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
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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.
    FEMS Yeast Research 12/2011; 11(8):631-42. DOI:10.1111/j.1567-1364.2011.00753.x · 2.82 Impact Factor
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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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    ABSTRACT: Redox pathways play a key role in pathogenesis. Glutathione, a central molecule in redox homeostasis in yeasts, is an essential metabolite, but its requirements can be met either from endogenous biosynthesis or from the extracellular milieu. In this report we have examined the importance of glutathione biosynthesis in two major human opportunistic fungal pathogens, Candida albicans and Candida glabrata. As the genome sequence of C. glabrata had suggested the absence of glutathione transporters, we initially investigated exogenous glutathione utilization in C. glabrata by disruption of the MET15 gene, involved in methionine biosynthesis. We observed an organic sulphur auxotrophy in a C. glabrata met15D strain; however, unlike its Saccharomyces cerevisiae counterpart, the C. glabrata met15D strain was unable to grow on exogenous glutathione. This inability to grow on exogenous glutathione was demonstrated to be due to the lack of a functional glutathione transporter, despite the presence of a functional glutathione degradation machinery (the Dug pathway). In the absence of the ability to obtain glutathione from the extracellular medium, we examined and could demonstrate that c-glutamyl cysteine synthase, the first enzyme of glutathione biosynthesis, was essential in C. glabrata. Further, although c-glutamyl cysteine synthase has been reported to be non-essential in C. albicans, we report here for what is believed to be the first time that the enzyme is required for survival in human macrophages in vitro, as well as for virulence in a murine model of disseminated candidiasis. The essentiality of c-glutamyl cysteine synthase in C. glabrata, and its essentiality for virulence in C. albicans, make the enzyme a strong candidate for antifungal development.
    Microbiology 02/2011; 157(2). DOI:10.1099/mic.0.045054-0 · 2.56 Impact Factor
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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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    ABSTRACT: The oligopeptide transporter (OPT) family of peptide and iron-siderophore transporters includes members from both prokaryotes and eukaryotes but with restricted distribution in the latter domain. Eukaryotic members were found only in fungi and plants with a single slime mold homologue clustering with the fungal proteins. All functionally characterized eukaryotic peptide transporters segregate from the known iron-siderophore transporters on a phylogenetic tree. Prokaryotic members are widespread, deriving from many different phyla. Although they belong only to the iron-siderophore subdivision, genome context analyses suggest that many of them are peptide transporters. OPT family proteins have 16 or occasionally 17 transmembrane-spanning α-helical segments (TMSs). We provide statistical evidence that the 16-TMS topology arose via three sequential duplication events followed by a gene-fusion event for proteins with a seventeenth TMS. The proposed pathway is as follows: 2 TMSs → 4 TMSs → 8 TMSs → 16 TMSs → 17 TMSs. The seventeenth C-terminal TMS, which probably arose just once, is found in just one phylogenetic group of these homologues. Analyses for orthology revealed that a few phylogenetic clusters consist exclusively of orthologues but most have undergone intermixing, suggestive of horizontal transfer. It appears that in this family horizontal gene transfer was frequent among prokaryotes, rare among eukaryotes and largely absent between prokaryotes and eukaryotes as well as between plants and fungi. These observations provide guides for future structural and functional analyses of OPT family members. Electronic supplementary material The online version of this article (doi:10.1007/s00232-011-9347-9) contains supplementary material, which is available to authorized users.
    Journal of Membrane Biology 02/2011; 240(2):89-110. DOI:10.1007/s00232-011-9347-9 · 2.46 Impact Factor
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