Quantitation of p53 nuclear relocation in response to stress using a yeast functional assay: effects of irradiation and modulation by heavy metal ions

Abstract

Many regulatory proteins undergo transient nuclear relocation under physical or chemical stress. This phenomenon is, however, difficult to assess due to the lack of sensitive and standardized biological assays. Here, we describe a new quantitative nuclear relocation assay (QNR), based on expression in yeasts of chimeric proteins in which an artificial transcription factor is fused to a target protein acting as driver for relocation. This assay combines the experimental versatility of yeast with quantitation of nuclear relocation at low levels of protein expression. We have assessed the nuclear relocation of yeast Yap1 and human p53, two transcription factors that relocate to the nucleus in response to oxidative-stress and DNA damage, respectively. We show that p53 efficiently drives the relocation of the chimeric reporter in response to irradiation and that this process requires the C-terminal nuclear export signal (NES). Cd²⁺ and Hg²⁺, two metal ions inducing DNA damage as well as conformational changes in p53, have opposite effects on p53 relocation in response to DNA damage. Whereas Hg²⁺ effects are synergistic to DNA damage, Cd²⁺ inhibits relocation and sequesters p53 into the cytoplasm. These results demonstrate the effectiveness of QNR to investigate the regulation of p53 shuttling in response to stress signals including suspected environmental carcinogens.