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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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Subcellular localization of Gex1 and Gex2. GEX1-GFP and GEX2-GFP strains were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (A) or very early in the exponential growth phase (EEP) (OD600 = 0.1) or in late exponential growth phase (LEP) (OD600 = 3) (B). GFP fluorescence was analyzed with the FITC filter set, and yeast morphology was studied with Nomarski optics. (C) GEX1-GFP or GEX1-HA/end3Δ cells were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (as described in A). Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analyzed by Western immunoblotting for the presence of GFP, HA, plasma membrane ATPase 1 (Pma1), and transmembrane subunit of the vacuolar ATPase (Vph1). (D) Wild-type cells transformed with pGEX1-HA or pGEX1-GFP were grown overnight in YNB with 2% galactose. Protein extracts from the strain producing Gex1-HA were fractionated on a sucrose density gradient as described in C. Cells producing Gex1-GFP were treated with CMAC to stain the vacuolar lumen, and images of GFP (FITC filter set), CMAC (DAPI filter set), and cell shape (Nomarski optics) were taken.
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Figure 2: Subcellular localization of Gex1 and Gex2. GEX1-GFP and GEX2-GFP strains were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (A) or very early in the exponential growth phase (EEP) (OD600 = 0.1) or in late exponential growth phase (LEP) (OD600 = 3) (B). GFP fluorescence was analyzed with the FITC filter set, and yeast morphology was studied with Nomarski optics. (C) GEX1-GFP or GEX1-HA/end3Δ cells were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (as described in A). Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analyzed by Western immunoblotting for the presence of GFP, HA, plasma membrane ATPase 1 (Pma1), and transmembrane subunit of the vacuolar ATPase (Vph1). (D) Wild-type cells transformed with pGEX1-HA or pGEX1-GFP were grown overnight in YNB with 2% galactose. Protein extracts from the strain producing Gex1-HA were fractionated on a sucrose density gradient as described in C. Cells producing Gex1-GFP were treated with CMAC to stain the vacuolar lumen, and images of GFP (FITC filter set), CMAC (DAPI filter set), and cell shape (Nomarski optics) were taken.

Mentions: We analyzed the distribution of Gex1 and Gex2 in strains with the corresponding genes tagged at the chromosomal locus (at the carboxy terminus of the encoded protein) with GFP or HA. The cells were grown to midexponential growth phase in the presence of BPS, and GFP fluorescence was analyzed by fluorescence microscopy (Figure 2, A and B). In the GEX1-GFP strain, fluorescence was detected at both the plasma and vacuolar membranes (Figure 2A). The distribution of the protein depended on the growth phase (Figure 2B). Very early in the exponential growth phase (optical density at 600 nm [OD600] = 0.1) Gex1-GFP was present mostly at the plasma membrane, whereas in late exponential growth phase (OD600 = 3) this protein was found mostly at the vacuolar membrane. As shown by SDS–PAGE (Figure 1A), GEX2-GFP expression levels were much lower than those for GEX1-GFP, and the corresponding protein was barely detected. Like Gex1-GFP, Gex2-GFP was present at both the plasma and vacuolar membranes. Given the technical difficulty of detecting Gex2 production and the 98% identity of the two genes, we hypothesized that the two proteins were probably involved in the same process and decided to concentrate our studies on Gex1, except in genetic studies, in which we used a strain with a double deletion of GEX1 and GEX2.


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)

Subcellular localization of Gex1 and Gex2. GEX1-GFP and GEX2-GFP strains were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (A) or very early in the exponential growth phase (EEP) (OD600 = 0.1) or in late exponential growth phase (LEP) (OD600 = 3) (B). GFP fluorescence was analyzed with the FITC filter set, and yeast morphology was studied with Nomarski optics. (C) GEX1-GFP or GEX1-HA/end3Δ cells were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (as described in A). Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analyzed by Western immunoblotting for the presence of GFP, HA, plasma membrane ATPase 1 (Pma1), and transmembrane subunit of the vacuolar ATPase (Vph1). (D) Wild-type cells transformed with pGEX1-HA or pGEX1-GFP were grown overnight in YNB with 2% galactose. Protein extracts from the strain producing Gex1-HA were fractionated on a sucrose density gradient as described in C. Cells producing Gex1-GFP were treated with CMAC to stain the vacuolar lumen, and images of GFP (FITC filter set), CMAC (DAPI filter set), and cell shape (Nomarski optics) were taken.
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Figure 2: Subcellular localization of Gex1 and Gex2. GEX1-GFP and GEX2-GFP strains were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (A) or very early in the exponential growth phase (EEP) (OD600 = 0.1) or in late exponential growth phase (LEP) (OD600 = 3) (B). GFP fluorescence was analyzed with the FITC filter set, and yeast morphology was studied with Nomarski optics. (C) GEX1-GFP or GEX1-HA/end3Δ cells were grown overnight in YPD supplemented with 200 μM BPS and collected at midexponential growth phase (as described in A). Cells were lysed and protein extracts were fractionated on a 20–60% sucrose density gradient. Aliquots of the various fractions were analyzed by Western immunoblotting for the presence of GFP, HA, plasma membrane ATPase 1 (Pma1), and transmembrane subunit of the vacuolar ATPase (Vph1). (D) Wild-type cells transformed with pGEX1-HA or pGEX1-GFP were grown overnight in YNB with 2% galactose. Protein extracts from the strain producing Gex1-HA were fractionated on a sucrose density gradient as described in C. Cells producing Gex1-GFP were treated with CMAC to stain the vacuolar lumen, and images of GFP (FITC filter set), CMAC (DAPI filter set), and cell shape (Nomarski optics) were taken.
Mentions: We analyzed the distribution of Gex1 and Gex2 in strains with the corresponding genes tagged at the chromosomal locus (at the carboxy terminus of the encoded protein) with GFP or HA. The cells were grown to midexponential growth phase in the presence of BPS, and GFP fluorescence was analyzed by fluorescence microscopy (Figure 2, A and B). In the GEX1-GFP strain, fluorescence was detected at both the plasma and vacuolar membranes (Figure 2A). The distribution of the protein depended on the growth phase (Figure 2B). Very early in the exponential growth phase (optical density at 600 nm [OD600] = 0.1) Gex1-GFP was present mostly at the plasma membrane, whereas in late exponential growth phase (OD600 = 3) this protein was found mostly at the vacuolar membrane. As shown by SDS–PAGE (Figure 1A), GEX2-GFP expression levels were much lower than those for GEX1-GFP, and the corresponding protein was barely detected. Like Gex1-GFP, Gex2-GFP was present at both the plasma and vacuolar membranes. Given the technical difficulty of detecting Gex2 production and the 98% identity of the two genes, we hypothesized that the two proteins were probably involved in the same process and decided to concentrate our studies on Gex1, except in genetic studies, in which we used a strain with a double deletion of GEX1 and GEX2.

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