(Received for publication, June 20, 1995; and in revised form, September 26, 1995)
From the
The copper toxicity of yeast lacking the CUP1 metallothionein is suppressed by overexpression of the CRS4 gene. We now demonstrate that CRS4 is equivalent to SOD1, encoding copper/zinc superoxide dismutase (SOD). While overexpression of SOD1 enhanced copper resistance, a deletion of SOD1, but not SOD2 (encoding manganese SOD), conferred an increased sensitivity toward copper. This role of SOD1 in copper buffering appears unrelated to its superoxide scavenging activity, since the enzyme protected against copper toxicity in anaerobic as well as aerobic conditions. The distinct roles of SOD1 in copper and oxygen radical homeostasis could also be separated genetically: the pmr1, bsd2, and ATX1 genes that suppress oxygen toxicity in sod1 mutants failed to suppress the copper sensitivity of these cells. The Saccharomyces cerevisiae SOD1 gene is transcriptionally induced by copper and the ACE1 trans-activator, and we demonstrate here that this induction of SOD1 promotes protection against copper toxicity but is not needed for the SOD1-protection against oxygen free radicals. Collectively, these findings indicate that copper/zinc SOD functions in the homeostasis of copper via mechanisms distinct from superoxide scavenging.
The transition metal copper represents a paradox to living
organisms because trace amounts of this ion are essential to promote
growth, yet concentrated levels of this same metal can drastically
impair cell growth and function. Although many hypotheses have been
proposed to explain copper toxicity in biological systems, a popular
model exploits the ability of the metal to participate in so-called
Fenton or Haber-Weiss chemistry(1, 2, 3) . In
this model, copper ions would catalyze the conversion of hydrogen
peroxide (HO
) to the powerful oxidant hydroxyl
radical (OH
), which has the capacity to damage
cellular components. Alternatively, copper toxicity may also be
mediated through the inappropriate binding of the metal to nitrogen,
oxygen, and sulfur ligands in biomolecules, thereby inactivating
enzymes and disrupting cellular function(4) .
In order to balance the growth inhibitory and stimulatory effect of copper ions, all organisms have evolved with various metal homeostasis factors that properly control the cellular accumulation, distribution, and detoxification of the metal. A number of copper transporters have been identified that act in the passage of copper across external membranes or in the intracellular delivery of the metal to cellular stores. For example, copper-transporting ATPases have been identified in bacteria(5) , yeast (6, 7) and man(8, 9, 10, 11) . All of these ATPases share a remarkable degree of homology, exemplifying the need to tightly conserve the homeostasis of essential copper ions. Non-ATPase copper transporters have also been identified in bacteria (12) and yeast(13) , and these share a common metal binding motif.
As another means of maintaining copper ion
homeostasis, many eukaryotic organisms contain one or more forms of a
metal binding metallothionein that acts to chelate, sequester, and
thereby detoxify copper ions. In the bakers' yeast, the CUP1
copper-containing metallothionein(14, 15, 16) has been demonstrated to not only protect against copper
but also to guard against the oxidative damage associated with
superoxide anion (17) . This metallothionein of Saccharomyces cerevisiae, and another encoded by the CRS5 gene(18) , are known to be transcriptionally regulated by
copper ions through the action of a copper and DNA binding
trans-activator,
ACE1(18, 19, 20, 21, 22) .
Interestingly, the ACE1 copper sensor factor has been shown to also
induce the S. cerevisiae SOD1 gene in response to elevated
copper(23, 24) . SOD1 encodes a copper- and
zinc-containing superoxide dismutase (SOD), ()and the
rationale for the co-induction of this free radical detoxifying enzyme
with the metallothioneins was not completely
understood(23, 24) . Nevertheless, these studies on S. cerevisiae CUP1 metallothionein and SOD have substantiated
the notion that considerable overlap exists between systems controlling
copper ion homeostasis and oxygen radical metabolism.
