast extract, peptone and dextrose) medium. S. pombe was maintained on YES (yeast extract, dextrose and supplements) medium. S. cerevisiae SD (synthetic-defined) minimal medium contained yeast nitrogen base, ammonium sulfate and dextrose supplemented with histidine, leucine and methionine (when required) at 50 mg/l . For S. pombe, SD EMM (Edinburgh minimal medium) was prepared as described previously . Glutathione was added as required. Growth, handling of bacteria and yeast, and all of the molecular techniques used in the present study were according to standard protocols 
HGT1, tagged with an HA tag at the C-terminus, was subcloned downstream of the TEF (transcriptional enhancer factor) promoter at BamHI and EcoRI sites of a modified p416TEF vector . This construct was used as a template for site-directed mutagenesis for creation of different site-directed mutants of Hgt1p, by a splice overlap extension strategy. The mutations were directly generated using different mutagenic oligonucleotides (Supplementary Table S2 at http://www.BiochemJ.org/bj/429/bj4290593add.htm), and the PCR products generated with these oligonucleotides were subcloned back into the TEF vector background (pTEF-His-HGT1m-HA) using appropriate restriction sites, for subsequent analyses. The resulting mutants were sequenced to confirm the presence of the desired nucleotide changes and rule out any undesired mutations introduced during the mutagenic procedure. For tagging the CaOPT1 gene with HA, we tagged the C-terminus of CaOPT1 with an HA epitope by PCR mutagenesis. CaOPT1 has a single CUG codon at position 371, predicted to code for serine in C. albicans and leucine in S. cerevisiae, and we also mutated this codon by site-directed mutagenesis so that it codes for serine in S. cerevisiae.
The dual ‘complementation-cum-toxicity’ plate assay for assessing HGT1 functionality
The dual complementation-cum-toxicity assay has been described previously [19,20]. The S. cerevisiae met15Δhgt1Δ strain (ABC 817) was transformed with a single-copy centromeric vector expressing wild-type or different mutants of TMD9 of HGT1 expressed downstream of the TEF promoter. Transformants were grown in minimal medium containing methionine and other supplements, without uracil, overnight. These cultures were re-inoculated in the same medium and allowed to grow until they reached exponential phase. An equal number of cells were harvested, washed with water and resuspended in sterile water to a D600 of 0.2. These were serially diluted 1:10, 1:100 and 1:1000. Cell resuspensions (10 μl) were spotted on to minimal medium containing different concentrations of glutathione (15, 30, 50, 100, 150 and 200 μM) or methionine (200 μM) as the sole organic sulfur source. The plates were incubated at 30 °C for 2–3 days and photographs were taken.
Glutathione transport assay
The S. cerevisiae ABC 817 strain (met15Δhgt1Δ) was transformed with different plasmids constructs bearing wild-type or HGT1 mutants under the TEF promoter and were grown in minimal medium containing methionine and other supplements, without uracil, overnight. These cultures were re-inoculated in the same medium and allowed to grow until they reached exponential phase. Cells were harvested, washed and placed on ice in a Mes-buffered medium, until the transport was initiated. Transport experiments were carried out with [35S]GSH as described previously . The results were expressed as nmol of glutathione/mg of protein per min. For the measurements of total protein, 100 μl of the above cell suspension (cell suspension volume used for the transport assay) was boiled with 15% sodium hydroxide for 10 min, followed by neutralization of total cell lysate by the addition of hydrochloric acid. A 100 μl aliquot of this crude cell lysate was incubated with 0.1% Triton X-100 for 10 min and total protein was estimated using Bradford reagent (Sigma) with BSA as a standard. For saturation kinetics, the initial rate of glutathione uptake was measured at a range of glutathione concentrations from 12.5 μM to 400 μM, with specific activity being kept constant at each concentration. The initial rate of glutathione uptake was determined by measuring the radioactive glutathione accumulated in the cells at 30 s and 180 s time points in the ABC 817 strain transformed with different plasmid constructs bearing wild-type or HGT1 mutants and KlHgt1, SjHgt1 or CnHgt1 under the TEF promoter or only empty vector, after subtracting the initial rate of glutathione uptake with vector alone from the initial rate of glutathione uptake with the different test constructs. Using Microsoft Excel, the Lineweaver–Burk best-fit plot was obtained, which was used to calculate the kinetic parameters. The average Km and Vmax values were calculated. The experiment was repeated a minimum of two times for each test construct in duplicate at each glutathione concentration. The corrected Vmax values indicated that the kinetic parameters were normalized to the protein expression levels.
