(Received for publication, June 3, 1994; and in revised form, September 21, 1994)
From the
The yeast Saccharomyces cerevisiae contains a plasma
membrane reductase activity associated with the gene product of the FRE1 locus. This reductase is required for Fe(III) uptake by
this yeast; transcription from FRE1 is repressed by iron
(Dancis, A., Klausner, R. D., Hinnebusch, A. G., and Barriocanal, J.
G.(1990) Mol. Cell. Biol. 10, 2294-2301). We
show here that Cu(II) is equally efficient at repressing FRE1 transcription and is an excellent substrate for the Fre1p
reductase. This reductase activity is required for 50-70% of the
uptake of Cu by wild type cells. Under conditions of low
Fre1-dependent activity, cells retain 30-70% of Cu(II) reductase
activity but only 8-25% of Fe(III) reductase activity. While
Fre1p-dependent activity is 100% inhibitable by Pt(II), this residual
Cu(II) reduction is insensitive to this inhibitor. The data suggest the
presence of a Fre1p-independent reductase activity in the yeast plasma
membrane which is relatively specific for Cu(II) and which supports
copper uptake in the absence of FRE1 expression. The gene
product of MAC1, which is required for regulation of FRE1 transcription, is also required for expression of Cu(II) reduction
activity. This is due in part to its role in the regulation of FRE1; however, it is required for expression of the putative
Cu(II) reductase, as well. Similarly, a gain-of-function mutation, MAC1
, which causes elevated and
unregulated transcription from FRE1 and elevated Fe(III)
reduction and
Fe uptake exhibits a similar phenotype with
respect to Cu(II) reduction and
Cu uptake. Ascorbate,
which reduces periplasmic Cu(II) to Cu(I), suppresses the dependence of
Cu uptake on plasma membrane reductase activity as is the
case for ascorbate-supported
Fe uptake. The close
parallels between Cu(II) and Fe(III) reduction, and
Cu and
Fe uptake, strongly suggest that Cu(II) uptake by yeast
involves a Cu(I) intermediate. This results in the reductive
mobilization of the copper from periplasmic chelating agents, making
the free ion available for translocation across the plasma membrane.
Limited detail is available about the mechanism(s) by which the
essential trace nutrient copper is taken up by eukaryotic cells.
Although all such cells studied in culture exhibit kinetically
saturable, that is, facilitated, uptake (1, 2, 3) only in the budding yeast Saccharomyces cerevisiae has a specific copper transport
function been identified. This appears to be due to the product of CTR1(4) . Significant features of the primary
structure of this protein include two to three membrane-spanning
domains and 11 repeats of the Met-X-Met sequence found also
in bacterial CopA and CopB proteins. These latter proteins have known
copper-handling functions(5, 6) .
In contrast, the
uptake of iron by yeast is much better understood in molecular terms.
High affinity accumulation of Fe(III) from the medium requires, first,
a reduction of extracellular Fe(III) to Fe(II) (7, 8, 9, 10) . This reduction is
catalyzed by a plasma membrane reductase
activity(7, 8, 9, 10, 11) .
The Fe(III) reductase activity requires, at the least, the product of
the FRE1 locus(9, 10) . The Fre1 protein is
homologous to the cytochrome b component of the
O
-generating respiratory burst oxidase found in
the plasma membrane of neutrophils(12) . Since this cytochrome
catalyzes the one-electron reduction of O
, it is likely
that Fre1p in yeast is directly responsible for catalyzing the
one-electron reduction of Fe(III). Deletion mutants in FRE1 can efficiently take up Fe(II), but not Fe(III), from the
medium(9, 10, 13) . This result indicates
that Fe(II) is the substrate for an iron transporter. Additional
evidence suggests that once inside the cell, the Fe(II) may be
reoxidized to Fe(III) in a reaction involving the product of the FET3 gene. This model of iron retention is supported by the
fact that a FET3 deletion mutant does not accumulate
iron(14) .
Transcription from FRE1 is strongly
repressed by iron in the medium(9, 10) . The
transcriptional activation of FRE1 upon iron withdrawal
requires, at the least, the product of the MAC1 gene(15) . Mac1p is a nuclear protein which contains in
its primary sequence a number of apparent metal-binding elements. While
a recessive, loss-of-function frameshift mutant, mac1-1,
fails to transcribe FRE1, a gain-of-function allele,
designated MAC1, is associated with
elevated and iron-independent transcription from this
locus(15) . This allele contains a His
Gln
substitution in one of the potential metal-binding motifs in Mac1p
suggesting that this region is functionally important. Strains carrying
this allele, which we show here is co-dominant with wild type, exhibit
strongly elevated reductase activity.