To identify additional factors involved in copper ion homeostasis, we developed a selection strategy to isolate S. cerevisiae genes that, when overexpressed, have the ability to suppress the copper toxicity associated with loss of the CUP1 metallothionein. This strategy led to the isolation of two genes, CRS4 and CRS5, that, when present in multi-copy, conferred a relatively high degree of copper resistance to yeast lacking CUP1. In our earlier studies, we discovered that CRS5 encodes an additional yeast metallothionein, only the second to be identified in S. cerevisiae(18) . Expecting to uncover a third S. cerevisiae metallothionein, we have here sequenced and characterized the CRS4 gene. Surprisingly, CRS4 was found to be identical to the S. cerevisiae SOD1 gene, encoding copper/zinc superoxide dismutase. The biological role of this enzyme in copper buffering in yeast is discussed herein.
Stocks of all strains were
maintained on YPD medium, and tests for copper ion and paraquat
sensitivity were carried out using a synthetic dextrose medium as
described previously(30) . Anaerobic cultures were maintained
by growth in an O-depleted culture jar (BBL GasPak) as
described previously(26) .
Construction of the pVC731-736 series of SOD1 containing plasmids was as follows: sequences corresponding to SOD1 residues -257 to +586 with respect to the translational start site were amplified by the polymerase chain reaction (PCR). The upstream SOD1 primer (shown in Fig. 7A) was either wild type (for construction of pVC734 and pVC736) or contained a CGC to TAT substitution at position -227 (for pVC731 and pVC733). The PCR products were ligated to the pCRII vector (Invitrogen) to generate pCR16 (wild-type promoter) and pCR15 (mutant ACE1 site); the inserts were then mobilized by digestion with BamHI and XhoI and inserted into the BamHI and SalI sites of the CEN vector, pRS315, to generate pVC731 and pVC734 and also into the 2-µm vector, pRS415, to generate pVC733 and pVC736.
Figure 7:
Mutations in the ACE1 binding site of the SOD1 gene promoter. A, the -257 to -225
primer utilized to construct the pVC736 and pVC733 SOD1 plasmids is shown. Asterisks mark the CGC to TAT
substitution made in pVC733. Both plasmids were used to transform sod1 yeast, and where indicated, cultures of transformed
cells were treated for 1 h with 60 µM CuSO
prior to isolation of total mRNA and analysis by Northern blot.
The blot was sequentially hybridized to SOD1 and ACT1-specific probes. When normalized against ACT1,
the pVC736 and pVC733 mRNA exhibited a 3.9- and 1.2-fold induction upon
copper treatment. B, paraquat sensitivity in the indicated
yeast strains was measured as in Fig. 6A. % Control
Growth was obtained by dividing the A
value
obtained with paraquat treated yeast over that of control untreated
cells. C, sensitivity toward copper toxicity was measured in
the indicated strains as in Fig. 4. Strains utilized were as
follows: SOD+, EG103; sod1
, KS107; sod1
sod2
, EG133. pVC733 and pVC736 indicate strains transformed with these SOD1 containing
episomal plasmids. All strains contain an amplified CUP1 locus.
Figure 6:
Paraquat and copper toxicity in yeast
lacking SOD1 and ACE1. The indicated strains of yeast were cultured in
a minimal medium supplemented with the indicated concentrations of
either paraquat (A) or CuSO (B), and
total cell growth was measured as in Fig. 4Fig. 5. % Control Growth was obtained by dividing the A
value obtained with paraquat treated yeast over that of control
untreated cells. The strains utilized are as follows: SOD+, EG103; sod1
, KS105; SOD+ ace1
, VC104; sod1
ace1
, VC121. All strains
contain an amplified CUP1 locus; the ace1
strains exhibit a striking copper sensitivity due to the loss of CUP1 gene induction by copper.
Figure 4:
Effects of sod1 and sod2
gene deletions on tolerance to heavy metals. The
indicated strains of yeast were cultured as described in Fig. 2in minimal medium that was either untreated (control) or supplemented with 60 µM CuSO
(copper) or 30 µM CdCl
(cadmium). Total growth was measured as in Fig. 2, and
results represent the averages of two to four independent trials (the
complete range of values obtained are represented by error
bars). Strains tested are as follows: SOD+, EG103; sod1
, KS101; sod2
, EG110; sod1
sod2
, KS100. All strains contain an amplified CUP1 locus and normally exhibit a high level of copper
resistance.