Preparation of cell extract and immunoblot analysis
Total crude cell preparation and immunoblot analysis was performed as described previously . Densitometry analysis of the unsaturated band signals was performed using the Scion Image software to quantify the protein expression levels in different mutants. The resulting signal intensity was normalized with respect to the band surface area (in square pixels) and expressed in arbitrary units. The relative protein expression levels in the mutant Hgt1p were represented as the percentage expression relative to wild-type Hgt1p, as the means of three independent blotting experiments.
Cellular localization of the mutant proteins by confocal microscopy
To localize Hgt1p and its different alanine mutants, indirect immunofluorescence was performed using a published protocol, modified as described previously . Images were obtained with an inverted LSM510 META laser-scanning confocal microscope (Carl Zeiss) fitted with a Plan-Apochromat ×100 (numerical aperture, 1.4) oil-immersion objective. The 488 nm line of an argon ion laser was directed over an HFT UV/488 beam splitter, and fluorescence was detected using an NFT 490 beam splitter in combination with a BP 505–530 band pass filter. Images obtained were processed using Adobe Photoshop version 5.5.
Multiple sequence analysis and phylogenetic analysis
Alanine-scanning mutagenesis of TMD9
The predicted TMD9 of Hgt1p contains 21 amino acids  that include three polar residues, Ser519, Asn522 and Gln526 (Figure 1). To assess the contribution of these individual residues in glutathione transport, each of the 18 non-alanine residues were mutated to alanine by site-directed mutagenesis. In addition, the three alanine residues (Ala509, Ala511 and Ala515) were mutated to glycine, to examine whether the side chains of these alanine residues might be functionally important. Each of the mutants was subjected to an initial functional characterization using a previously designed sensitive plate assay, termed a dual complementation-cum-toxicity assay [19,20]. The assay is based on the dual behaviour of HGT1 expressed under the strong constitutive TEF promoter in a met15Δhgt1Δ strain. The met15Δhgt1Δ strain is an organic sulfur auxotroph owing to met15Δ, and is also deficient in glutathione uptake owing to hgt1Δ. At a low glutathione concentration (15 μM), HGT1 expressed under the TEF promoter complements the growth defect, whereas at higher glutathione concentrations (50 μM or higher), the HGT1 expressed under the TEF promoter shows toxicity owing to excess glutathione accumulation . Thus the p416TEF plasmid bearing the different mutants of TMD9 (or wild-type) were individually transformed into the met15Δhgt1Δ strain and mutants were analysed for their ability to confer complementation and/or toxicity on the cells over a range of glutathione concentrations (Figure 2). As seen in Figure 2, the different mutations exhibited a differential effect on the ability of the cells to grow on glutathione at low and high concentrations as compared with the wild-type Hgt1p. Hence, based on their growth behaviour in the dual complementation-cum-toxicity assay, the mutants were characterized into four groups. The ‘severe effect’ group included those in which complementation was defective at a glutathione concentration of 15 μM. The ‘moderate effect’ group included those in which complementation was observed at 15 μM, but toxicity was not seen at higher glutathione concentrations. The ‘mild effect’ group was those in which complementation was observed at 15 μM, but which show limited toxicity at higher concentrations. The ‘very mild to no effect’ group were mutants in which complementation was observed at 15 μM, and toxicity was seen at a higher concentration of glutathione (mutant very similar to wild-type protein) (Table 1).