While Fe(III) reductase
activity in iron-repressed wild type cells, or in fre1 or mac1-1 mutant strains, is strongly depressed, it is not
absent(9, 10, 15) . This indicates the
likelihood that there are Fre1p-independent reduction reactions
catalyzed by one or more proteins of the yeast plasma membrane. Indeed,
yeast plasma membrane reductase activities have been well
established(11, 16, 17, 18) .
Although Cu(II) also appears to be a substrate for Fre1p, in cells
strongly repressed in Fre1p activity, there is significantly more
residual Cu(II) versus Fe(III) reductase
activity(15) . Thus, a Fre1p-independent, Cu(II) reductase
activity could be present in yeast. Using both genetic and biochemical
approaches, in this paper we test the hypothesis that S. cerevisiae possesses a Cu(II)-specific reductase activity which supplies
Cu(I) to the putative copper transporter, Ctr1p.
This general conclusion is demonstrated
also by the spore analysis shown in Fig. 1. On the plates shown
are sets of tetrads from crosses between wild type (F113) and fre1-containing (F
Fe) strains and a MAC1
-containing mutant. The YPD plates on which
the spores were grown were overlaid with TTC. The elevated reductase
activity in MAC1
FRE1 colonies is
clearly demonstrated by the immediate (2-4 min) reduction of the
TTC to a dark red, insoluble formazan. The spores from the wild type
(F113)
MAC1
cross show the expected 2:2
segregation of this phenotype (left plate, Fig. 1). In
contrast, the spores from the fre1
(F
Fe)
MAC1
cross show a characteristic
tetratype segregation in which non-parental ditypes (e.g., fre1
MAC1
) lack color because they lacked
Fre1p (independently scored by the URA3 marker used to disrupt FRE1) (right plate, Fig. 1). Note that the
plates contained 50 µM CuSO
which suppressed
mitochondrial reduction of the TTC. This TTC overlay is commonly used
as a screen for mitochondrial function in yeast(25) .
Figure 1:
Segregation of MAC1 reductase activity and suppression
by the fre1
allele. Strain DF5 up1 (MAC1
FRE1) was crossed with
strains F113 (wild type, MAC1
FRE1) and F
FE (MAC1 fre1
), the two diploids sporulated and the
resulting tetrads dissected. The spores here were grown on YPD plates
containing 50 µM CuSO
which were subsequently
overlaid with a TTC-containing agar solution at 30 °C (see
``Experimental Procedures''). The presence of the MAC1
FRE1 genotype is
indicated by the bright red formazan which precipitates in the culture
within 2-4 min (seen as a dark colony in the
photograph). The MAC1
fre1
genotype remains white due to the suppression of MAC1
by fre1
.
A
positive role for Mac1p in FRE1 expression is indicated by the
fact that the MAC1 allele is dominant or
co-dominant with wild type. While reflected in all of the phenotypes
associated with this gain-of-function mutation(15) , this
dominance is illustrated in Fig. 2by the relative copper
sensitivity of homozygous MAC1 and MAC1
and heterozygous MAC1 MAC1
diploids. MAC1
homozygotes are growth arrested between 200
and 300 µM CuSO
in synthetic media, or about
6-fold more copper sensitive than wild type, while heterozygotes are
about 4-fold more sensitive in the background used for this comparison.
The MAC1
allele is suppressed by over-expression
of wild type MAC1, however(15) .
Figure 2:
The MAC1 allele is co-dominant with wild type. The MAC1
allele is scored here as
sensitivity to Cu
. Strain DF5 up1 (MAT
MAC1
) was crossed with strains BCup1a (MATa MAC1
) and wild type S144 (MATa MAC1), while S144 was crossed with parental
wild type strain DF5
(MAT
MAC1). Fresh
colonies of the resulting three diploids, one heterozygous and two
homozygous at the MAC1 locus, were streaked on YPD plates
containing the concentration of CuSO
indicated. The
photograph was taken following 48 h of growth at 30
°C.
Figure 3:
Repression of FRE1 transcription
by iron and copper. Total RNA was isolated from log-phase cultures of
strain DF5 grown in metal-free, defined media containing the
additions as noted in the figure ([FeCl
],
[CuCl
] = 40 µM when added).
Total RNA (20 µg/lane) was size-fractionated by electrophoresis in
a 1% formaldehyde-agarose gel, capillary transferred to Immobilon, and
probed with
P-labeled random-primed fragments of FRE1, and of ACT1 (actin) as control. The photograph
is of a film exposed for 14 h and is overexposed with respect to ACT1 mRNA in order to illustrate the relative levels of the FRE1 transcript in the four conditions. As determined by
densitometric analysis (LKB Ultrascan XL Laser Densitometer), the
relative levels of FRE1 mRNA in the four samples were (left to
right) 13, 1, 1.5 and 1.8, respectively.