Figure 5:
Suppressors of oxidative damage and the
effect on copper toxicity in sod1 yeast. The indicated
yeast strains were cultured in liquid minimal medium supplemented with
the indicated concentrations of CuSO
as in Fig. 4. % Control Growth was obtained by dividing the A
value obtained with copper-treated yeast over
that of control untreated cells. Strains of yeast utilized are as
follows: A, sod1
-pATX1, KS107 transformed with
the p18 construct harboring ATX1(40) ; SOD+-pATX1, EG103 transformed with p18; B, sod1
pmr1
, VC119; SOD+ pmr1
,
PJKP1-2; C, sod1
bsd2
, VC120; SOD+ bsd2
, XL103
b2. All strains contain an
amplified CUP1 locus. Results represent averages of two to
three independent trials where range
10%. In the various
experiments, control untreated cells grew to an A
of 2.5-4.0.
Figure 2:
Resistance to copper toxicity in yeast
transformed with the CRS4 gene. Growth of the indicated yeast
strains was tested in the presence of increasing concentrations of
CuSO. Liquid cultures seeded to an optical density at 600
nm of 0.1 were grown for 16 h at 30 °C, and total growth was
determined turbidimetrically at an optical density at 600 nm. Where
indicated, strains were transformed either with YEp13 vector
or with YEpCRS4, the p28 CRS4 containing plasmid. The
relevant genotypes of the yeast strains tested were as follows: A, 51.2c = cup1
::URA3; B, VC-sp6
= cup1
. Results represent the
averages of 2-3 independent trials with range
15%.
Construction of the sod1::LEU2 and sod1
::TRP1 gene replacement plasmids and deletion of the SOD1 chromosomal locus were performed as follows. A fragment
containing SOD1 sequences -703 to +18, with respect
to the translational start, was amplified by PCR and inserted into
vector pCRII (Invitrogen). This construct was digested with SacI and PstI to liberate the SOD1 sequences, which were then subcloned into the SacI and PstI sites of pRS305. Downstream SOD1 sequences
+314 to +586 (with respect to translational start) were
introduced at the HindIII and PstI sites of this
construct by inserting a HindIII-PstI fragment from
pVC16, generating the sod1
::LEU2 deletion construct,
pKS1. The sod1
::TRP1 deletion construct pKS3 was
constructed by subcloning the ApaI-SacI fragment from
pKS1 into the ApaI and SacI sites of pRS404. To
delete the chromosomal SOD1 gene, the pKS1 or pKS2 plasmid was
linearized with PstI and used to transform haploid leu2 or trp1 yeast, respectively, by electroporation. Correct
deletion of SOD1 sequences +18 to +314 was confirmed
using PCR.
Identification of CRS4 as SOD1 was
accomplished by sequencing both strands of p28hp and p28
sac
using the Sequenase method (U. S. Biochemical Corp.) modified for
double-stranded DNA. Sequence information was stored by the DNA Strider
software package and upon a search of the N.C.B.I data base, CRS4 was found to encompass the S. cerevisiae SOD1 and URA8 open reading frames. Northern and Southern blot analyses
were conducted as described previously(33) .
Figure 1:
Copper resistance in cup1 yeast overexpressing CRS4 and CRS5. The cup1
yeast 51.2C was transformed with the multi-copy
vector YEp13 (31) or with vector harboring either the CRS5 (YEpCRS5) or CRS4 (YEpCRS4) gene. Cells
were plated onto minimal medium supplemented with 25 µM CuSO
where indicated (+Cu) and were
grown for 2 days either in air (+OXYGEN) or in anaerobic
culture jars (-OXYGEN).
