(Received for publication, October 25, 1996, and in revised form, January 17, 1997)
From the Division of Toxicological Sciences,
Department of Environmental Health Sciences, Johns Hopkins University
School of Public Health, Baltimore, Maryland 21205, the ¶ Cell
Biology and Metabolism Branch, NICHHD, National Institutes of Health,
Bethesda, Maryland 20892, and the § Department of Chemistry,
Northwestern University, Evanston, Illinois 60208-3113
The ATX1 gene of Saccharomyces
cerevisiae was originally identified as a multi-copy suppressor
of oxidative damage in yeast lacking superoxide dismutase. We now
provide evidence that Atx1p helps deliver copper to the copper
requiring oxidase Fet3p involved in iron uptake. atx1
null mutants are iron-deficient and are defective in the high affinity
uptake of iron. These defects due to ATX1 inactivation are
rescued by copper treatment, and the same has been reported for strains
lacking either the cell surface copper transporter, Ctr1p, or the
putative copper transporter in the secretory pathway, Ccc2p. Atx1p
localizes to the cytosol, and our studies indicate that it functions as
a carrier for copper that delivers the metal from the cell surface
Ctr1p to Ccc2p and then to Fet3p within the secretory pathway. The iron
deficiency of atx1 mutants is augmented by mutations in
END3 blocking endocytosis, suggesting that a parallel
pathway for intracellular copper trafficking is mediated by
endocytosis. As additional evidence for the role of Atx1p in iron
metabolism, we find that the gene is induced by the same iron-sensing
trans-activator, Aft1p, that regulates CCC2 and
FET3.
The yeast Saccharomyces cerevisiae represents an excellent model system for the study of heavy metal metabolism. In the past few years, several genes involved in the transport and trafficking of copper and iron have been identified in this yeast. CTR1 encodes a high affinity transporter for copper ions (1, 2). Once copper enters the cell via Ctr1p, it can transported to various distinct cellular locations. First copper can be transported to the mitochondria where the metal is needed to activate cytochrome oxidase. This pathway requires both Ctr1p and a small cytosolic protein, Cox17p (2, 3). Secondly, copper is needed to activate cytosolic copper-binding proteins such as the copper and zinc requiring superoxide dismutase (Sod1p). Accordingly, ctr1 mutants are defective in Sod1p activity (2). Ctr1p is also necessary for efficient copper-regulated expression of the Cup1 metallothionein, and thus may be needed for copper transport to the nucleus (2). Additionally, Ctr1p is involved in the delivery of copper ions to the secretory pathway. One protein requiring this mode of copper delivery is Fet3p, a multi-copper oxidase that is needed for high affinity iron uptake (1, 4-6). The copper-loaded Fet3p functions as part of a complex with the iron permease Ftr1p to mediate iron uptake at the plasma membrane (7, 8). Because of this requirement for copper-Fet3p in iron transport, cells lacking Ctr1p are defective in iron uptake (1, 4).
An additional component of the copper delivery pathway to Fet3p is CCC2 (5, 9), the yeast homologue of the human Wilson (10-12) and Menkes (13-15) genes. Like its mammalian counterparts, Ccc2p possesses sequences common to P-type ATPases and also copies of the conserved copper-binding motif MTCXXC (9). These reputed ATPases of yeast and man are believed to function in the translocation of copper across intracellular membranes into the secretory pathway. Indeed, the Menkes protein has been localized by Gitlin and co-workers to a compartment in the Golgi (16). Although CCC2 functions in copper delivery, the CCC2 transcript falls under the transcriptional control of the iron-responsive trans-activator Aft1p (17, 18). This is consistent with the role of Ccc2p in copper delivery to Fet3p involved in iron uptake. By contrast, CTR1 is involved in global copper trafficking and is regulated by the copper-responsive activator, Mac1p.1
The mechanism(s) by which Ccc2p and the Menkes/Wilson gene products obtain their copper for translocation into the secretory pathway has been unknown. By virtue of its cytotoxic nature, copper is unlikely to exist free in the cytosol. One hypothesis is that a cytosolic carrier for copper delivers the metal to the ATPase for transport into the secretory pathway. The studies presented here focus on one likely candidate for such a copper carrier: the small copper homeostasis factor, Atx1p.