Iron uptake is known to require
reduction of Fe(III)(9, 10, 13) . One can
make two predictions about Fe(III) uptake with respect to
the effect of citrate. The same predictions would apply for Cu(II) if
Cu(II) uptake is similarly reduction-dependent. First, in
wild type, citrate (and lactate, data not shown) would inhibit uptake
of both
Fe(III) and
Cu(II) because the
reduction step would be suppressed by citrate chelation of the higher
valence state. The data in Table 3confirm this prediction
(compare left two columns to right two). The second prediction is that
Fe(III) and
Cu(II) uptake would be less dependent on cell-based reduction (whether Fre1p-dependent or
independent) in the absence of citrate than in its presence. This is
observed also (e.g., compare wild type to FRE1 deleted strain, fre1
, first two rows) suggesting
that in the absence of stabilization of the higher valency Cu(II) and
Fe(III) states by citrate, nonspecific reduction can support uptake.
The uptake data in Table 3will be discussed in detail below.
In Table 2, with respect to metal reduction: 1) in wild type, iron (40 µM) in the growth medium reduces Fe(III) reduction from 520 ± 35 to 40 ± 3 nmol/min/mg protein (92% repression; range, 91-93%, n = 4). In contrast, Cu(II) reduction is reduced from 7.1 ± 0.6 to 2.2 ± 0.2 nmol/min/mg protein (69% repression; range, 63-74%, n = 4).
2) Deletion of FRE1 results in a loss of 75% of Fe(III) reductase activity but only 30% of Cu(II) reductase activity. Similarly, the mac1-1 mutant exhibits a loss of 88% Fe(III), but only 69% of Cu(II) reductase activity (ranges: 85-91 versus 65-73% of wild type activity, n = 5).
3) Under iron-derepressing conditions (Fre1p
activity high), Pt(II) inhibits Fe(III) and Cu(II) reduction, although
Cu(II) reduction is slightly less sensitive (at p < 0.1).
Pt(II) is known to inhibit Fe(III) reduction by Frelp, and Fe(III)
uptake, but not Fe(II) uptake(13) . In contrast, under iron
repressing conditions, or in an equivalent condition of low Fre1p-based
Fe(III) reductase activity (e.g., in fre1 and mac1-1 strains), Cu(II) reduction is either insensitive
to inhibition by Pt(II) or is only slightly inhibited. Under these
conditions any residual Fe(III) reduction is 100% inhibited by Pt(II).
These data consistently suggest that S. cerevisiae contains a Cu(II)-specific reductase which is not (completely) repressible by iron. Furthermore, this reductase is distinguishable from the Fre1p-dependent reductase by its insensitivity to Pt(II) inhibition. Lastly, copper in the growth medium is equivalent to iron in repressing Fre1p reductase activity. This equivalence is consistent with the similar effects of copper and iron on FRE1 transcription (Fig. 3).
In Table 3, with respect to metal uptake: 1)
in general, iron in the growth medium has a significantly weaker
repressing effect on Cu versus
Fe
uptake, irrespective of whether uptake is measured in the absence or
presence of citrate. However, as the complement to 3) above, the
largest difference in this relative repression is under the condition
of overall suppression of metal reduction, i.e., in the
presence of citrate. Thus, when a metal is easily reducible (minus citrate) the role of a specific reductase in metal uptake is
diminished. When metal reduction is made more difficult by
stabilization of the higher valence state, the role of a specific
reductase is magnified. Conversely, when metal reductase activity is
high, as in the MAC1
mutant, the effect of
citrate is diminished; the elevated reductase activity provides a
``leveling'' effect on the thermodynamic consequence of
citrate chelation of the metal ion. This is similar to the conclusion
suggested by the data in Table 1as noted above.
2) There is
one significant exception to this pattern, namely, that in
iron-repressed MAC1 mutant,
Fe
uptake in citrate is strongly repressed. The data in Table 2show that Fe(III) reduction is not repressed in this
strain under this condition. Therefore, this result confirms a previous
conclusion that Fe(III) reduction and
Fe uptake are
differentially regulated by iron (13) and indicates also that
Mac1p is not associated with regulation of iron uptake.
Whether the repression by iron of uptake in this strain is due to
repression of transport activity per se, or of the
Fet3p-dependent activity required for iron accumulation (14) is
not known.
