Differential roles played by the native cysteine residues of the yeast glutathione transporter, Hgt1p.
Hgt1p, a high-affinity glutathione transporter from the yeast Saccharomyces cerevisiae, belongs to the structurally uncharacterized oligopeptide transporter (OPT) family. To initiate structural studies on Hgt1p, a cysteine-free (cys-free) Hgt1p was generated. This cys-free Hgt1p was nonfunctional and pointed to a critical role being played by the native cysteine residues of Hgt1p. To investigate their role, genetic and biochemical approaches were undertaken. Functional suppressors of the cys-free Hgt1p were isolated, and yielded double revertants bearing C622 and C632. Subsequent biochemical characterization of the individual C622S/A or C632S/A mutations revealed that both these cysteine residues were, in fact, individually indispensable for Hgt1p function and were required for trafficking to the plasma membrane. However, despite their essentiality, the presence of only these two native cysteines in Hgt1p generated a very weak glutathione transporter with minimal functional activity. Hence, the remaining 10 cysteines were also contributing towards Hgt1p activity, although they were not found to be singly responsible or crucial for Hgt1p functional activity. These residues, however, contributed cumulatively towards the stability and the functionality of Hgt1p, without affecting the trafficking to the cell surface. The study reveals differential roles for the cysteines of Hgt1p and provides first insights into the structural features of an OPT family member.
Kaur
Jaspreet
J
Institute of Microbial Technology, Chandigarh, India.
Srikanth
Chittur V
CV
Bachhawat
Anand K
AK
D019943:Amino Acid Substitution / Q000235:genetics; D003545:Cysteine / Q000235:genetics*; D004252:DNA Mutational Analysis; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008954:Models, Biological; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013489:Suppression, Genetic
D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D050505:Mutant Proteins; C491110:OPT1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C086860:glutathione transporter; D003545:Cysteine