We originally isolated ATX1 (ni-oidant) as a multi-copy suppressor of oxygen toxicity in yeast devoid of copper/zinc superoxide dismutase (Sod1p) (19). Cells lacking Sod1p exhibit a number of metabolic defects when grown in air, including aerobic auxotrophies for lysine and methionine (20-23). Overexpression of ATX1 suppresses all the Sod1p-linked defects in a manner dependent upon cellular copper uptake (19). ATX1 encodes an 8.2-kDa polypeptide homologous to the MerP mercury transport proteins of bacteria (19). Close homologues to ATX1 were also identified in Caenorhabditis elegans, and more recently, in humans (33). In each case, the Atx1-like protein contains a single copy of the MTCXXC metal binding motif also found in Ccc2p and the Wilson and Menkes gene products (19). In the present study, we provide strong evidence that yeast Atx1p is localized to the cytosol and functions in a copper trafficking pathway (Ctr1p-Atx1p-Ccc2p) mediating delivery of copper to Fet3p.
The
S. cerevisiae strain SL202 was obtained by deleting the
SOD1 gene of YPH250 (24) using a
sod1::LEU2 plasmid (25). Strains SL108 and
SL214-SL217 were constructed by creating an atx1
mutation in AA255 (26), SL202, YPH250 (24), RH144-3D, and SM2186,
respectively (isogeneic wild type and end3ts
mutant strains; kind gifts of S. Michaelis), using either the atx1
::HIS3 plasmid described below (for SL214)
or the atx1
::LEU2 plasmid (remaining strains)
described earlier (19). M2P is the AFT-1up
derivative of wild type CM360 (2). Strain 3 is a ccc2 mutant strain obtained from T. Dunn (5). Strains EG103 and the
corresponding atx1
derivative SL103
A1 have been
described (19, 23).
The pDA1-HIS atx1::HIS3 deletion plasmid was
constructed by mobilizing the atx1
deletion fragment from
plasmid pDA1J (19) through digestion with XhoI and
SacI, and then by inserting this fragment into the
XhoI and SacI sites of the HIS3
integrating vector, pRS403 (24). p413-A1 represents an ATX1
CEN vector constructed by amplifying ATX1 sequences
397 to +800 by the polymerase chain reaction
(PCR),2 inserting the product into the
pCRII vector (Invitrogen), and subcloning the ATX1 fragment
into the BamHI and XhoI sites of pRS413 (24).
To insert ATX1 into the overexpression vector pET11d, the
ATX1 coding region was amplified by PCR using mutagenic
primers to introduce unique restriction sites at both the 5
(NcoI) and 3
(BamHI) ends of the gene. The PCR
product and pET11d vector were both digested with NcoI and
BamHI, the desired fragments were purified from low melt
agarose gels and subsequently ligated to produce the pET11d-ATX1
expression vector.
Stocks of yeast strains were maintained on a standard yeast
extract-peptone-dextrose (YPD) medium (27). Iron dependence tests were
carried out as described (8) on synthetic dextrose (SD) minimal medium
plates containing 1-3.0 mM ferrozine
(3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine;
Sigma) and supplemented with ferrous ammonium sulfate
(Sigma) as needed. Agarose substituted for agar to
minimize contamination of iron. Measurements of ferrous iron uptake
were conducted in triplicate samples essentially as described (1). All
yeast transformations were carried out by electroporation (28).
Transformants of strains containing sod1 mutations
required initial cultivation in anaerobic culture jars (23).
Atx1p was purified from an E. coli strain carrying a T7 expression vector containing the ATX1 gene. Complete details regarding Atx1p overexpression and purification will be published later.3 Purified Atx1p was judged to be greater than 95% homogeneous by SDS-PAGE and further analyzed by electrospray ionization-mass spectrometry to confirm the size of the intact protein.
The purified Atx1p was used to produce anti-Atx1p antibodies in rabbits by Cocalico Biologicals, Inc. The antibody was purified using an ImmunoPure IgG (Protein A) purification kit, according to manufacturer's specifications (Pierce).
