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 (GSH) is considered the most important redox buffer of the cell. To better characterize its essential function during oxidative stress conditions, we studied the physiological response of H2O2-treated yeast cells containing different amounts of GSH. We showed that the transcriptional response of GSH-depleted cells is severely impaired, despite an efficient nuclear accumulation of the transcription factor Yap1. Moreover, oxidative stress generates high genome instability in GSH-depleted cells, but does not activate the checkpoint kinase Rad53. Surprisingly, scarce amounts of intracellular GSH are sufficient to preserve cell viability under H2O2 treatment. In these cells, oxidative stress still causes the accumulation of oxidized proteins and the inactivation of the translational activity, but nuclear components and activities are protected against oxidative injury. We conclude that the essential role of GSH is to preserve nuclear function, allowing cell survival and growth resumption after oxidative stress release. We propose that cytosolic proteins are part of a protective machinery that shields the nucleus by scavenging reactive oxygen species before they can cross the nuclear membrane.Free Radical Biology & Medicine 10/2013; · 5.27 Impact Factor
Article: Glutathione transporters.[Show abstract] [Hide abstract]
ABSTRACT: BACKGROUND: Glutathione (GSH) is synthesized in the cytoplasm but there is a requirement for glutathione not only in the cytoplasm, but in the other organelles and the extracellular mileu. GSH is also imported into the cytoplasm. The transports of glutathione across these different membranes in different systems have been biochemically demonstrated. However the molecular identity of the transporters has been established only in a few cases. SCOPE OF REVIEW: An attempt has been made to present the current state of knowledge of glutathione transporters from different organisms as well as different organelles. These include the most well characterized transporters, the yeast high-affinity, high-specificity glutathione transporters involved in import into the cytoplasm, and the mammalian MRP proteins involved in low affinity glutathione efflux from the cytoplasm. Other glutathione transporters that have been described either with direct or indirect evidences are also discussed. MAJOR CONCLUSIONS: The molecular identity of a few glutathione transporters has been unambiguously established but there is a need to identify the transporters of other systems and organelles. There is a lack of direct evidence establishing transport by suggested transporters in many cases. Studies with the high affinity transporters have led to important structure-function insights. GENERAL SIGNIFICANCE: An understanding of glutathione transporters is critical to our understanding of redox homeostasis in living cells. By presenting our current state of understanding and the gaps in our knowledge the review hopes to stimulate research in these fields. This article is part of a Special Issue entitled Cellular functions of glutathione.Biochimica et Biophysica Acta 11/2012; · 4.66 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