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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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Influence of GEX1 and GEX2 on the PKA and MAPK pathways. (A) WT cells transformed with pADH1-MSN2-GFP alone or in combination with the pØ or pGEX1-HA plasmids, and gex1Δ gex2Δ cells transformed with pADH1-MSN2-GFP, were grown overnight to midexponential growth phase. Slides were exposed only once to ensure that Msn2-GFP was not targeted to the nucleus due to light exposure. (B) WT and gex1Δ gex2Δ cells and WT cells transformed with the pØ or pGEX1-HA plasmid were sequentially diluted fivefold and spotted onto glucose-containing medium for WT and gex1Δ gex2Δ and onto galactose-containing medium for the other strains. Plates were incubated for 3 d, and glycogen accumulation was assayed by gently pouring an iodine solution over the spots. (C) WT, gex1Δ gex2Δ, and cells transformed with the pØ (1) and pGEX1-HA (2) plasmids were grown overnight in glucose-containing or galactose-containing (for strains bearing plasmids) medium, and extracts of the cells were prepared and subjected to Western immunoblotting. Gex1-HA was detected with a monoclonal anti-HA antibody, Mpk1-GFP was detected with a monoclonal anti-GFP antibody, and the phosphorylated forms of Mpk1 were detected with a polyclonal anti–phospho-p44/42 MAPK antibody. PGK was detected with a polyclonal antibody and was used as a loading control. (D) The cells described in B were subjected to fivefold dilution and spotted onto glucose- or galactose-containing medium with the indicated concentrations of cell wall–stressing agents Congo red (CR) and calcofluor white (CFW).
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Figure 7: Influence of GEX1 and GEX2 on the PKA and MAPK pathways. (A) WT cells transformed with pADH1-MSN2-GFP alone or in combination with the pØ or pGEX1-HA plasmids, and gex1Δ gex2Δ cells transformed with pADH1-MSN2-GFP, were grown overnight to midexponential growth phase. Slides were exposed only once to ensure that Msn2-GFP was not targeted to the nucleus due to light exposure. (B) WT and gex1Δ gex2Δ cells and WT cells transformed with the pØ or pGEX1-HA plasmid were sequentially diluted fivefold and spotted onto glucose-containing medium for WT and gex1Δ gex2Δ and onto galactose-containing medium for the other strains. Plates were incubated for 3 d, and glycogen accumulation was assayed by gently pouring an iodine solution over the spots. (C) WT, gex1Δ gex2Δ, and cells transformed with the pØ (1) and pGEX1-HA (2) plasmids were grown overnight in glucose-containing or galactose-containing (for strains bearing plasmids) medium, and extracts of the cells were prepared and subjected to Western immunoblotting. Gex1-HA was detected with a monoclonal anti-HA antibody, Mpk1-GFP was detected with a monoclonal anti-GFP antibody, and the phosphorylated forms of Mpk1 were detected with a polyclonal anti–phospho-p44/42 MAPK antibody. PGK was detected with a polyclonal antibody and was used as a loading control. (D) The cells described in B were subjected to fivefold dilution and spotted onto glucose- or galactose-containing medium with the indicated concentrations of cell wall–stressing agents Congo red (CR) and calcofluor white (CFW).

Mentions: Dechant et al. (2010) recently demonstrated that cytosolic pH regulates the PKA glucose signaling pathway and a genome-wide screen for mutants overexcreting glutathione identified mutants of the PKA pathway as having the highest levels of glutathione excretion (22–25 times higher than that of wild-type cells; Perrone et al., 2005). We showed that Gex1-HA overproduction induced acidification of the cytosol and a decrease in glutathione content. We therefore investigated the possible role of Gex1 and Gex2 in the PKA pathway. For this purpose, we transformed the gex1Δ gex2Δ deletion mutant and the strain overproducing Gex1-HA with a plasmid encoding the stress-responsive transcription factor Msn2 tagged with GFP (Görner et al., 1998). Msn2 is targeted to the nucleus in response to various stresses, including oxidative stress and acidification of the cytosol, through the action of several signaling cascades, including the PKA pathway. The disruption of GEX1 and GEX2 induced a defect in the targeting of Msn2-GFP to the nucleus, and Gex1-HA overproduction induced a relocalization of Msn2-GFP to the nucleus, consistent with the induction by Gex1-HA of cytosol acidification (Figure 7A). Msn2 and Msn4 have been shown to increase the expression of genes involved in glycogen synthesis (Smith et al., 1998). Thus defects in Msn2 targeting to the nucleus lead to a decrease in glycogen accumulation. We assessed glycogen accumulation in the cells, using an iodine solution (Figure 7B). The disruption of GEX1 and GEX2 resulted in lower glycogen content than for the wild-type strain, confirming a defect in the targeting of Msn2 to the nucleus. The wild-type strain transformed with an empty plasmid behaved like the wild-type strain transformed with pGEX1-HA because glycogen is hardly synthesized when cells are grown on galactose as the sole carbon source.


