DYNAMIC REGULATION OF COPPER UPTAKE AND DETOXIFICATION GENES IN SACCHAROMYCES-CEREVISIAE

The essential yet toxic nature of copper demands tight regulation of t he copper homeostatic machinery to ensure that sufficient copper is pr esent in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nu tritional copper sensor Mac1p regulates the copper-dependent expressio n of the high affinity Cu(I) uptake genes CTR1, CTR3, and FRE1, while the toxic copper sensor Ace1p regulates the transcriptional activation of the detoxification genes CUP1, CRS5, and SOD1 in response to coppe r. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presenc e of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightf old the original basal level whereas the Ace1p-mediated transcriptiona l activation of CUP1 was rapid and potent but transient. CUP1 expressi on driven by an Ace1p DNA binding domain-herpes simplex virus VP16 tra nsactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activate d DNA binding domain. In vivo dimethyl sulfate footprinting analysis o f the CUP1 promoter demonstrated transient occupation of the metal res ponse elements by Ace1p which paralleled CUP1 mRNA expression. Analysi s of a Mac1p mutant, refractile for copper-dependent repression of the Cu(I) transport genes, showed an aberrant pattern of CUP1 expression and copper sensitivity These studies (i) demonstrate that the nutritio nal and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to a ppropriately regulate copper ion homeostasis and (ii) establish the re quirement for a wild-type Mac1p for survival in the presence of toxic copper levels.