3) Mac1p positively regulates Cu uptake as
it does
Fe uptake, but, in contrast to the latter process,
its role in
Cu uptake is not iron-dependent. The first
conclusion is indicated by the differences in
Cu uptake by
wild type, mac1-1, and MAC1
strains (cf., last two columns, bottom of Table 3). The
second conclusion is shown particularly by the lack of significant
repression by iron of the
Cu uptake by the mac1-1 and MAC1
strains. The
former strain has none of the known iron-repressible, Fre1p-dependent
Cu(II) reductase activity while the latter strain has an elevated and
iron-independent level of this activity (Table 2). In both
strains, iron strongly represses
Fe uptake (last two
columns, top of Table 3) while it has little effect on
Cu uptake (last two columns, bottom of Table 3).
Metal ions like Cu(II/I) and Fe(III/II) are commonly taken
into the yeast cell as ``free'' ions, uncomplexed to any
potential ligand present in the
periplasm(3, 27, 28, 29) . How the
ligand displacement associated with this first step of metal ion uptake
is catalyzed is a fundamental part of the uptake mechanism. The
coordination chemistry of Fe(II/III) and Cu(I/II) offers clues to the
mechanism of this displacement step. First, the lower valency states of
many transition metal ions are often more exchange labile. For example,
the water exchange rate at Fe(III) is 3 10
s
, while at Fe(II) it is 3
10
s
(30) . Second, formation constants
for Fe(III) and Cu(II) are most often altered strongly by reduction of
the metal ion, e.g., the formation constant for the citrate
complex of Fe(III) is 10
times that for the Fe(II)
complex(7) . Thus, the one-electron reduction of Fe(III) or
Cu(II) would strongly facilitate the displacement of the ligand to
which the metal ion was bound in the medium, making the metal available
for transport across the plasma membrane. The reductive exchange of
copper into ceruloplasmin (31) and the reductive mobilization
of copper from this protein (32) appear to illustrate this
suggestion.
This model of reductive mobilization of Fe(III) and
Cu(II) from periplasmic chelating agents can be related to geologic
aerobiosis in which the more soluble and exchange labile Fe(II) and
Cu(I) in the pre-biotic O-free environment were converted
to Fe(III) and Cu(II). At neutral pH, these two higher valency ions
readily undergo hydrolysis to essentially insoluble
hydroxides(30) . In essence, reduction of the metal ion
increased metal bioavailability. A complementary adaptation was the
biosynthesis and secretion of siderophores(33) . Some yeasts
may employ both means to accumulate Fe(III)(34) .
Biochemical evidence supports the hypothesis that Fe(III) uptake in
yeast involves a reduction step. In particular, iron in the medium
regulates the amount of the Fe(III) reductase activity, which is
induced in iron-deficient
conditions(7, 8, 9, 10, 13) ;
Fe(II) uptake has been shown to be independent of this
activity(9, 13) . Also, inhibition of reductase by
Pt(II) has an inhibitory effect on Fe(III) uptake without altering
Fe(II) uptake, a result which also indicates the primary role of
Fe(III) reduction in uptake(13) . Lastly, Fe(III) uptake was
shown to be independent of the stability constant of the Fe(III)
complex used as substrate for uptake, but was shown to be inhibited by
a chelator with high affinity for Fe(II) such as ferrozine or
BPS(7, 13) . Genetic evidence has directly implicated
Fe(III) reduction as the initial event of Fe(III) uptake by yeast.
Thus, fre1 strains take up Fe(III) very poorly while they
accumulate Fe(II) with rates equivalent to wild
type(9, 13) . In summary, the biochemical and genetic
data provide strong evidence for the reduction dependence of
high-affinity uptake of ``free'' Fe(III) by S.
cerevisiae.
The reductase activity in the plasma membrane of S. cerevisiae was first associated with a family of
transmembrane NADH dehydrogenases using ferricyanide as
substrate(16) . Indeed, the extensive survey of substrates
performed here using both wild type and Fre1pminus strains
establishes that, at the least, the Fre1p reductase is very
nonspecific. In particular, our data show that Cu(II) is an excellent
substrate for this reductase and show further that about 50-70%
of Cu uptake in FRE1-derepressed, wild type cells
(in the absence of ascorbate) is supported by Fre1p-dependent activity.
Previous studies indicated that copper accumulation by the yeast Debaryomyces hansenni might involve metal reduction(17, 18) . The fact that ascorbate enhances copper transport from ceruloplasmin into human K562 cells suggests that reductive accumulation of copper is a general feature of copper uptake by eukaryotes(35) . In fact, the biochemical and genetic results presented here suggest that Cu(II) uptake by S. cerevisiae follows a mechanistic path which may have some components in common with the pathway for Fe(III) accumulation, that is, the data support the general conclusion that Cu(II) uptake can depend on Fre1p.
