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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 cadmium uptake. (A) WT and gex1Δ gex2Δ cells were grown in YPD, subjected to serial fivefold dilution, and spotted onto solid glucose medium (glu) containing the indicated concentrations of cadmium (CdSO4), BPS, nickel (NiSO4), and copper (CuSO4). The same experiment was carried out with WT cells transformed with the pØ or pGEX1-HA plasmids, except that cells were spotted onto complete galactose (gal)-containing medium. (B) WT and gex1Δ gex2Δ cells were grown in the presence of 1 μM cadmium for 6 h. WT cells bearing the pØ or pGEX1-HA plasmids were grown as in A, except that the carbon source was galactose instead of glucose. Iron, copper, and cadmium contents were then quantified for all strains by the inductively coupled plasma mass spectrometry method as carried out at the Service Central d'Analyse du CNRS (Solaize, France). The diagrams presented illustrate the consequence of gex1gex2 deletions or Gex1 overproduction on cadmium content.
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Figure 3: Gex1 and Gex2 are involved in cadmium uptake. (A) WT and gex1Δ gex2Δ cells were grown in YPD, subjected to serial fivefold dilution, and spotted onto solid glucose medium (glu) containing the indicated concentrations of cadmium (CdSO4), BPS, nickel (NiSO4), and copper (CuSO4). The same experiment was carried out with WT cells transformed with the pØ or pGEX1-HA plasmids, except that cells were spotted onto complete galactose (gal)-containing medium. (B) WT and gex1Δ gex2Δ cells were grown in the presence of 1 μM cadmium for 6 h. WT cells bearing the pØ or pGEX1-HA plasmids were grown as in A, except that the carbon source was galactose instead of glucose. Iron, copper, and cadmium contents were then quantified for all strains by the inductively coupled plasma mass spectrometry method as carried out at the Service Central d'Analyse du CNRS (Solaize, France). The diagrams presented illustrate the consequence of gex1gex2 deletions or Gex1 overproduction on cadmium content.

Mentions: Gex1 and Gex2 belong to the MFS subfamily and may be involved in transporting metals other than iron. We therefore assessed the ability of the gex1Δ gex2Δ strain and of the strain overproducing Gex1 to grow on media containing high concentrations of divalent metals (Cd2+, Ni2+, Cu2+, Mn2+, Mg2+, Co2+, Zn2+) and in the presence of the iron chelator BPS (Figure 3A and unpublished data). The gex1Δ gex2Δ mutant grew poorly only in the presence of the toxic heavy metal cadmium (Cd2+). The strain overproducing Gex1-HA was more resistant to this metal than the wild-type strain transformed with an empty plasmid. We then quantified the cadmium, iron, and copper contents of cells grown in a Cd-supplemented medium (Figure 3B). No significant differences were found in the iron and copper contents of the various strains, whereas a clear difference between strains was observed for cadmium. The gex1Δ gex2Δ strain had a much higher cadmium content than wild-type cells, and the cells overproducing Gex1-HA had a lower intracellular cadmium content. Gex1-HA was targeted to the plasma membrane when overproduced from the pGEX1-HA plasmid (Figure 2D). A decrease in cadmium content may therefore be indicative of an increase in cadmium efflux at the plasma membrane mediated by Gex1-HA. These results are consistent with the growth phenotypes observed in the presence of cadmium, and they suggest that Gex1 may mediate cadmium export at the plasma membrane. The topology of membrane-bound proteins is acquired in the endoplasmic reticulum and remains constant, regardless of the final site to which the protein is targeted. Thus a protein with multiple membrane spans and amino and carboxy termini facing the cytosol will present the same topology at the plasma membrane and at the vacuolar membrane (schemes in Figure 3B). Gex1 at the vacuolar membrane may therefore mediate cadmium import into the vacuolar lumen and participate in cadmium detoxification.


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 cadmium uptake. (A) WT and gex1Δ gex2Δ cells were grown in YPD, subjected to serial fivefold dilution, and spotted onto solid glucose medium (glu) containing the indicated concentrations of cadmium (CdSO4), BPS, nickel (NiSO4), and copper (CuSO4). The same experiment was carried out with WT cells transformed with the pØ or pGEX1-HA plasmids, except that cells were spotted onto complete galactose (gal)-containing medium. (B) WT and gex1Δ gex2Δ cells were grown in the presence of 1 μM cadmium for 6 h. WT cells bearing the pØ or pGEX1-HA plasmids were grown as in A, except that the carbon source was galactose instead of glucose. Iron, copper, and cadmium contents were then quantified for all strains by the inductively coupled plasma mass spectrometry method as carried out at the Service Central d'Analyse du CNRS (Solaize, France). The diagrams presented illustrate the consequence of gex1gex2 deletions or Gex1 overproduction on cadmium content.
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Related In: Results  -  Collection

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Figure 3: Gex1 and Gex2 are involved in cadmium uptake. (A) WT and gex1Δ gex2Δ cells were grown in YPD, subjected to serial fivefold dilution, and spotted onto solid glucose medium (glu) containing the indicated concentrations of cadmium (CdSO4), BPS, nickel (NiSO4), and copper (CuSO4). The same experiment was carried out with WT cells transformed with the pØ or pGEX1-HA plasmids, except that cells were spotted onto complete galactose (gal)-containing medium. (B) WT and gex1Δ gex2Δ cells were grown in the presence of 1 μM cadmium for 6 h. WT cells bearing the pØ or pGEX1-HA plasmids were grown as in A, except that the carbon source was galactose instead of glucose. Iron, copper, and cadmium contents were then quantified for all strains by the inductively coupled plasma mass spectrometry method as carried out at the Service Central d'Analyse du CNRS (Solaize, France). The diagrams presented illustrate the consequence of gex1gex2 deletions or Gex1 overproduction on cadmium content.
Mentions: Gex1 and Gex2 belong to the MFS subfamily and may be involved in transporting metals other than iron. We therefore assessed the ability of the gex1Δ gex2Δ strain and of the strain overproducing Gex1 to grow on media containing high concentrations of divalent metals (Cd2+, Ni2+, Cu2+, Mn2+, Mg2+, Co2+, Zn2+) and in the presence of the iron chelator BPS (Figure 3A and unpublished data). The gex1Δ gex2Δ mutant grew poorly only in the presence of the toxic heavy metal cadmium (Cd2+). The strain overproducing Gex1-HA was more resistant to this metal than the wild-type strain transformed with an empty plasmid. We then quantified the cadmium, iron, and copper contents of cells grown in a Cd-supplemented medium (Figure 3B). No significant differences were found in the iron and copper contents of the various strains, whereas a clear difference between strains was observed for cadmium. The gex1Δ gex2Δ strain had a much higher cadmium content than wild-type cells, and the cells overproducing Gex1-HA had a lower intracellular cadmium content. Gex1-HA was targeted to the plasma membrane when overproduced from the pGEX1-HA plasmid (Figure 2D). A decrease in cadmium content may therefore be indicative of an increase in cadmium efflux at the plasma membrane mediated by Gex1-HA. These results are consistent with the growth phenotypes observed in the presence of cadmium, and they suggest that Gex1 may mediate cadmium export at the plasma membrane. The topology of membrane-bound proteins is acquired in the endoplasmic reticulum and remains constant, regardless of the final site to which the protein is targeted. Thus a protein with multiple membrane spans and amino and carboxy termini facing the cytosol will present the same topology at the plasma membrane and at the vacuolar membrane (schemes in Figure 3B). Gex1 at the vacuolar membrane may therefore mediate cadmium import into the vacuolar lumen and participate in cadmium detoxification.

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