Yeast Fractionation and Immunofluorescence MicroscopyFor Western blot analysis, yeast cells were initially grown overnight in selecting SD medium to confluence, then diluted in 50 ml of SD medium to an A600 nm of 0.3 and allowed to grow for an additional 6 h. Cells were then broken by glass bead homogenization in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM each of EDTA and EGTA, 20 µg/ml leupeptin, 10 µg/ml pepstatin A, 1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride. Extracts were subjected to 15% SDS-PAGE and Western analysis using the purified anti-Atx1 IgG at a 1:500 dilution. The secondary antibody consisted of anti-rabbit IgG conjugated to horseradish peroxidase (Amersham) diluted 1:10,000. Detection employed the ECL kit (Amersham), according to manufacturer's specifications. For resolution of membrane and soluble components, extracts were prepared by osmotic shock as described previously (29), and subjected to centrifugation at 100,000 × g in a Beckman 50 Ti-50 rotor. The pellets were resuspended in lysis buffer to the same volume, and all samples were prepared for SDS-PAGE and Western blot analysis, as above.
For detection of Atx1p by immunofluorescence, the atx1
strain SL108 transformed with pRS-A1 (2µ ATX1) was grown
in 10 ml of selecting SD medium to a final
A600 nm of 1.0. Cells were fixed with
formaldehyde, permeabilized by zymolyase treatment, and prepared for
antibody staining (30). Incubation with rabbit anti-Atx1p (diluted
1:250) proceeded for 2 h. Detection was carried out with a
fluorescein isothiocyanate (FITC)-labeling kit (Boehringer Mannheim)
and utilized a goat anti-rabbit secondary antibody (diluted 1:500)
coupled to FITC. Nucleic acids were stained by DAPI
(Sigma), and FITC and DAPI staining was monitored by
fluorescence microscopy.
The CTR1 and CCC2 genes of yeast are
involved in distinct copper transport steps (1, 5). Mutants at these
loci are defective in the high affinity uptake of ferrous iron (1, 5)
and require iron supplemented medium for growth. To study whether
ATX1 participates in the same pathway of copper trafficking,
we tested the effects of an atx1 gene deletion on iron
dependent growth. An isogeneic pair of atx1
and wild type
strains was plated onto a minimal medium prepared with the
iron-specific chelator ferrozine (31, 32), and growth was monitored. As
shown in Fig. 1A, the atx1
strain exhibited no growth on this medium, and this defect was reversed
by transforming the mutant with a CEN vector harboring wild
type ATX1. The growth inhibition of atx1
strains on ferrozine was also completely reversed by supplementation
with iron concentrations sufficient to reverse the effects of the iron
chelator (Fig. 1A).
We also obtained direct measurements of ferrous iron uptake. As shown
in Fig. 1B, iron uptake in a wild type strain was reduced by
treatment with the BCS copper-specific chelator, confirming the copper
dependence of high affinity iron transport (1, 4, 5). Consistent with
previous results (5), ferrous iron uptake was undetectable in a strain
containing a ccc2 gene deletion and this deficiency was
completely rescued by supplementation with copper (Fig. 1B).
The atx1 strain also exhibited a reduction in iron uptake
that was corrected by copper. However, the atx1
mutation
resulted in an incomplete blockage (65-70% inhibition) of iron
uptake, compared with the total inhibition of transport observed with
ccc2
strains (Fig. 1B). Nevertheless, the
effects of atx1 mutations on the uptake and dependence on
iron suggested that Atx1p operates in the same pathway as Ctr1p and
Ccc2p to deliver copper to Fet3p. This notion is confirmed in the
accompanying paper, where an atx1 mutant strain is shown to
be deficient in the production of copper-Fet3p (33).
To understand further how ATX1 functions in copper metabolism, we examined the intracellular localization of the encoded polypeptide. Atx1p is a small protein that lacks transmembrane domains or other localization signals (19), suggesting that it may be cytosolic.
To monitor Atx1p production, we prepared anti-Atx1p antibodies.
Purified Atx1p protein was obtained following expression in E. coli and used to produce polyclonal rabbit antibodies. The efficacy of this anti-Atx1 was then assessed by Western blot. As seen
in Fig. 2A, a single major band exhibiting an
apparent molecular mass of 7.0 kDa was visualized from a strain
harboring ATX1 on a 2µ vector, but was absent in the
isogeneic atx1 strain. Atx1p produced from a
CEN vector or the single-copy chromosomal gene could also be
visualized (Fig. 2B), but yielded weak signals, suggesting
that ATX1 may be normally expressed at low levels in yeast.
In all cases, Atx1p migrated slightly faster on SDS gels than would be
expected from its predicted molecular mass of 8.2 kDa, and this has
also been observed with purified Atx1p produced in E. coli.