While any of the activities seen with Cu(II) in regard to FRE1 and its gene product could be gratuitous, this does not make biologic sense. The functions of trace metals, like those of the vitamins, are diverse and are specific to the metal ion. Thus, the requirement for any one metal is quite distinct from that for another and, given this fact, the regulation of the metabolism of one metal can be expected to be quite separate also(27) . The fact that copper, along with iron, regulates FRE1 mRNA levels suggests a strong physiologic link between these two metals in S. cerevisiae, perhaps one related to the proposed model of Fe(II) retention within the yeast cell. In this model, iron retention requires the reoxidation of the Fe(II) taken into the cell to Fe(III) in a reaction catalyzed by the Fet3 protein(14) . Sequence homology indicates that Fet3p is strongly related to the family of multi-copper oxidases, a family which includes ceruloplasmin. Data have shown that a fet3-containing mutant, or a copper-deficient wild type, fails to accumulate iron whether present in the medium as Fe(II) or Fe(III). Thus, copper deficiency leads to iron deficiency. Intriguingly, a similar linked deficiency has been observed in mammals, perhaps due to a reduced activity of ceruloplasmin(36, 37) .
An
important experimental observation described here is that reduction of
a metal ion by a specific cell activity can be obscured if the
metal ion is not stabilized in its higher valence state by chelation in
the periplasm, that is, failing to stabilize Cu(II) by chelation by
citrate makes all cell and/or medium reductants equal. Consequently, in
the absence of such stabilization, Cu uptake becomes
relatively insensitive to changes in cell reductase activity (cf. Table 3to Table 2). Previous Cu(II) uptake
studies in this laboratory (3, 27) and others (2) which did not stabilize Cu(II) thus failed to detect
changes in cell reductase activity as reflected in changes in uptake
rate.
The role of Mac1p in the regulation of putative Cu(II)
reduction and Cu transport closely parallels its apparent
role in iron metabolism. That is, the data in Table 4indicate
that while ascorbate-supported
Cu uptake is relatively
independent of Mac1p (transporter activity), Pt(II)-inhibited
Cu uptake was not (Cu(II) reductase activity dependent on
Fre1p). Similarly, while the data show that Mac1p plays a role in the
regulation of Fre1-dependent activity ( Fig. 3and Table 2), it does not appear to regulate the activity of the
putative Fe(II) transporter or any cell activity associated with Fe(II)
accumulation. This latter conclusion is based on the fact that in a Mac1
strain, iron in the medium, which in this
mutant does not repress FRE1 transcription (15) or
Fre1p activity (Table 2), represses
Fe uptake (Table 3). Thus, Fe(III) reduction is strongly catalyzed in this
strain under this condition, but Fe(II) uptake/accumulation is strongly
repressed. This difference could be due to a Mac1-independent
inactivation of an Fe(II) transporter by iron. Alternatively, it could
be due to inactivation of the FET3 locus by iron(14) .
As described above, Fet3p is essential for accumulation of iron by
yeast. In this model, FET3 transcriptional regulation by iron,
unlike iron regulation of FRE1, would be Mac1p-independent.
Biologically, this delineation of Mac1p function is consistent with the
conclusion that while in yeast Fre1p plays a role in the metabolism of
both Fe(III) and Cu(II) as suggested above, the role of Fet3p is
specific to iron metabolism in this organism(14) .
This work
does not address the mechanism of Mac1p function explicitly. However,
the dominant phenotypes associated with MAC1 do
suggest that Mac1p functions within a complex with other cellular
components. Since Mac1p is consistently associated with activation of
gene expression by some change in cellular nutrient or stress
condition(15) , it most likely acts as a positive regulatory
element. In this model, Mac1p
is constitutively
activated with respect to transcriptional regulation at FRE1 and is suppressed (competed out) only by over-expression of wild
type Mac1p (15) . Consistent with a model in which Mac1p is a
constituent of a macromolecular complex is the presence in Mac1p of a
consensus zinc finger or CCHC box(38) :
YXCX
CX
HX
C. Preliminary studies (15) have failed to detect any DNA binding activity for Mac1p,
however, indicating that if this structure does represent a zinc
finger, it may be involved in a protein-protein interaction in a
hetero-oligomeric complex (which itself might bind to DNA). The
mechanism of action of Mac1p in the regulation of metal metabolism, and
in other stress responses(15) , in S. cerevisiae is
the subject of continuing investigation.