Hgt1p topology showing that the location of the TMDs and residues Phe523 and Gln526 in TMD9 critical for the transport activity of Hgt1p falls on the hydrophilic face of the transmembrane domain
Functional characterization of alanine mutants of TMD9 of Hgt1p
|Mutant||Complementation (15 μM GSH)||Toxicity (>50 μM GSH)||Transport activity (%)|
|Severe effect (complementation defective)|
|Moderate effect (no toxicity)|
|Mild effect (mild toxicity at high GSH concentrations)|
|Mutant||Complementation (15 μM GSH)||Toxicity (>50 μM GSH)||Transport activity (%)|
|Severe effect (complementation defective)|
|Moderate effect (no toxicity)|
|Mild effect (mild toxicity at high GSH concentrations)|
On the basis of this functional plate assay, only F523A, in addition to Q526A, showed a very severe defect in functionality, and the A515G, I524A, P525A and I529A mutants showed a moderate effect on functional activity of Hgt1p. Five mutants had a mild effect (W510A, F512A, I516A, S518A and I528A), and the remaining mutants (A509G, A511G, V513A, I514A, L517A, S519A, L520A, V521A, N522A and G527A) had no, or an insignificant, effect being close to or comparable with wild-type (Table 1), and were not pursued further.
Functional evaluation of mutants by radioactive-uptake assay
Although the plate assay is a very sensitive reflection of the functionality of the mutants, it was still necessary to confirm their functionality biochemically, by direct radioactive uptake assays. The mutants falling in the ‘severe’, ‘moderate’ or ‘mild’ category were evaluated by measuring the initial rate of [35S]GSH uptake in the met15Δhgt1Δ strain, transformed with the different mutants of TMD9 of Hgt1p. We found that the results from the uptake assay were similar to the genetic assay and defined groups of activity similar to the plate assay (Table 1). Out of the 21 mutants of TMD9, F523A, like the previously described Q526A, displayed substantially reduced transport activity. Four mutants (A515G, I524A, P525A and L529A) led to a moderate effect on functional activity of Hgt1p. Five mutants had a mild effect (W510A, F512A, I516A, S518A and I528A). The remaining mutants showed little or no effect in the genetic assay and were not analysed (Table 1). Analysis of the steady-state protein levels of these mutants revealed that the protein expression levels of the mutants ranged between 50 and 90% relative to the wild-type protein levels (Supplementary Figures S1A and S1B at http://www.BiochemJ.org/bj/429/bj4290593add.htm). Only mutants I524A and P525A showed a significant fall in protein levels, and it is likely that the decreased protein levels in these mutants could account for the loss in functionality for these, but not for the other, mutants.
Analysis of cell-surface trafficking of mutants with loss in functional activity
As Hgt1p is a plasma-membrane-localized transporter , it was important to examine the defective mutants expressed under the TEF promoter for their subcellular localization. This was carried out by indirect immunofluorescence using an anti-HA monoclonal antibody as a primary antibody followed by the Alexa Fluor® 488-conjugated secondary antibody images captured by confocal microscopy as described previously . A signal was observed at the plasma membrane of the cells transformed with wild-type Hgt1p as well as the defective mutants (Supplementary Figure S2 at http://www.BiochemJ.org/bj/429/bj4290593add.htm). Interestingly, although F512A and I524A were predominantly localized to the plasma membrane, they also showed a small amount of intracellular signal in addition to the cell-surface signal in a few of the cells, suggesting a partial defect in trafficking of these mutants. However, the remaining mutants, W510A, F512A, A515G, I516A, S518A, F523A, I524A, P525A, Q526A, I528A and L529A, localized correctly to the plasma membrane.
Kinetic analysis of F523A reveals a role in substrate binding and translocation
The only mutant in TMD9, other than the previously described Q526A, that was severely affected in function was F523A. Among the mutants that were moderately affected in function were A515G, I524A, P525A and L529A. In a previous study using similar criteria, we had observed that mutants that were falling into the ‘moderately effected’ group which were amenable to kinetic analysis showed no significant change in either substrate binding or catalysis . Furthermore, of the four mutants falling into this category in the present study, mutants I524A and P525A had significantly lower protein level expression, which might account for much of the defect. We therefore did not follow up this category of mutants for kinetic analysis and only focused on F523A, the single mutant other than the previously described Q526A that showed a severe affect in activity. As this mutant displayed almost normal levels of protein expression, and was also localized to the cell surface, it suggested a more direct role in glutathione binding and translocation. To gain further insight into the mechanism by which the F523A mutant resulted in decreased transport of glutathione, this mutant was further characterized by carrying out detailed kinetic analysis. The Km and Vmax values for wild-type Hgt1p were estimated to be 48.1±10.5 μM and 57.8±3.9 nmol of glutathione/mg of protein per min . Kinetic characterization of the mutant revealed that the F523A mutant had a 4-fold increased Km for GSH (Km=182.2±39.9 μM) and also exhibited a loss in Vmax value (corrected Vmax=29.2±3.0 nmol of glutathione/mg of protein per min). These findings suggest that this residue is likely to play a critical role in interacting with the substrate. Although an approx. 2-fold higher Vmax value was observed in the wild-type as compared with the F523A, this might be partially explained by the approx. 1.25-fold higher protein levels seen in the wild-type as compared with the mutant (Figure 3B).
