Gln-222 in transmembrane domain 4 and Gln-526 in transmembrane domain 9 are critical for substrate recognition in the yeast high affinity glutathione transporter, Hgt1p.
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.
Kaur
Jaspreet
J
Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India.
Bachhawat
Anand K
AK
D000595:Amino Acid Sequence; D005978:Glutathione / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
D009004:Monosaccharide Transport Proteins; C491110:OPT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D005978:Glutathione