GEX1 and GEX2 are induced under conditions of iron depletion and Gex1 is regulated principally by Aft2. (A) GEX1-GFP/GEX2-HA or GEX1-HA/GEX2-GFP were grown in YPD or YPD supplemented with 200 μM BPS to midexponential growth phase. Total protein extracts were prepared and analyzed by Western immunoblotting with antibodies directed against GFP, HA, and phosphoglycerate kinase (PGK) as a loading control. (B) GEX1-GFP/GEX2-HA and GEX1-HA/GEX2-GFP cells carrying pAFT1, pAFT1–1^up, pAFT2, or pAFT2–1^up were grown overnight in YNB without doxycycline. Total yeast extracts were analyzed by Western immunoblotting for GFP, HA and PGK.

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.

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.

Glutathione distribution is dependent on GEX1 and GEX2 expression. WT, gex1Δ gex2Δ, WT + pØ, and WT + pGEX1-HA cells were grown in the presence or absence of 1 μM cadmium for 6 h (with galactose as a carbon source for the plasmid-bearing strains). The cells were centrifuged and total glutathione (reduced plus oxidized), and oxidized glutathione disulfide (GSSG) levels were determined in crude cell extracts (A) and in the medium (B) in the enzymatic recycling assay (see Materials and Methods). The values obtained were normalized with respect to protein content, determined in the BCA protein assay. The ratio GSH:GSSG for cell extracts is presented in the table (A). All data points in the figure represent means of at least six determinations. Asterisk indicates significant differences (P < 0.005, Student's t test).

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.

The overproduction of Gex1-HA induces acidification of the cytosol and the vacuole. (A) WT, gex1Δ gex2Δ, and nha1Δ cells transformed with pADH1-pHluorin were grown overnight to midexponential growth phase in glucose-containing medium. WT cells cotransformed with pADH1-pHluorin and the pØ or the pGEX1-HA plasmids and nhx1Δ cells cotransformed with pADH1-pHluorin and pØ plasmids were grown to midexponential phase in galactose-containing medium. Fluorescence was visualized with the FITC filter set (characteristics given in Materials and Methods), and cell shape was studied with Nomarski optics. (B) Fluorescence intensity values (arbitrary units) were obtained for an emission wavelength of 508 nm for two excitation wavelengths: 410 and 470 nm (I₄₁₀ and I₄₇₀) (the values correspond to at least 70 measurements). (C) WT and gex1Δ gex2Δ cells were grown in YPD. WT cells transformed with the pØ or pGEX1-HA plasmids were grown overnight in galactose-containing medium. All strains were stained with quinacrine, analyzed for quinacrine fluorescence, and studied with Nomarski optics. The diagram illustrates a proposed mode of action for Gex1-HA.

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).