Figure 3.

Labile heme and heme signaling is increased, but total heme is attenuated in response to Pb²⁺ toxicity. A, yeast cell viability, as measured by solution turbidity at an optical density (O.D.) of 600 nm, is diminished by exposure to Pb²⁺ in a dose-dependent manner. B, at the LD₅₀ dose of Pb²⁺ (500 μm), cells hyperaccumulate up to 10 mm Pb²⁺, as measured by TXRF. C, a dose of 500 μm Pb²⁺ diminishes total heme to levels similar to 500 μm SA, but Pb²⁺ increases labile heme 2-fold, whereas SA does not. D, Hap1p activity in response to heme depletion by SA or Pb²⁺ is measured using the pCYC1-eGFP Hap1 reporter construct (pCYC1). To control for the Hap1-independent effects of Pb²⁺ on eGFP expression, we measured eGFP fluorescence in response to Pb²⁺ or SA using an allele of eGFP driven by the heme/Hap1-independent promoter, GPD (pGPD). The ratio of pCYC1 to pGPD eGFP expression (pCYC1/pGPD) is a measure of heme/Hap1-specific activation of CYC1. All data represent the mean ± S.D. (error bars) of triplicate cultures, and the statistical significance relative to untreated cells was assessed using a two-sample t test. *, p < 0.05; **, p < 0.005; ***, p < 0.001; n.s., not significant.