Multiple sequence alignment for the protein sequences of the PT members of the OPT family
Functional analysis of Phe523 mutants
The drastic fall in the activity of the F523A mutant and the 4-fold increase in Km for GSH in this mutant suggested that Phe523 played an important role in binding and transport activity of the protein. To investigate the nature of interactions being made by Phe523 that are important for GSH recognition and transport, we created different mutants where we changed the phenylalanine residue to tryptophan, tyrosine or isoleucine and evaluated them for their ability to transport glutathione. Functionality of the transporter was examined by both the genetic assay as well as by measurement of radioactive uptake. All of these mutants, F523W, F523I and F523Y, exhibited complementation at a glutathione concentration of 15 μM, but, with increasing GSH concentrations, F523Y and F523I showed toxicity at higher GSH concentration, thus showing a behaviour similar to that of wild-type protein, whereas F523W was less active as it showed less toxicity at higher concentrations. F523I exhibited moderate toxicity as compared with Phe523 (Supplementary Figure S3A at http://www.BiochemJ.org/bj/429/bj4290593add.htm). These results were further validated by the radioactive-uptake assay, where F523W displayed 25–30% GSH uptake (F523A had 18–20%) relative to the wild-type protein at 100 μM GSH. In contrast, F523Y exhibited approx. 85% and F523I exhibited 65% transport activity in the radiolabelled GSH uptake as compared with wild-type protein (Supplementary Figure S3B). These observations suggest that the presence of an aromatic residue was not mandatory for the activity, although it did influence the transport.
Sequence comparisons of the TMD9 regions of the fungal OPTs
The observation that Phe523 and Gln526 in TMD9 were critical for substrate recognition in Hgt1p prompted us to examine the occurrence of these residues as well as other possible conserved residues in TMD9 of fungal OPT members of the PT clade. Previously Gln526 was found to be present in the two reported glutathione transporters, Hgt1 and Pgt1, and was absent from the other functionally characterized non-glutathione transporters, suggesting that Gln526 might be a residue specific for high-affinity glutathione transporters. Multiple sequence alignment of the PT clade of the fungal OPT family revealed that in TMD9 only two residues, Phe525 and Gly527, were conserved across the family. However, whereas P525A mutants led to a significant fall in protein expression levels, implicating a role for this residue primarily in protein stability, the G527A mutant surprisingly showed no apparent role in glutathione uptake despite its wide conservation in the family.
When we examined the conservation pattern of Phe523 and Gln526 of TMD9, we observed that these residues are simultaneously conserved in only a small subset of OPT family members. Other than S. cerevisiae, these members include the S. pombe glutathione transporter, Pgt1, as well as still uncharacterized homologues, present in six other yeasts which include K. lactis, Zygosaccharomyces rouxii, Candida lusitaniae, Pichia pastoris, P. guilliermondii and S. japonicus (Figure 3). Importantly, the phenylalanine and glutamine residues are not found in the fungal OPT members that have been clearly shown not to play any role in glutathione transport, such as OPT2 of S. cerevisiae, Isp4 and SPCC1840.12 of S. pombe [1,6]. It therefore appeared possible that Phe523 and Gln526 might be critical determinants for recognition of glutathione as a substrate.