Overexpression of Atx1p in E. coli resulted in a full-length protein lacking its N-terminal methionine as determined by electrospray ionization-mass spectrometry.4
A cell fractionation experiment was conducted to test whether Atx1p is a soluble cytosolic protein. Extracts were prepared by gentle lysis as described (29) from cells harboring ATX1 on a CEN vector and were subjected to high speed centrifugation to resolve soluble and membrane-associated cellular constituents. As a control for these experiments, we monitored fractionation of the Atx2-HA protein, which is known to localize to vesicles in the secretory pathway (29). As seen in Fig. 2C, Atx1p was totally recovered in the supernatant following a 100,000 × g centrifugation, whereas this same treatment caused precipitation of the Golgi-localized Atx2-HA protein. The Golgi protein was only solubilized upon treatment of extracts with Triton X-100 (Fig. 2C). This fractionation study demonstrated that Atx1p is a soluble protein and is most likely cytosolic.
Indirect immunofluorescence microscopy was used to study Atx1p
localization further. Cells harboring the ATX1 2µ plasmid
were fixed and permeabilized, and were probed with the rabbit anti-Atx1 antibody and a secondary anti-rabbit antibody conjugated to FITC. By
FITC straining, Atx1p exhibited uniform staining throughout the cytosol
and was excluded from the nucleus, as defined by co-staining with DAPI
(Fig. 3). Atx1p produced from the CEN vector
showed a similar pattern of staining, although the immunofluorescence signal was much weaker in this case (data not shown). Our
immunofluorescence experiments together with the cell fractionation
study strongly indicated that Atx1p is a cytosolic protein. The
complete absence of Atx1p in the nucleus rules against a possible role
for the protein as a copper-transcription factor.
The Interacting Roles of Ccc2p, Atx1p, and Endocytosis in Copper Trafficking
The localization of Atx1p to the cytosol suggested
that this protein may be involved in the cytosolic trafficking of the
metal to Ccc2p and then to Fet3p. As an alternative scenario, Atx1p and
Ccc2p could act in parallel, redundant pathways to separately deliver
copper to Fet3p. If this latter possibility were true, Ccc2p and Atx1p
should cross-compensate for one another. We therefore tested whether
multi-copy CCC2 and ATX1 could suppress the
effects of an atx1 and ccc2
null mutation,
respectively. As seen in Fig. 4B, multi-copy
ATX1 was incapable of suppressing the iron deficiency of a
ccc2 mutant, even though the Atx1p protein was clearly
overproduced under these conditions (Fig. 2 and Ref. 19). This result
indicated that Atx1p does not function in parallel with Ccc2p. However,
in contrast to results obtained with multi-copy ATX1,
overexpressed CCC2 was capable of suppressing the iron
dependence of an atx1
mutant (Fig. 4A). The
ability of multi-copy CCC2 to compensate for ATX1
(but not the converse) supports the notion that Ccc2p functions
downstream of Atx1p. These studies also show that an Atx1p-independent
pathway can substitute for Atx1p when Ccc2p is overproduced.
Additional evidence for the Atx1p-independent pathway of copper
transport was obtained through iron uptake studies. As seen in Fig.
1B, ferrous iron uptake was reduced in the
atx1 deletion strain, in comparison to the total
elimination of iron transport associated with a deletion of
CCC2. This partial effect of atx1 mutations was
also seen in a growth test for iron dependence. atx1
strains exhibited no growth on ferrozine-containing medium that was not
supplemented with additional iron (Fig. 1A). However, the
addition of 50 µM ferrous iron to the ferrozine medium
supported growth of the atx1
strain, but not of the
ccc2
mutant (Fig. 5). These observations
suggested that a "back up" system for copper delivery exists in
atx1
mutants. Possible candidates for this auxiliary
system included Sod1p and the Cup1p and Crs5p metallothioneins, as all
three are small soluble copper-binding proteins (34-36). However, we
observed that ferrous iron uptake was not eliminated in an
atx1
strain also containing mutations in SOD1
(Fig. 1B) or in a strain containing triple deletions in
ATX1, CUP1, and CRS5 (data not shown).
Thus the metallothioneins and Sod1p do not aid in copper delivery to
Fet3p.
