Differential roles played by the native cysteine residues of the yeast glutathione transporter, Hgt1p

Institute of Microbial Technology, Chandigarh, India.
FEMS Yeast Research (Impact Factor: 2.82). 06/2009; 9(6):849-66. DOI: 10.1111/j.1567-1364.2009.00529.x
Source: PubMed


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.

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    • "Transport of glutathione through the plasma membrane of yeast cells can be facilitated through transporters of the OPT family. The high-affinity transporters for glutathione, Hgt1p and Pgt1, have been localized in the plasma membrane of S. cerevisiae and Schizosaccharomyces pombe, respectively, (Bourbouloux et al., 2000; Thakur et al., 2007; Kaur et al., 2009) and are responsible for the uptake of glutathione from the growth media. "
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    ABSTRACT: Glutathione is an important antioxidant in most prokaryotes and eukaryotes. It detoxifies reactive oxygen species and is also involved in the modulation of gene expression, in redox signaling, and in the regulation of enzymatic activities. In this study, the subcellular distribution of glutathione was studied in Saccharomyces cerevisiae by quantitative immunoelectron microscopy. Highest glutathione contents were detected in mitochondria and subsequently in the cytosol, nuclei, cell walls, and vacuoles. The induction of oxidative stress by hydrogen peroxide (H(2) O(2) ) led to changes in glutathione-specific labeling. Three cell types were identified. Cell types I and II contained more glutathione than control cells. Cell type II differed from cell type I in showing a decrease in glutathione-specific labeling solely in mitochondria. Cell type III contained much less glutathione contents than the control and showed the strongest decrease in mitochondria, suggesting that high and stable levels of glutathione in mitochondria are important for the protection and survival of the cells during oxidative stress. Additionally, large amounts of glutathione were relocated and stored in vacuoles in cell type III, suggesting the importance of the sequestration of glutathione in vacuoles under oxidative stress.
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    • "Laboratory of R. Kaur pBSK E. coli expression vector (Stratagene) p416TEF-ScGSH1 S. cerevisiae GSH1 gene cloned in BspDI and HindIII sites of p416TEF Sharma et al. (2000) p416TEF-ScHGT1 S. cerevisiae HGT1 gene cloned in BamHI and EcoRI sites of p416TEF Kaur et al. (2009) "
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    • "Bacterial and archaeal homologues of the OPT family have yet to be characterized biochemically, but as shown here, they are prevalent throughout the prokaryotic world (Kaur et al. 2009). A high-resolution three-dimensional X-ray structure of an OPT family homologue has yet to be solved. "
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