FIG 5.
CgRpn4 regulates ergosterol levels in C. glabrata cells during fluconazole stress, affecting permeability and drug accumulation. (A) KUE100 and KUE100_Δcgrpn4 C. glabrata cells were harvested after 15 h of growth in RPMI 1640 medium (control) or after 4 h or 12 h of fluconazole stress. Ergosterol was extracted and quantified by HPLC. Cholesterol was used as an internal standard to evaluate the yield of ergosterol extraction. The displayed ergosterol contents are representative of results from at least six independent experiments. Error bars represent the corresponding standard deviations. *, P < 0.05; **, P < 0.01. (B) Comparison of plasma membrane permeabilities of exponential-phase KUE100 (squares) and KUE100_Δcgrpn4 (diamonds) cells under control conditions or upon 1 h of fluconazole stress. The estimation of plasma membrane permeability is based on the fluorescence intensity values exhibited by yeast cells upon the passive accumulation of propidium iodide. Error bars represent the corresponding standard deviations. **, P < 0.01; ***, P < 0.001. (C) Time course accumulation of radiolabeled [3H]fluconazole in strains KUE100 (squares) and KUE100_Δcgrpn4 (diamonds) during cultivation in liquid BM in the presence of 150 mg/liter unlabeled fluconazole. Accumulation values are the averages from at least three independent experiments. Error bars represent the corresponding standard deviations. **, P < 0.01.