Endocytosis is another method by which copper could be delivered to
intracellular locations. We therefore tested the role of endocytosis as
the secondary means of trafficking copper in atx1
strains. These studies utilized a temperature-sensitive end3
mutation known to block endocytosis at 37 °C (37). end3 mutants are defective in both receptor-mediated and fluid-phase endocytosis, yet other vesicle-mediated processes are not affected (37). A temperature-sensitive mutation in END3 did not cause growth inhibition on the low iron medium (Fig. 5), consistent with
other studies showing that end3 mutants are not defective in
high affinity ferrous iron uptake.1 However, at the
non-permissive temperature, this end3 mutation did enhance
the iron deficiency of an atx1
strain. At 37 °C, but
not at 25 °C, an atx1
end3 double mutant exhibited the
same iron dependence of a ccc2 mutant and showed no growth
on ferrozine medium supplemented with only 50 µM ferrous
ammonium sulfate (Fig. 5). Growth of the atx1
end3 double
mutant was stimulated by 350 µM ferrous ammonium sulfate
and also by 500 µM CuSO4 (Fig. 5), indicating
that iron deficiency resulted from inadequate copper delivery to Ccc2p
and Fet3p. Hence, the apparent residual trafficking of copper to Fet3p
in atx1 mutants involves endocytosis.
The genes required for
high affinity ferrous iron uptake in yeast fall under the control of
the iron-sensing trans-activator, Aft1p (17, 18). Included in this list
of iron regulated genes are FRE1 (encoding ferric reductase;
Ref. 38), FTR1 (iron permease), FET3, and
CCC2 (17). In comparison, CTR1, which is involved in global copper homeostasis, is regulated by Mac1p (39), a copper-sensing trans-activator.1 Upon inspection of
ATX1 upstream sequences we noted a single Aft1p consensus
sequence TGCACCC at nucleotides 110 to
116 (Fig. 6A). However, possible recognition sequences
for the Mac1p trans-activator could not be found. This observation
suggested that ATX1 may fall under the Aft1p regulon.
Northern blot analysis was used to test the regulation of
ATX1 by copper and iron. For studies on copper,
ATX1 expression was monitored in strains treated with the
copper chelator bathocuproine sulfonate under conditions known to
induce the CTR1 and CTR3 copper transport genes
(2, 40). We also tested the effects of null and hyper-active alleles of
MAC1 (39) on ATX1 mRNA levels. However, in
all cases, ATX1 gene expression remained unaffected by
copper depletion or by mutations in MAC1 (data not shown),
indicating that ATX1 is not copper-regulated. To examine the
possible regulation by iron and Aft1p, ATX1 gene expression
was monitored in strains containing either an aft1 null
mutation or a hyper-active allele of AFT1
(AFT1-1up; Ref. 18). As shown in Fig.
6B, ATX1 mRNA was up-regulated in a strain
containing the hyper-active AFT1-1up allele, as
was FET3 mRNA known to be a target for Aft1p-regulation (17). Densitometric tracings revealed that the induced level of
ATX1 and FET3 mRNA in the
AFT1-1up strain compared with the isogeneic
wild type was 3.0- and 3.6-fold, respectively. Surprisingly however,
the basal level of ATX1 gene expression was unaffected in
strains containing an aft1 null mutation (Fig.
6B) or in wild type strains treated with repressing concentrations of iron that inactivate Aft1p (data not shown; Ref. 17).
In comparison, FET3 expression was virtually eliminated in
the aft1
strain (Fig. 6B). CCC2
mRNA levels are also known to be markedly decreased in an
aft1
strain (17). Hence unlike CCC2 and
FET3, ATX1 is not absolutely dependent on Aft1p,
and other trans-acting factors must be involved in ATX1
regulation.
In bakers' yeast, multiple pathways of copper trafficking emerge from the plasma membrane copper transporter, Ctr1p. Copper taken up by Ctr1p is delivered to the mitochondria where the metal is needed for electron transport, to a compartment needed for activation of cytosolic copper proteins (such as Sod1p), and to the secretory pathway (1, 2, 4, 5, 7, 8). The studies presented herein describe a small copper carrier, Atx1p, that functions in the Ctr1 pathway that delivers copper to secretory compartments.
Several lines of evidence support a role for Atx1p in the trafficking
of copper from Ctr1p to the secretory pathway. Like the copper
transporters Ctr1p and Ccc2p, Atx1p is required for activation of
Fet3p, a multi-copper oxidase involved in iron transport (1, 4, 5, 8).
