Differential roles played by the native cysteine residues of the yeast glutathione transporter, Hgt1p.
ABSTRACT 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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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.FEMS Yeast Research 12/2011; 11(8):631-42. · 2.46 Impact Factor
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ABSTRACT: Candida albicans lacks the ability to survive within its mammalian host in the absence of endogenous glutathione biosynthesis. To examine the ability of this yeast to utilize exogenous glutathione, we exploited the organic sulfur auxotrophy of C. albicans met15Δ strains. We observed that glutathione is utilized efficiently by the alternative pathway of glutathione degradation (DUG pathway). The major oligopeptide transporters OPT1-OPT5 of C. albicans that were most similar to the known yeast glutathione transporters were not found to contribute to glutathione transport to any significant extent. A genomic library approach to identify the glutathione transporter of C. albicans yielded OPT7 as the primary glutathione transporter. Biochemical studies on OPT7 using radiolabeled GSH uptake revealed a K(m) of 205 μm, indicating that it was a high affinity glutathione transporter. OPT7 is unusual in several aspects. It is the most remote member to known yeast glutathione transporters, lacks the two highly conserved cysteines in the family that are known to be crucial in trafficking, and also has the ability to take up tripeptides. The transporter was regulated by sulfur sources in the medium. OPT7 orthologues were prevalent among many pathogenic yeasts and fungi and formed a distinct cluster quite remote from the Saccharomyces cerevisiae HGT1 glutathione transporter cluster. In vivo experiments using a systemic model of candidiasis failed to detect expression of OPT7 in vivo, and strains disrupted either in the degradation (dug3Δ) or transport (opt7Δ) of glutathione failed to show a defect in virulence.Journal of Biological Chemistry 12/2011; 286(48):41183-94. · 4.65 Impact Factor
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ABSTRACT: Glutathione is a thiol-containing tripeptide that plays important roles in redox-related processes. The first step in glutathione biosynthesis is catalyzed by γ-glutamylcysteine synthetase (γ-GCS). The crystal structure of E. coli γ-GCS has revealed the presence of a disulphide bond. As the disulphide-bonding cysteines, Cys372 and Cys395 are not well conserved among γ-GCS enzymes in this lineage, we have initiated a biochemical genetic strategy to investigate the functional importance of these and the other cysteines. In a cysteine-free γ-GCS that was non- functional, suppressor analysis yielded combinations of cysteine and aromatic residues at the position of the disulphide bond, and one mutant that lacked any cysteines. Kinetic analysis of the wild type and mutant enzymes revealed that the disulphide bond was not involved in determining the affinity of the enzyme towards its substrate, but had an important role in determining the stability of the protein, and its catalytic efficiency. We show that in vivo the γ-GCS enzyme can also exist in a reduced form and that the mutants lacking the disulphide bond show a decrease in half-life. These results demonstrate a novel means of regulation of γ-GCS by the redox environment that works by an alteration in its stability.Biochemical Journal 11/2012; · 4.65 Impact Factor