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Gex1 is a yeast glutathione exchanger that interferes with pH and redox homeostasis.

Dhaoui M, Auchère F, Blaiseau PL, Lesuisse E, Landoulsi A, Camadro JM, Haguenauer-Tsapis R, Belgareh-Touzé N - Mol. Biol. Cell (2011)

Bottom Line: Gex1 was found mostly at the vacuolar membrane and, to a lesser extent, at the plasma membrane.The deletion mutant accumulated intracellular glutathione, and cells overproducing Gex1 had low intracellular glutathione contents, with glutathione excreted into the extracellular medium.Finally, the imbalance of pH and glutathione homeostasis in the gex1Δ gex2Δ and Gex1-overproducing strains led to modulations of the cAMP/protein kinase A and protein kinase C1 mitogen-activated protein kinase signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Ubiquitine et Trafic Intracellulaire, Institut Jacques Monod, UMR 7592 CNRS-Université Paris-Diderot, France.

ABSTRACT
In the yeast Saccharomyces cerevisiae, glutathione plays a major role in heavy metal detoxification and protection of cells against oxidative stress. We show that Gex1 is a new glutathione exchanger. Gex1 and its paralogue Gex2 belong to the major facilitator superfamily of transporters and display similarities to the Aft1-regulon family of siderophore transporters. Gex1 was found mostly at the vacuolar membrane and, to a lesser extent, at the plasma membrane. Gex1 expression was induced under conditions of iron depletion and was principally dependent on the iron-responsive transcription factor Aft2. However, a gex1Δ gex2Δ strain displayed no defect in known siderophore uptake. The deletion mutant accumulated intracellular glutathione, and cells overproducing Gex1 had low intracellular glutathione contents, with glutathione excreted into the extracellular medium. Furthermore, the strain overproducing Gex1 induced acidification of the cytosol, confirming the involvement of Gex1 in proton transport as a probable glutathione/proton antiporter. Finally, the imbalance of pH and glutathione homeostasis in the gex1Δ gex2Δ and Gex1-overproducing strains led to modulations of the cAMP/protein kinase A and protein kinase C1 mitogen-activated protein kinase signaling pathways.

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Gex1 and Gex2 are involved in the oxidative stress response. (A) WT and gex1Δ gex2Δ cells were spotted onto solid glucose medium (glu) containing the indicated concentrations of H2O2. This experiment was repeated with wild-type cells (WT) bearing the pØ or pGEX1-HA plasmids, except that the cells were spotted onto galactose (gal)-containing plates. (B) GEX1-GFP cells were cultured overnight in YPD containing H2O2 (0.1 mM). GEX1-GFP cells transformed with pYAP1 and pYAP2 were grown overnight in YNB supplemented with glucose as a carbon source. GFP fluorescence was visualized with the FITC filter set, and cell shape was studied with Nomarski optics. (C) Total protein extracts from the same cells as in B and GEX1-GFP cells grown overnight in YPD supplemented with 200 μM BPS were prepared and analyzed by Western immunoblotting with antibodies directed against GFP and PGK as a loading control.
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Figure 5: Gex1 and Gex2 are involved in the oxidative stress response. (A) WT and gex1Δ gex2Δ cells were spotted onto solid glucose medium (glu) containing the indicated concentrations of H2O2. This experiment was repeated with wild-type cells (WT) bearing the pØ or pGEX1-HA plasmids, except that the cells were spotted onto galactose (gal)-containing plates. (B) GEX1-GFP cells were cultured overnight in YPD containing H2O2 (0.1 mM). GEX1-GFP cells transformed with pYAP1 and pYAP2 were grown overnight in YNB supplemented with glucose as a carbon source. GFP fluorescence was visualized with the FITC filter set, and cell shape was studied with Nomarski optics. (C) Total protein extracts from the same cells as in B and GEX1-GFP cells grown overnight in YPD supplemented with 200 μM BPS were prepared and analyzed by Western immunoblotting with antibodies directed against GFP and PGK as a loading control.

Mentions: Low glutathione ratio (GSH:GSSG) may indicate that cells are subject to oxidative stress. We assessed the sensitivity of the various strains to oxidative stress generated by treatment with H2O2 (Figure 5A). We found that gex1Δ gex2Δ cells were hypersensitive to H2O2 and cells overproducing Gex1-HA were resistant to H2O2. This may indicate a role for Gex1 in protecting cells against exposure to H2O2. We analyzed Gex1-GFP levels after exposure to H2O2 and found that 40% of cells displayed faint GFP fluorescence after treatment with 0.1 mM H2O2 (Figure 5, B and C). Western immunoblotting analysis of Gex1-GFP levels after H2O2 treatment showed that H2O2 induced Gex1-GFP synthesis less efficiently than BPS treatment (levels after H2O2 treatment were only one-seventh those after BPS exposure). Finally, Gex1-GFP was not induced following overproduction of the transcription factors required for oxidative stress tolerance, Yap1 and Yap2 (Figure 5, B and C). Thus GEX1 was involved in protecting cells against the oxidative stress induced by H2O2 treatment, and GEX1 expression was not dependent on the stress-responsive factors Yap1 and Yap2 but may have been dependent on the iron-responsive transcription factor Aft2.


