FIGURE 1.

AICAR accumulation and toxicity in yeast. A, addition of extracellular AICAr does not result in a massive intracellular AICAR accumulation in yeast. After an overnight preculture, wild-type cells (Y175) were diluted and kept in exponential phase for 24 h in SDcasaWAU medium. External AICAr (5 mm) was then added (red line) or not (blue line) for 20 min, and metabolites were extracted and separated by liquid chromatography (only the 60–65-min elution time region of the chromatogram is presented). AICAR intracellular concentration was determined by measuring yeast cell volume as described (1). The inset corresponds to a zoom of the indicated region. Internal AICAR values correspond to three independent extractions, and standard deviation is indicated. B, schematic representation of purine and histidine pathways in yeast. Ade, adenine; Hypox, hypoxanthine; PM, plasma membrane; PRPP, 5-phosphoribosyl-1-pyrophosphate. Only the enzymes mentioned in the text are shown (in blue). C, intracellular AICAR accumulation is strongly increased in the ade16 ade17 ade8 his1 mutant. The cells (Y6986) were grown in SDcasaWAU medium, treated (green line) or not (orange line) with AICAr (5 mm, 20 min), and the metabolites were extracted and separated as in Fig. 1A. Internal AICAR values correspond to three independent extractions, and standard deviation is indicated. N.D., not detectable. D, effect of external AICAr on growth of the ade8 his1 (Y2660) or ade16 ade17 ade8 his1 (Quadruple; Y6986) strains. The cells were grown overnight, serially diluted, and spotted on SDcasaWAU medium containing external AICAr. Plates were imaged after an incubation of 2 days at the indicated temperature.