Functional analysis of HGT1 orthologues from K. lactis, P. guilliermondii and S. japonicus containing phenylalanine and glutamine in TMD9 identifies these as high-affinity glutathione transporters
To investigate whether the orthologues of HGT1 in K. lactis (KlHGT1), P. guilliermondii (PgHGT1) and S. japonicus (SjHGT1), representatives of members containing the corresponding Phe523 and Gln526 residues of TMD9, might also function as high-affinity glutathione transporters, we amplified and cloned each of these ORFs under the strong constitutive TEF promoter of S. cerevisiae. We observed complementation of the mutant strains by KlHGT1 and SjHGT1 indicating that these two ORFs were able to transport glutathione. We have observed previously that a strong correlation exists between the ability of transporters (or mutants) to complement at low concentrations of glutathione and their affinity for the substrate [6,19]. Thus complementation at a low concentration of glutathione (15 μM) requires a transporter to have a low Km (high affinity) for the substrate. KlHGT1 and SjHGT1 could complement the growth defect of the met15Δhgt1Δ strain of S. cerevisiae even at low (15 μM) glutathione concentrations (Supplementary Figure S4A at http://www.BiochemJ.org/bj/429/bj4290593add.htm). These results clearly suggest that the KlHGT1 and SjHGT1 encode high-affinity glutathione transporters in K. lactis and S. japonicus respectively. In the case of PgHGT1, however, it did not complement the S. cerevisiae met15Δhgt1Δ strain. We therefore expressed PgHGT1 in S. pombe [under the strong constitutive ADH (alcohol dehydrogenase) promoter]. PgHGT1 was able to restore the growth of the S. pombe cys1aΔpgt1Δ strain (cysteine auxotroph and lacking the glutathione transporter) on glutathione. Furthermore, PgHGT1 complemented the growth defect even at low glutathione concentrations, suggesting that PgHGT1 also functions as a high-affinity glutathione transporter (Supplementary Figure S4B).
To confirm whether these transporters displayed a high affinity for glutathione, we determined the Km of the two transporters that were functional in S. cerevisiae KlHGT1 and SjHGT1. We observed that KlHGT1 had a Km of 64.3±8.7 μM, whereas SjHGT1 had a slightly higher Km of 204.1±56 μM. These transporters were therefore clearly functioning as high-affinity glutathione transporters.
Q526E and the double mutant Q526E/F523I show increased GSH transport activity as compared with Q526A
Multiple sequence alignment of S. cerevisiae HGT1 with other fungal OPT family members revealed an isoleucine residue in the position of phenylalanine (Phe523) and a glutamate residue in the position of glutamine (Gln526) (Figure 3). As a phenylalanine to isoleucine is an acceptable amino acid change at position 523 for Hgt1p activity, we examined whether Q526E could also display glutathione transport activity. Glutamine is an amide of glutamate, and the change from glutamine to glutamate changes the charge at this position, although the side-chain group is essentially the same.
We observed that the Q526E mutant restored the activity relative to Q526A, albeit to a very limited extent, but when we introduced both F523I and Q526E creating a double mutant Q526E/F523I, it was able to support growth of the met15Δhgt1Δ strain even at a GSH concentration of 15 μM (Supplementary Figure S5A at http://www.BiochemJ.org/bj/429/bj4290593add.htm). In terms of radioactive uptake, 35–45% of activity was observed in this double mutant (Q526E/F523I) compared with wild-type protein (Figure 4A), and this was despite the mutant showing reduced protein expression levels relative to the wild-type (Figure 4B). The single and double mutants were all correctly localized to the cell surface (Supplementary Figure S5B).
