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Winge, et al., Current Opinion in Chemical
Biology 1998, 2216221
Metalloregulatory pathways in S. cerevisiae. (a)
Two distinct transcription factors (Ace1 and
Mac1) mediate Cu activation and Cu inhibition of
gene expression. Ace1 mediates Cu activation of
expression of genes involved in Cu-ion
detoxification. In Cu deficient cells, Mac1
stimulates transcription of genes the products of
which are involved in Cu ion uptake. (b) Aft1 and
(c) Zap1 mediate expression of genes in Fe- and
Zn-deficient yeast, respectively. The candidate
DNA-binding domains are in white, whereas
candidate transactivation domains are shaded.
Genes expressed by the specific factors are
listed in italics.
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Winge, et al., Current Opinion in Chemical
Biology 1998, 2216221
A schematic representation of the either known or
predicted domains of the metal-responsive
transcription factors in yeast. Positions of
cysteine residues are shown by dots. Cysteine
substitutions that result in constitutive
(non-metal responsive) activity of each factor
are designated by . Only cysteine residues are
shown for simplicity.
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Primary structural and functional domain
comparison of two classes of copper
metalloregulatory transcription factors involved
in copper metabolism. The nutritional
copper-sensing protein Cuf1 (S. pombe) exhibits
51 and 45 sequence identity with the
amino-terminal 63 and 62 amino acid residues that
are part of the copper-activated DNA-binding
domains of the toxic copper sensors Ace1 (S.
cerevisiae) and Amt1 (C. glabrata), respectively.
While Cuf1 also shows 48 sequence identity with
the REP-II domain at the carboxyl terminus of the
nutritional copper sensor Mac1 (S. cerevisiae).
The amino acid sequence numbers refer to the
position relative to the first amino acid of the
protein. The dots () indicate positions of
cysteine residues. The squares () depict
histidine residues that are required for function
in Mac1. The black-shaded regions indicate the
location of the (R/K)GRP motif that is part of a
conserved minor groove binding domain (33).
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Fig. 1.   Cellular localization of Mac1. (A) A
C-terminal fusion of Mac1 with the GFP (Mac1-GFP)
activates expression of a CTR1-lacZ reporter
gene. (B) Fluorescence of cells harboring the
Mac1-GFP fusion cultured in Cu-deficient (30 µM
bathocuproine sulfonate, BCS) and Cu-replete
(100 µM CuSO4) medium. Cells harboring the fusion
gene and GFP under the control of the GAL1
promoter were grown at 30C in synthetic complete
media containing galactose in place of dextrose.
GFP was excited at 488 nm and detected at 520 nm
DAPI was excited at 345 nm and detected at
465 nm.
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Fig. 3.   Copper-induced stabilization of Mac1.
(A) Fusions of a myc epitope to either Mac1 or
Mac1up1 activate expression of a CTR1-lacZ
reporter gene. Expression of Mac1-myc (B) or
Mac1up1-myc (C) was induced with galactose for
3 h prior to addition of glucose. Cultures were
then split one aliquot was untreated and 100 µM
CuSO4 was added to the other. Samples from each
culture were removed at 45 min intervals. Cell
extracts were separated by SDS-PAGE, transferred
to nitrocellulose and then immunoblotted with a
monoclonal anti-c-myc antibody and an anti-actin
antibody (to control for gel loading)
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Fig. 6.   Model of the Cu-induced intramolecular
interaction. DNA-binding and transactivation
domains are shown by hatching and shaded boxes,
respectively. The dots in the figures represent
cysteinyl residues. Laran T. Jensen and
Dennis R. Winge. Identification of a
copper-induced intramolecular interaction in the
transcription factor Mac1 from Saccharomyces
cerevisiae. The EMBO Journal Vol. 17,pp.
5400-5408, 1998
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Figure 1. Metal selectivity and sensitivity for
repression of the CTR3 promoter. Strain DTY1,
transformed with pRSCTR3-lacZ, was grown as
described under "Experimental Procedures." Total
RNA from control (0), CuSO4 (10, 1 µM
500, 100, 1 nM 100 pM) and CdCl2 (10, 1 µM
500, 100, 1 nM 100 pM) cultures (A) as well as
control (0), AgNO3 (10, 1 µM 100, 1 nM 100 pM),
and HgCl2 (10, 1 µM 100, 1 nM 100 pM) cultures
(B) was isolated. RNase protection analyses (A
and B) show the down-regulation of the CTR3
promoter-lacZ fusion in the presence of these
metal ions. The lacZ and ACT1 mRNA steady-state
levels are indicated with arrows. Results
illustrated are representative of three
independent experiments. C and D, graphic
representations of the RNase protection analyses
by PhosphorImage quantitation. Solid lines
indicate the response of the CTR3-lacZ fusion
gene to copper (C) and silver (D), whereas dashed
lines show that metal concentrations 3 orders of
magnitude greater than for CuSO4 were required
for cadmium (C) and mercury (D) to repress the
CTR3 promoter to 50 maximal repression.
 
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Gross, et al., (2000). J. Biol. Chem. 275
32310-32316
S1 nuclease protection assays to quantify mRNA
levels of three genes up-regulated by Mac1. The
upper band for each sample is the specified gene.
The lower band in each case, indicated by the
arrow, is the calmodulin (CMD1) loading control.
fp lanes show undigested, free probes. A, RNA was
isolated from wild-type (wt) CM66J cells cultured
in LCCM in the presence and absence of 100 µM BCS
or MAC1up1 (UP) cells. B, RNA was isolated from
wild-type cells or mac1 cells cultured in LCCM in
the presence or absence of 100 µM BCS.
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Fig. 2. S1 nuclease protection assays to quantify
mRNA levels of genes down-regulated by Mac1. The
upper band for each sample is the specified gene.
The lower band in each case, indicated by an
arrow, is the calmodulin (CMD1) loading control.
fp lanes show undigested, free probes. A, RNA was
isolated from cells cultured in LCCM in the
absence or presence of 100 µM BCS. B, RNA was
isolated from wild-type (wt) cells or MAC1up1
(UP) cells. without or harboring a ZRE/lacZ
fusion gene. C, RNA was isolated from wild-type
CM66J cells (wt) or MAC1up1 (UP) cells in the
absence or presence of 10 µM ZnSO4
Fig. 3. S1 nuclease protection assay of genes
up-regulated in copper-treated cells. A,
independent RNA isolates 1 and 2 from cells
cultured in the absence and presence of 100 µM
CuSO4 for 30 min (used in microarray experiments
1 and 2 Table II). B, RNA isolated from CM66J
wild-type cells cultured in low iron medium (Fe),
with 100 µM bathophenantroline sulfonate (BPS),
or supplemented with 1.5 µM ferric chloride. In
each case, mid-log phase cells were incubated in
the presence and absence of 100 µM Cu(II) for 30
min. C, RNA isolated from from CM3260 wild-type
cells (wt) or aft1 cells incubated in the
presence and absence of Cu(II) for 30 min.
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