To examine the extent to which CRS4 protects cells from copper toxicity, a liquid culture test was
utilized to obtain measurements of the mean inhibitory concentration
for the metal (concentration required to inhibit total cell growth by
50%). On the YEp13 yeast episomal plasmid, CRS4 is amplified
to 5-10 copies/cell (Southern blot analyses, not shown), and in cup1 yeast, this elevation in CRS4 gene dosage
increased the mean inhibitory concentration for copper by approximately
400% (Fig. 2A). To test whether this increased
resistance to copper is specific to cup1
strains, we also
assayed for CRS4 function in a cup1
strain harboring a single copy of the metallothionein gene. As
shown in Fig. 2B, these strains are normally more
resistant to copper toxicity than are cup1
yeast, and
this resistance was increased further by overexpression of CRS4; however, the CRS4-containing plasmid only
increased the mean inhibitory concentration by approximately 35% in cup1
cells (Fig. 2B). The apparent
nonadditive effects of CRS4 and CUP1 suggested that
these two genes may play functionally redundant roles in copper
detoxification.
Figure 3:
Identification of the CRS4 gene.
Plasmid p28 was isolated from a yeast genomic YEp13 library as a clone
that confers copper resistance to cup1 yeast. The
remaining constructs represent fragments of the p28 insert subcloned
into the 2-µm yeast vectors YEp13, pRS423, or pRS425 as described
under ``Experimental Procedures.'' The constructs were used
to transform strain 51.2c, and all were found to support growth of this cup1
strain on minimal medium plates containing 25
µM CuSO
(as in Fig. 1). Positions and
directions of the SOD1 and URA8 open reading frames
are indicated. Arrows on the pVC736 fragment indicate primers
used for PCR amplification. RI, EcoRI; Sp, SphI; S1, SacI; H1, HpaI.
The S. cerevisiae SOD1 gene (35) encodes a copper and zinc containing SOD that is known to
scavenge superoxide anion (O) radicals
and is thought to play a critical role in protecting cells against
oxidative damage. Yeast strains containing mutations in the SOD1 gene exhibit a number of aerobic defects including a deficiency in
lysine biosynthesis and in sulfur metabolism and a sensitivity toward
atmospheric oxygen and paraquat, a
O
-generating
agent(25, 26, 35, 36, 37, 38, 39) .
All of these defects are dependent upon the presence of oxygen,
indicating that SOD plays a pivotal role in protecting cellular
constituents against oxygen-related toxicity.
To explore the role of SOD1 in copper buffering, we compared the growth of a sod1 mutant lacking the endogenous SOD1 gene to
that of a SOD1 wild-type strain. Resistance to copper was also
tested in a sod2
mutant lacking the mitochondrial
manganese containing SOD and a sod1
sod2
double
mutant strain lacking both forms of SOD. The strains utilized for these
studies also contain a wild-type tandemly amplified CUP1 locus
and are considerably more resistant to copper toxicity than are cup1
and cup1
yeast strains. As
shown in Fig. 4, the strains containing a sod1
single or sod1
sod2
double mutation exhibited
an increased inhibition of growth relative to wild-type yeast in 60
µM copper medium, and this increased sensitivity toward
copper was not observed with sod2
single mutants. In
comparison, strains containing a mutation in SOD2 exhibited an
elevated sensitivity toward 30 µM cadmium that was not
observed with sod1 mutants (Fig. 4). In other studies,
the sod1
and sod2
mutants consistently
exhibited impaired growth relative to wild-type strains at 30-90
µM CuSO
and 5-50 µM CdCl
, respectively (not shown). The increased copper
sensitivity of sod1
mutants was also observed with
anaerobically grown cells (not shown), consistent with our observation
that overexpression of SOD1 suppressed copper toxicity in both
aerobic and anaerobic conditions (Fig. 1). Thus the SOD1 protein
appears to play a role in copper buffering in addition to oxygen
radical scavenging.