Strains containing mutations in ATX1 are deficient in
ferrous iron uptake and show iron-dependent growth. Furthermore, experiments in the accompanying paper (33) demonstrate that atx1 mutants are defective in production of
copper-Fet3p, in vivo. Our localization of Atx1p to the
cytosol, together with our genetic epistasis experiment, indicates that
Atx1p functions as a cytosolic carrier for copper, shuttling the metal
from the cell surface Ctr1p to the copper translocating ATPase, Ccc2p, in the secretory pathway. Consistent with its role as a soluble copper
carrier, purified Atx1p specifically coordinates copper ions in
vitro.4 It is noteworthy that the bacterial homologue
to Atx1p, MerP, likewise functions as a small metal carrier and is
believed to help shuttle mercury ions across the periplasmic space to
the MerT mercury transporter in the cell membrane (41, 42).
Ccc2p and Fet3p can also obtain copper ions in an
ATX1-independent manner. atx1 mutants exhibited
partial inhibition of ferrous iron uptake. In comparison, iron uptake
was eliminated in ccc2 mutants. Our studies show that the
apparent residual copper trafficking in atx1 mutants
involves endocytosis. A blockage in endocytosis together with a
mutation in atx1 conferred a dependence on iron for growth
that was indistinguishable from that observed with ccc2
mutations. The mechanism by which endocytosis could facilitate intracellular copper transport is currently not known. One possibility is that Ctr1p vesicles are involved. Ctr1p has been shown to be internalized to vesicles by endocytosis (43). A mutation in CTR1 blocks not only the Atx1p-dependent pathway
of copper delivery, but also the endocytosis pathway, since
ctr1
mutants do not exhibit the back-up system for copper
delivery that is evident in atx1
mutants (1). We
therefore propose that Ctr1p may bypass Atx1p through vesicle fusion
events and directly deliver copper to Ccc2p, as illustrated in the
model of Fig. 7. In any case, the contribution of
endocytosis to cytosolic copper trafficking appears minor in comparison
to the predominant role of Atx1p.
Atx1p is not a global facilitator of intracellular copper transport. Our other studies5 show that Atx1p does not function in copper delivery to cytochrome oxidase in the mitochondria or to Sod1p in the cytosol. How then does cytosolic Atx1p favor Ccc2p/Fet3p in the secretory pathway over other copper-requiring targets? It is noteworthy that Atx1p and Ccc2p share the same MTCXXC consensus metal-binding motif that serves to coordinate copper ions. It is therefore possible that this motif mediates the direct delivery of copper from Atx1p to Ccc2p and may also facilitate interactions between the two proteins.
ATX1 was originally identified, not as a copper trafficking
protein, but rather as a multi-copy suppressor of oxidative damage in
sod1 mutants (19). Is the apparent anti-oxidant activity of Atx1p related to its role in iron transport? Our studies suggest not. We observed that multi-copy ATX1 can still suppress
Sod1p deficiency in strains containing deletions in either
CCC2 or FET3.5 Hence,
suppression of oxidative damage does not require copper delivery to
Fet3p. However, ATX1 suppression of sod1
is
dependent on the Ctr1p copper transporter (19). It is therefore
conceivable that multi-copy ATX1 suppresses
sod1
by increasing delivery of redox active copper ions
to a target in the cytosol where Sod1p is normally needed. This target
may represent a non-Sod1 copper complex that is capable of neutralizing
oxygen radicals.
Our gene regulation studies also suggest that the role of Atx1p may not be restricted to iron uptake. Aft1p is an iron-sensing trans-activator that is essential for the expression of genes involved in iron uptake, including the FRE1 ferric reductase gene, the FTR1 ferrous permease, FET3, and CCC2 (17). We demonstrate here that ATX1 can also fall under Aft1p control; however, unlike other Aft1p-regulated genes, ATX1 does not show an absolute requirement for Aft1p. The bulk of ATX1 expression is unaffected by a null mutation in AFT1, and another trans-activator must be responsible. Our preliminary studies suggest that Atx1p also falls under the control of the oxygen-sensing trans-activator, Yap1p (44). The regulation of ATX1 by both iron status and oxidative stress would be consistent with the dual roles for copper-Atx1p in protection against oxygen radical toxicity and in the delivery of copper to Fet3p.
We are indebted to Dr. S. Michaelis for the
end3 mutant, to Dr. T. Dunn for strain 3, and to Dr. D. Kosman for the YPH250aft1 strain and for helpful discussions.