Gex1 is a yeast glutathione exchanger that interferes with pH and redox homeostasis.

Dhaoui M, Auchère F, Blaiseau PL, Lesuisse E, Landoulsi A, Camadro JM, Haguenauer-Tsapis R, Belgareh-Touzé N - Mol. Biol. Cell (2011)

Gex1 and Gex2 are involved in the oxidative stress response. (A) WT and gex1Δ gex2Δ cells were spotted onto solid glucose medium (glu) containing the indicated concentrations of H2O2. This experiment was repeated with wild-type cells (WT) bearing the pØ or pGEX1-HA plasmids, except that the cells were spotted onto galactose (gal)-containing plates. (B) GEX1-GFP cells were cultured overnight in YPD containing H2O2 (0.1 mM). GEX1-GFP cells transformed with pYAP1 and pYAP2 were grown overnight in YNB supplemented with glucose as a carbon source. GFP fluorescence was visualized with the FITC filter set, and cell shape was studied with Nomarski optics. (C) Total protein extracts from the same cells as in B and GEX1-GFP cells grown overnight in YPD supplemented with 200 μM BPS were prepared and analyzed by Western immunoblotting with antibodies directed against GFP and PGK as a loading control.
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Related In: Results  -  Collection

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Figure 5: Gex1 and Gex2 are involved in the oxidative stress response. (A) WT and gex1Δ gex2Δ cells were spotted onto solid glucose medium (glu) containing the indicated concentrations of H2O2. This experiment was repeated with wild-type cells (WT) bearing the pØ or pGEX1-HA plasmids, except that the cells were spotted onto galactose (gal)-containing plates. (B) GEX1-GFP cells were cultured overnight in YPD containing H2O2 (0.1 mM). GEX1-GFP cells transformed with pYAP1 and pYAP2 were grown overnight in YNB supplemented with glucose as a carbon source. GFP fluorescence was visualized with the FITC filter set, and cell shape was studied with Nomarski optics. (C) Total protein extracts from the same cells as in B and GEX1-GFP cells grown overnight in YPD supplemented with 200 μM BPS were prepared and analyzed by Western immunoblotting with antibodies directed against GFP and PGK as a loading control.
Mentions: Low glutathione ratio (GSH:GSSG) may indicate that cells are subject to oxidative stress. We assessed the sensitivity of the various strains to oxidative stress generated by treatment with H2O2 (Figure 5A). We found that gex1Δ gex2Δ cells were hypersensitive to H2O2 and cells overproducing Gex1-HA were resistant to H2O2. This may indicate a role for Gex1 in protecting cells against exposure to H2O2. We analyzed Gex1-GFP levels after exposure to H2O2 and found that 40% of cells displayed faint GFP fluorescence after treatment with 0.1 mM H2O2 (Figure 5, B and C). Western immunoblotting analysis of Gex1-GFP levels after H2O2 treatment showed that H2O2 induced Gex1-GFP synthesis less efficiently than BPS treatment (levels after H2O2 treatment were only one-seventh those after BPS exposure). Finally, Gex1-GFP was not induced following overproduction of the transcription factors required for oxidative stress tolerance, Yap1 and Yap2 (Figure 5, B and C). Thus GEX1 was involved in protecting cells against the oxidative stress induced by H2O2 treatment, and GEX1 expression was not dependent on the stress-responsive factors Yap1 and Yap2 but may have been dependent on the iron-responsive transcription factor Aft2.

Bottom Line: Gex1 was found mostly at the vacuolar membrane and, to a lesser extent, at the plasma membrane.The deletion mutant accumulated intracellular glutathione, and cells overproducing Gex1 had low intracellular glutathione contents, with glutathione excreted into the extracellular medium.Finally, the imbalance of pH and glutathione homeostasis in the gex1Δ gex2Δ and Gex1-overproducing strains led to modulations of the cAMP/protein kinase A and protein kinase C1 mitogen-activated protein kinase signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Ubiquitine et Trafic Intracellulaire, Institut Jacques Monod, UMR 7592 CNRS-Université Paris-Diderot, France.

ABSTRACT
In the yeast Saccharomyces cerevisiae, glutathione plays a major role in heavy metal detoxification and protection of cells against oxidative stress. We show that Gex1 is a new glutathione exchanger. Gex1 and its paralogue Gex2 belong to the major facilitator superfamily of transporters and display similarities to the Aft1-regulon family of siderophore transporters. Gex1 was found mostly at the vacuolar membrane and, to a lesser extent, at the plasma membrane. Gex1 expression was induced under conditions of iron depletion and was principally dependent on the iron-responsive transcription factor Aft2. However, a gex1Δ gex2Δ strain displayed no defect in known siderophore uptake. The deletion mutant accumulated intracellular glutathione, and cells overproducing Gex1 had low intracellular glutathione contents, with glutathione excreted into the extracellular medium. Furthermore, the strain overproducing Gex1 induced acidification of the cytosol, confirming the involvement of Gex1 in proton transport as a probable glutathione/proton antiporter. Finally, the imbalance of pH and glutathione homeostasis in the gex1Δ gex2Δ and Gex1-overproducing strains led to modulations of the cAMP/protein kinase A and protein kinase C1 mitogen-activated protein kinase signaling pathways.

Show MeSH
Related in: MedlinePlus