Functional analysis of Q526E and F523I/Q526E mutants
Investigation of the putative HGT1 orthologues from Cryptoccocus neoformans and C. albicans reveals CnHGT1, but not CaOPT1, as a high-affinity glutathione transporter: enlarging the clusters of glutathione transporters in fungi
To investigate whether the orthologues of HGT1 which contained isoleucine and glutamate in place of Phe523 and Gln526 in TMD9 might also function as high-affinity glutathione transporters, we expressed the C. neoformans orthologue CnHGT1 under the TEF promoter of S. cerevisiae and examined its ability to complement the growth defect of the met15Δhgt1Δ strain. Even at 15 μM glutathione, complementation could be observed and suggested that CnHGT1 is also a glutathione transporter (Supplementary Figure S4C). Kinetic analysis revealed it to be a high-affinity glutathione transporter (Km=82.5±15.5 μM)
CaOPT1 is the apparent C. albicans orthologue of Hgt1p (they are the respective best hit of each other as seen from Reverse BLAST analysis), and also shows significant sequence similarity to Hgt1p. CaOPT1 has been described as an OPT that can transport tetra- and octa-peptides , but its role in glutathione transport has never been evaluated. When we examined the TMD9 of CaOPT1 it lacked the important phenylalanine and glutamine residues (Figure 3), but, being the apparent orthologue of Hgt1p, it was of interest to evaluate whether CaOPT1 might function as a glutathione transporter. CaOPT1 was cloned and expressed downstream of the TEF promoter and when transformed into the S. cerevisiae met15Δhgt1Δ strain it failed to complement the growth defect at lower glutathione concentrations (15–30 μM). However, at significantly higher concentrations of glutathione (greater than 150 μM) growth was observed (Supplementary Figure S6A at http://www.BiochemJ.org/bj/429/bj4290593add.htm). This observation suggested that CaOPT1 is only a weak transporter of glutathione. We measured the uptake of radiolabelled glutathione ([35S]GSH) (at 100 μM GSH) in the met15Δhgt1Δ strain of S. cerevisiae. CaOPT1p resulted in very low glutathione accumulation in the cells, 5–6-fold lower than Hgt1p (Figure 5A). The level of transport was too low to enable us to determine the kinetic parameters. The lower activity of the CaOPT1 was not due to improper expression in an heterologous system (Figure 5B), nor was it due to a defect in localization to the cell surface, as seen by immunoblotting (Supplementary Figure S6B). The little transport that was observed when normalized to expression levels indicated extremely poor glutathione transport capabilities compared with HGT1.
CaOPT1 of C. albicans is not an efficient glutathione transporter
The identification of Phe523 of HGT1 as a second residue together with Gln526 of TMD9 as playing an important role in determining the affinity of the transporter towards its substrate, glutathione, has allowed us to define two key residues important for substrate specificity of Hgt1p. It has also led to a means to identify the substrate specificity of other members of the OPT family since the phenylalanine and glutamine residues at these positions appeared as a possible signature motif for recognizing high-affinity glutathione transporters of the OPT family. Sequence retrieval and comparison reveal that yeasts of seven other different genera also had orthologues of Hgt1p that contained these same residues in the corresponding positions of TMD9. Functional analysis of the putative orthologues from three of the yeasts of this group, K. lactis, P. guilliermondii and S. japonicus, reveal that these were indeed coding for high-affinity glutathione transporters. The Km of SjHGT1 was a little higher (204.1 μM) than those of the other transporters, but the growth at 15 μM clearly classified it as a high-affinity glutathione transporter. The orthologous proteins with phenylalanine and glutamine in TMD9 were observed to form a clear cluster in phylogenetic analysis that we refer to as the ‘phenylalanine and glutamine cluster’ or the ‘ScHGT1 cluster’. As subsequent analysis with Hgt1p revealed that replacement of phenylalanine with isoleucine and glutamine with glutamate also led to functional activity, and these residues were found in the corresponding position of phenylalanine and glutamine in many proteins, we also investigated a putative orthologue from C. neoformans belonging to this group, CnHGT1, and found that CnHGT1 also functioned as a high-affinity glutathione transporter. Interestingly, phylogenetic analysis with members of the OPT family [that included all members present in the TCDB (transporter classification database)] reveals that proteins with the glutamate and isoleucine residues also cluster together (‘glutamate and isoleucine cluster’ or the ‘CnHGT1 cluster’) (Figure 6). The observation that the double mutant containing glutamate and isoleucine changes in HGT1 was more effective in transporting glutathione than the single mutants suggests the need for the co-evolution of these residues for glutathione transport in these orthologues.