As shown in Fig. 5A, sod1 mutants still exhibited a striking sensitivity
toward copper toxicity when the anti-oxidant gene ATX1 was
overexpressed. Furthermore, a pmr1 mutation was also incapable
of reversing the copper toxicity of sod1
cells, since the sod1
pmr1
mutant still exhibited impaired growth in
30-70 µM concentrations of copper compared with SOD1 wild-type yeast containing a pmr1
mutation (Fig. 5B). A mutation in BSD2 was likewise
ineffective in suppressing the copper sensitivity associated with loss
of SOD1 (Fig. 5C). BSD2 gene
mutations are themselves associated with an increase in sensitivity
toward copper(28) , and an additive effect on copper toxicity
was observed with the combination of bsd2 and sod1 mutations (Fig. 5C). Hence, perturbations in the
function of ATX1, PMR1, and BSD2 can
suppress only the oxidative defects and not the copper toxicity of sod1
mutants. These results provide genetic evidence that SOD1 plays separate physiological roles in copper ion
buffering and oxygen radical detoxification.
We also
tested the significance of ACE1 in SOD1 function by
inactivating the ACE1 binding site in the SOD1 promoter. A
pair of 2-µm-containing constructs were made that contain SOD1 sequences -257 to +586, with respect to the
translational start site. These constructs, pVC733 and pVC736, were
identical, except pVC733 contained a CGC to TAT triple mutation in the
ACE1 binding site of the SOD1 promoter (Fig. 7A;
Refs. 20, 21, 23, 24). Both constructs were used to transform a
sod1 strain and were monitored for copper induction by
Northern blot analysis. As shown in Fig. 7A, the two SOD1 constructs were expressed to equal degrees in the absence
of copper; however, only the pVC736 construct containing the wild-type
ACE1 binding site responded to copper through induction of SOD1 mRNA. To test whether this increase in SOD1 mRNA is
essential to its role in buffering oxygen radicals versus metals, sod1
strains harboring the pVC733 mutated,
and the pVC736 wild-type constructs were tested for sensitivity to both
oxygen and copper. Lysine auxotrophy is an excellent marker for oxygen
toxicity in yeast lacking copper/zinc
SOD(26, 35, 36, 38, 39) ,
and both pVC733 and pVC736 were fully capable of complementing the
lysine auxotrophy of sod1
cells (not shown). The
wild-type and mutant constructs were also equally effective in
complementing the paraquat sensitivity of sod1
yeast (Fig. 7B). Hence, the ACE1 binding site does not appear
to impact on the ability of SOD1 to protect against oxidative
damage. However, in contrast to results obtained with paraquat (Fig. 7B), the pVC736 wild-type construct was
substantially more effective in complementing the copper sensitivity of sod1
yeast than was the pVC733 mutant construct, and this
result was observed in the background of both sod1
and sod1
sod2
yeast (Fig. 7C). Identical
experiments were conducted with the same SOD1 wild-type and
mutant constructs expressed on a yeast CEN vector, and
although the CEN constructs were less efficient at
complementing the sod1
mutation, the same qualitative
results were obtained; the wild-type SOD1 construct was
considerably more effective in suppressing copper toxicity (data not
shown). Together with the studies on ace1
yeast (Fig. 6), these findings strongly indicate that the role of SOD1 in copper buffering, but not in oxygen radical
detoxification, is dependent upon the ACE1 trans-activator and a
functional ACE1 binding site in the SOD1 promoter.
The studies presented here strongly suggest that copper/zinc
SOD serves a physiological role in copper ion buffering. We demonstrate
that the S. cerevisiae SOD1 gene can act as a multicopy
suppressor of copper toxicity. SOD1 was only one of two loci
found to have the capacity to suppress the strong copper sensitivity
associated with loss of the S. cerevisiae CUP1 metallothionein. The other, CRS5, encodes an additional
metallothionein(18) . SOD1 not only protects against
copper toxicity as a multicopy locus, but the single copy chromosomal
gene also participates in copper buffering since a sod1 gene deletion was associated with an increased sensitivity toward
copper. Of the two forms of SOD, copper buffering appears specific to
the copper/zinc containing enzyme; a null mutation in the SOD2 gene encoding the mitochondrial manganese SOD was not associated
with an increased sensitivity toward copper.
