Dynamic regulation of copper uptake and detoxification genes in Saccharomyces cerevisiae

Abstract

The essential yet toxic nature of copper demands tight regulation of the copper homeostatic machinery to ensure that sufficient copper is present in the cell to drive essential biochemical processes yet prevent the accumulation to toxic levels. In Saccharomyces cerevisiae, the nutritional copper sensor Mac1p regulates the copper-dependent expression 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 copper. In this study, we characterized the tandem regulation of the copper uptake and detoxification pathways in response to the chronic presence of elevated concentrations of copper ions in the growth medium. Upon addition of CuSO4, mRNA levels of CTR3 were rapidly reduced to eightfold the original basal level whereas the Ace1p-mediated transcriptional activation of CUP1 was rapid and potent but transient. CUP1 expression driven by an Ace1p DNA binding domain-herpes simplex virus VP16 transactivation domain fusion was also transient, demonstrating that this mode of regulation occurs via modulation of the Ace1p copper-activated DNA binding domain. In vivo dimethyl sulfate footprinting analysis of the CUP1 promoter demonstrated transient occupation of the metal response elements by Ace1p which paralleled CUP1 mRNA expression. Analysis 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 nutritional and toxic copper metalloregulatory transcription factors Mac1p and Ace1p must sense and respond to copper ions in a dynamic fashion to appropriately regulate copper ion homeostasis and (ii) establish the requirement for a wild-type Mac1p for survival in the presence of toxic copper levels.

FIG. 1

Expression of CTR3 and CUP1 in response to 1, 10, and 100 μM CuSO₄. (A) Exponential-phase cultures of S. cerevisiae DTY1 were treated with 1, 10, and 100 μM CuSO₄. Five-milliliter samples were taken after 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min of Cu treatment. RNA extracted from each sample was analyzed by RNase protection assays. Each reaction contained 30 μg of total RNA. CTR3, CUP1, and ACT1 RNAs are indicated by arrows. (B) Quantitation of CTR3 mRNA expression in response to Cu. (C) Quantitation of CUP1 mRNA expression in response to Cu. All values in panels B and C were normalized against those for ACT1 mRNA as an internal control.

FIG. 2

Induction of Cup1p in response to 100 μM CuSO₄. Exponential-phase cultures of S. cerevisiae DTY1 were labeled with 5 μCi of [³⁵S]cysteine (800 Ci/mmol) per ml for 30 min and then treated with 100 μM CuSO₄. Five-milliliter samples were taken at 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min of Cu treatment. Total protein extracts were analyzed on a 20% nondenaturing polyacrylamide gel. Each lane contains 30 μg of total protein. The gel was fixed with 10% acetic acid and 30% methanol for 1 h, fluorographed with En³Hance (Dupont), dried under vacuum, and exposed to Kodak BioMax Film with an intensifying screen at −80°C.

FIG. 3

Western blot analysis of Ace1p levels during Cu ion treatment. Exponential-phase cultures of S. cerevisiae DTY1 were treated with 1, 10, or 100 μM CuSO₄. Five-milliliter samples were taken after 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, and 90 min of Cu treatment. Thirty micrograms of total protein extract was analyzed by immunoblotting with polyclonal antibodies raised against purified recombinant Ace1p from E. coli (rACE1p).

FIG. 4

Transcription of CTR3 and CUP1 by ACE1-VP16 in response to 100 μM CuSO₄. Exponential-phase cultures of strains MPY2 and MPY3 harboring a wild-type ACE1 gene and a gene fusion between the Ace1p DNA binding domain and the VP16 activation domain integrated at the his3 locus, respectively, were treated with 100 μM CuSO₄. Five-milliliter samples were taken after 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min of Cu treatment. Thirty micrograms of total RNA was used in RNase protection experiments for each sample, using ACT1 mRNA as an internal control. CTR3, CUP1, and ACT1 RNase protection products are indicated by arrows.

FIG. 5

In vivo DMS footprinting analysis of the CUP1 promoter. (A) Exponential-phase cultures of strain DTY1 were treated with 100 μM Cu. After 0, 15, 30, and 60 min of Cu treatment, 250-ml samples were taken and analyzed by in vivo DMS footprinting to assess the occupancy of the MREs by Ace1p. The positions of MREs 1 through 4 are indicated schematically, and G residues within MREs are indicated by dots. (B) Quantitation of the relative accessibility to DMS modification of representative G residues from the proximal MRE (MRE 1) closest to the CUP1 transcriptional start site. These values were normalized against the bands at positions −212 and −243 with respect to the transcriptional start site, whose intensities remained constant throughout the time course of Cu ion treatment.

FIG. 6

Effect of increasing Cu ion concentration on CUP1 regulation. Exponential-phase cultures of strain DTY1 were treated with 1 or 5 mM CuSO₄. Five-milliliter samples were taken after 0, 15, 30, 45, 60, 90, and 120 min of Cu treatment. RNA was extracted from each sample and analyzed by RNase protection assays. Each reaction contained 20 μg of total RNA. CUP1 and ACT1 mRNAs are indicated by arrows. Quantitation of CUP1 mRNA is shown in the graph.

FIG. 7

Expression of CTR3 and CUP1 in a MAC1^up1 strain. (A) Exponential-phase cultures of strain DTY205, which harbors the dominant MAC1^up1 allele, were treated with 1, 10, and 100 μM Cu. Five-milliliter samples were taken after 0, 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, and 120 min of Cu ion treatment and analyzed by RNase protection assays. Thirty micrograms of total RNA was analyzed in each reaction. The CTR3, CUP1, and ACT1 RNase protection products are indicated by arrows. (B) Quantitation of CTR3 expression in response to Cu. (C) Quantitation of CUP1 expression in response to Cu. The values shown in panels B and C were normalized against values for ACT1 as an internal control.

FIG. 8

Copper sensitivity of MAC1 and MAC1^up1 strains. Yeast strains of the indicated relevant genotypes were plated on SC-His medium containing increasing concentrations of CuSO₄. Strains DTY1 and DTY205 were transformed with the pRS313 vector to allow growth on SC-His plates. Both strains contain the CTR1 and CTR3 genes and harbor a wild-type MAC1 and MAC1^up1 alleles, respectively. MPY18 strains lack both high-affinity Cu transport genes and harbor a chromosomal deletion of the mac1Δ allele. These strains were transformed with a plasmid expressing either the MAC1 or MAC1^up1 allele on a pRS313-based centromeric vector. The MAC1 genes were both tagged with a single copy of the hemagglutinin epitope at the 3′ end of the open reading frame.