Unrooted phylogenetic tree of the OPT family
The demonstration that the closest homologue of Hgt1p in C. albicans, CaOPT1, lacks the corresponding phenylalanine/isoleucine and glutamine/glutamate residues, and does not display any significant capability of glutathione transport further underlines the importance of these residues in glutathione recognition. The present study should also help to resolve much of the confusion that has surrounded the yeast members of the OPT family. CaOPT1, the first member of the OPT family to have been investigated, was described as an OPT. In contrast, Hgt1p of S. cerevisiae, which shows significant similarity to CaOPT1, was described as a high-affinity glutathione transporter, and simultaneously also as an OPT . However, the higher affinity for glutathione and the transcriptional regulation of the protein by sulfur limitation  has clearly underlined its true physiological role in glutathione transport. It was therefore imperative to assess the role of CaOPT1 in glutathione transport as this had not been investigated previously. CaOPT1 was found to have valine and leucine residues in the corresponding positions in TMD9 to phenylalanine and glutamine, and on the basis of the findings of the present study it is clear that their true substrate specificity might indeed be other than glutathione. [CaOPT1 has a single CUG codon that codes for serine rather than leucine. However, introducing this change in CaOPT1 expressed in S. cerevisiae led only to a further decrease in glutathione transport capability (results not shown).] Although previous studies on the OPT members CaOPT1–CaOPT5 in C. albicans have revealed that several of the CaOPTs are up-regulated during conditions of nitrogen limitation and were capable of utilizing different oligopeptides of different sequence and sizes, the affinities for the different substrates were not examined . Several plant homologues of the OPT family in the PT clade have also been evaluated for glutathione transport, but it has been observed that none of the plant transporters are high-affinity glutathione transporters, even though some of them can transport glutathione at lower affinity. The substrate specificity of these transporters has thus not yet been clearly defined, although in many cases they also have been shown to transport different oligopeptides. Interestingly, none of the plant clones contained the phenylalanine and glutamine residues in TMD9. The clear demonstration that TMD9 plays a key role in substrate specificity will thus greatly facilitate and accelerate work on assigning the substrate specificity of not only the fungal, but also the plant, OPTs.
Although the present study has focused on residues in TMD9 that are important for glutathione recognition and translocation in HGT1 and would be of great use in assigning function to the rapidly increasing numbers of OPT members, one needs to bear in mind that in addition to TMD9, other TMDs and residues within these other TMDs might also participate in forming the channel that would be critical for substrate binding and translocation. This is, as yet, only the first TMD of any OPT subjected to such a detailed analysis. Thus the complete signature motif for glutathione recognition would only be available when the other TMDs have been similarly examined. A previous study had implicated TMDs 1, 4 and 9  and it would be important to investigate TMDs 1 and 4 in greater detail, as well as other TMDs (TMDs 5 and 8) that were not targeted in the previous study, among others.
Although hydrophilic residues in TMDs are in many cases known to play a key role in forming the aqueous channel for substrate translocation of hydrophilic substrates, and was in fact the basis for the original strategy where polar charged amino acids of the TMDs of HGT1 were targeted leading to the identification of Gln526, the identification of the aromatic residue Phe523 as important for substrate specificity was initially surprising. However, several reports have identified aromatic amino acid residues in TMDs of transporters of even hydrophilic substrates as being critical in substrate recognition and transport activity [31–33]. Thus, for example, two aromatic residues in TMD10 of the high-affinity galactose transporter, Gal2, Tyr446 and Trp455, were found to be critical for galactose recognition .
The interactions involving Phe523 in substrate recognition are unclear. However, the observation that F523Y and F523I mutants were functional although they showed lower activity compared with the wild-type protein, whereas the F523W showed a severe loss in activity, suggested that both size, as well as hydrophobicity, of the residue were important at that position. Clearly, more structural insights are required. However, in the absence of any structures currently available in even remote members of this family, current insights on the functioning of these transporters would have to be gathered from biochemical and genetic analysis of these transporters. It is hoped that future studies directed towards these goals would yield a clearer picture on the mechanism of substrate recognition and translocation by these important class of transporters.
Anil Thakur performed all of the experiments. Anand Bachhawat supervised the project. Both authors contributed to the experiment design, analysis of the data and the preparation of the manuscript.
We thank Pragya Yadav for help in cloning the CaOPT1 alleles, Akhilesh Kumar for making the CUG to UCG mutation in CaOPT1 and for HA tagging of CaOPT1. We thank Mr Deepak Bhatt and Dr Alok Mondal for their help in acquiring confocal images. We thank Mr Manish Datt for drawing the side-view of TMDs using the PyMol Viewer. We also thank Jaspreet Kaur for suggestions and critical input during the course of this study. All individuals acknowledged are from the Institute of Microbial Technology, Chandigarh, India.
This work was supported in part by Grant-in-Aid projects to A.K.B. from the Department of Science and Technology, Government of India [grant number SR/SO/BB-10/2009] and Department of Biotechnology, Government of India [grant number SR/SO/BB-24/2004]. A.T. was the recipient of a Research Fellowship from the Council of Scientific and Industrial Research, Government of India.