We provide several
lines of evidence that the apparent role of copper/zinc SOD in copper
buffering is distinct and perhaps unrelated to the well established
role of this enzyme in free radical detoxification. First, the
previously reported defects associated with loss of S. cerevisiae
SOD1 (aerobic growth inhibition, methionine and lysine auxotrophy,
increased mutation rate) were absolutely dependent on the presence of
oxygen(25, 28, 35, 36, 37, 38, 39) .
In contrast, the copper sensitivity associated with sod1 mutants was observed independent of atmospheric oxygen. The roles
of SOD1 in copper and oxygen radical homeostasis can also be
separated at the genetic level. A mutation in the PMR1 or BSD2 genes or overexpression of ATX1 will suppress
the oxygen-related defects associated with loss of SOD1 function(26, 27, 28, 40) ;
however, these same genetic perturbations failed to overcome the
increased copper sensitivity of sod1
mutants. We
initially found this result surprising, since all three of these
suppressors are themselves involved in copper homeostasis. PMR1 encodes a P-type ATPase homologue that is believed to function in
the delivery of manganese, copper, and calcium ions into the
Golgi(27) ; BSD2 is involved in the homeostasis of
copper and cadmium(28) , and ATX1 appears to encode a
small copper carrier(40) . Evidently these suppressors increase
the local concentration and availability of redox active transition
metals but do not offer any metal buffering function.
It has been
proposed that SOD can circumvent the Haber-Weiss reaction (the two-step
copper or iron catalyzed production of OH) by
scavenging the O
that would normally
provide the reducing equivalents for the transition
metal(1, 2, 3, 41) . However,
O
scavenging cannot account for the
protective effect of SOD1 with respect to copper ions since SOD1 protects against copper toxicity under anaerobic conditions.
Instead, we propose that the copper resistance associated with SOD1 gene dosage reflects the ability of the encoded enzyme to directly
bind and sequester the metal and thereby help prevent the dysfunctional
binding of copper to biological metal ligands. In this model, SOD1 has the potential to function as a metallothionein, as previously
proposed by Ciriolo et al.(42) . Fee and co-workers (43) have proposed that bacterial forms of SOD can similarly
function in iron metabolism.
In the budding yeast, several lines of
evidence suggest functional relatedness between the metallothioneins
and copper/zinc SOD. Not only does SOD1 suppress
metallothionein-related defects (this study), but the converse holds
true; overexpression of CUP1 metallothionein will suppress
defects associated with loss of SOD1(17) .
Furthermore, SOD1 and the CUP1 and CRS5 metallothioneins are coordinantly induced at the transcriptional
level by copper ions and the copper trans-activator
ACE1(18, 19, 20, 23, 24) .
Originally, the rationale for copper induction of SOD1 was not
clear, as the gene is constitutively expressed at a relatively high
level and ACE1 only serves to increase this expression by a few times.
We demonstrate here that the copper induction of SOD1 by ACE1
is not essential to the anti-oxidant role of the enzyme. SOD1 still protects against oxidative damage in yeast containing an ace1 deletion and with SOD1 genes lacking a
functional ACE1 binding site. In contrast, the functional ACE1 factor
and ACE1 binding sequences were found to be necessary for the
protection against copper toxicity afforded by SOD1. We
therefore propose that the constitutive level of copper/zinc SOD is
sufficient to protect cells against oxidative damage by efficiently
scavenging O
. However, when exposed to
toxic copper levels, the cell responds by further increasing SOD1
levels. This elevation in SOD1, together with the induction of the
metallothioneins, provides an effective method for metal buffering.
It is noteworthy that mutations in the human SOD1 gene have
been associated with a familial form of amyotrophic lateral sclerosis
(FALS), a fatal neurodegenerative disorder affecting the motor neurons (44) . All FALS mutations are dominant, and in certain
instances, the disease cannot be explained simply by a loss of SOD1
O scavenging
activity(45, 46, 47, 48, 49, 50) .
The apparent role of copper/zinc SOD1 in copper buffering may in fact
have important implications with regard to the etiology of
FALS(47) .