Abstract
Hgt1p, a member of the oligopeptide transporter family, is a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae. We have explored the role of polar or charged residues in the putative transmembrane domains of Hgt1p to obtain insights into the structural features of Hgt1p that govern its substrate specificity. A total of 22 charged and polar residues in the predicted transmembrane domains and other conserved regions were subjected to alanine mutagenesis. Functional characterization of these 22 mutants identified 11 mutants which exhibited significant loss in functional activity. All 11 mutants except T114A had protein expression levels comparable with wild type, and all except E744A were proficient in trafficking to the cell surface. Kinetic analyses revealed differential contributions toward the functional activity of Hgt1p by these residues and identified Asn-124 in transmembrane domain 1 (TMD1), Gln-222 in TMD4, Gln-526 in TMD9, and Glu-544, Arg-554, and Lys-562 in the intracellular loop region 537-568 containing the highly conserved proline-rich motif to be essential for the transport activity of the protein. Furthermore, mutants Q222A and Q526A exhibited a nearly 4- and 8-fold increase in the K(m) for glutathione. Interestingly, although Gln-222 is widely conserved among other functionally characterized oligopeptide transporter family members including those having a different substrate specificity, Gln-526 is present only in Hgt1p and Pgt1, the only two known high affinity glutathione transporters. These results provide the first insights into the substrate recognition residues of a high affinity glutathione transporter and on residues/helices involved in substrate translocation in the structurally uncharacterized oligopeptide transporter family.
Highlights
Gopeptide transporter (OPT)3 family, that contains a large number of fungal, plant, and prokaryotic members [2]
In the current study we have focused on the polar and charged residues in the transmembrane domains of Hgt1p to explore their role in substrate recognition
As replacement of Gln-222 in TMD4 and Gln-526 in TMD9 with alanine resulted in a significant decrease in the affinity of the transporter for glutathione, it suggested that the two residues might directly participate in glutathione recognition as a substrate
Summary
Chemicals and Reagents—All the chemicals used in this study were obtained from commercial sources and were of analytical grade. The resulting plasmid pTEFM1-His-HGT1-HA was used as a template to introduce two silent mutations to generate the restriction sites-MscI (1366) and XbaI (1584) in the wild-type HGT1 gene using the QuikChange site-directed mutagenesis kit with respective mutagenic oligonucleotides pairs given in supplemental Table 1 This modification generated a recombinant HGT1 gene that was unaltered in the encoded protein product but had additional restriction sites to facilitate cloning and screening of mutant DNA (Fig. 1). For the prediction of transmembrane strain transformed with different plasmid constructs bearing domains in Hgt1p, five different transmembrane prediction wild type or HGT1 mutants under the TEF promoter were software programs were used: HMMTOP [28], TMHMM [29], grown in minimal media containing methionine and other sup- MEMSAT [30], PHDHTM [31], and TopPRED II [32]. The resulting signal intensity was normalized with ically disfavors a helix formation and, were predicted to TABLE 1 Polar/charged residues in the transmembrane domains of Hgt1p and residues selected for alanine scanning in this study
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