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)

Influence of GEX1 and GEX2 on the PKA and MAPK pathways. (A) WT cells transformed with pADH1-MSN2-GFP alone or in combination with the pØ or pGEX1-HA plasmids, and gex1Δ gex2Δ cells transformed with pADH1-MSN2-GFP, were grown overnight to midexponential growth phase. Slides were exposed only once to ensure that Msn2-GFP was not targeted to the nucleus due to light exposure. (B) WT and gex1Δ gex2Δ cells and WT cells transformed with the pØ or pGEX1-HA plasmid were sequentially diluted fivefold and spotted onto glucose-containing medium for WT and gex1Δ gex2Δ and onto galactose-containing medium for the other strains. Plates were incubated for 3 d, and glycogen accumulation was assayed by gently pouring an iodine solution over the spots. (C) WT, gex1Δ gex2Δ, and cells transformed with the pØ (1) and pGEX1-HA (2) plasmids were grown overnight in glucose-containing or galactose-containing (for strains bearing plasmids) medium, and extracts of the cells were prepared and subjected to Western immunoblotting. Gex1-HA was detected with a monoclonal anti-HA antibody, Mpk1-GFP was detected with a monoclonal anti-GFP antibody, and the phosphorylated forms of Mpk1 were detected with a polyclonal anti–phospho-p44/42 MAPK antibody. PGK was detected with a polyclonal antibody and was used as a loading control. (D) The cells described in B were subjected to fivefold dilution and spotted onto glucose- or galactose-containing medium with the indicated concentrations of cell wall–stressing agents Congo red (CR) and calcofluor white (CFW).
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Related In: Results  -  Collection

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Figure 7: Influence of GEX1 and GEX2 on the PKA and MAPK pathways. (A) WT cells transformed with pADH1-MSN2-GFP alone or in combination with the pØ or pGEX1-HA plasmids, and gex1Δ gex2Δ cells transformed with pADH1-MSN2-GFP, were grown overnight to midexponential growth phase. Slides were exposed only once to ensure that Msn2-GFP was not targeted to the nucleus due to light exposure. (B) WT and gex1Δ gex2Δ cells and WT cells transformed with the pØ or pGEX1-HA plasmid were sequentially diluted fivefold and spotted onto glucose-containing medium for WT and gex1Δ gex2Δ and onto galactose-containing medium for the other strains. Plates were incubated for 3 d, and glycogen accumulation was assayed by gently pouring an iodine solution over the spots. (C) WT, gex1Δ gex2Δ, and cells transformed with the pØ (1) and pGEX1-HA (2) plasmids were grown overnight in glucose-containing or galactose-containing (for strains bearing plasmids) medium, and extracts of the cells were prepared and subjected to Western immunoblotting. Gex1-HA was detected with a monoclonal anti-HA antibody, Mpk1-GFP was detected with a monoclonal anti-GFP antibody, and the phosphorylated forms of Mpk1 were detected with a polyclonal anti–phospho-p44/42 MAPK antibody. PGK was detected with a polyclonal antibody and was used as a loading control. (D) The cells described in B were subjected to fivefold dilution and spotted onto glucose- or galactose-containing medium with the indicated concentrations of cell wall–stressing agents Congo red (CR) and calcofluor white (CFW).
Mentions: Dechant et al. (2010) recently demonstrated that cytosolic pH regulates the PKA glucose signaling pathway and a genome-wide screen for mutants overexcreting glutathione identified mutants of the PKA pathway as having the highest levels of glutathione excretion (22–25 times higher than that of wild-type cells; Perrone et al., 2005). We showed that Gex1-HA overproduction induced acidification of the cytosol and a decrease in glutathione content. We therefore investigated the possible role of Gex1 and Gex2 in the PKA pathway. For this purpose, we transformed the gex1Δ gex2Δ deletion mutant and the strain overproducing Gex1-HA with a plasmid encoding the stress-responsive transcription factor Msn2 tagged with GFP (Görner et al., 1998). Msn2 is targeted to the nucleus in response to various stresses, including oxidative stress and acidification of the cytosol, through the action of several signaling cascades, including the PKA pathway. The disruption of GEX1 and GEX2 induced a defect in the targeting of Msn2-GFP to the nucleus, and Gex1-HA overproduction induced a relocalization of Msn2-GFP to the nucleus, consistent with the induction by Gex1-HA of cytosol acidification (Figure 7A). Msn2 and Msn4 have been shown to increase the expression of genes involved in glycogen synthesis (Smith et al., 1998). Thus defects in Msn2 targeting to the nucleus lead to a decrease in glycogen accumulation. We assessed glycogen accumulation in the cells, using an iodine solution (Figure 7B). The disruption of GEX1 and GEX2 resulted in lower glycogen content than for the wild-type strain, confirming a defect in the targeting of Msn2 to the nucleus. The wild-type strain transformed with an empty plasmid behaved like the wild-type strain transformed with pGEX1-HA because glycogen is hardly synthesized when cells are grown on galactose as the sole carbon source.

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