©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for Cu(II) Reduction as a Component of Copper Uptake by Saccharomyces cerevisiae(*)

(Received for publication, June 3, 1994; and in revised form, September 21, 1994)

Richard Hassett Daniel J. Kosman (§)

From the Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo, New York 14214

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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(2)-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(2)-generating respiratory burst oxidase found in the plasma membrane of neutrophils(12) . Since this cytochrome catalyzes the one-electron reduction of O(2), 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 fre1Delta 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.


EXPERIMENTAL PROCEDURES

Materials

Chemicals

Standard media components were from Difco. The reagents for preparing the completely defined synthetic medium used for growth of copper-free cultures and the MES (^1)for buffering the uptake medium were AnalaR or AristaR grade chemicals (British Drug House) purchased from Gallard-Schlesinger. Media supplements and reagents for the reductase assays were purchased from Sigma. Potassium tetrachloroplatinate was purchased from Johnson Matthey (Ward Hill, MA). Cu(NO(3))(2) was provided by the Buffalo Materials Research Center, SUNY at Buffalo. FeCl(3) was obtained from ICN. Solutions were prepared in doubly deionized, doubly (glass) distilled water.

Medium for Cell Growth

Yeast stocks were maintained on yeast extract (1%)-bacto-peptone (2%)-dextrose (2%) medium (YPD). For most experiments cultures were grown in a synthetic, complete medium prepared without sulfate (2, 19) from which iron and copper were extracted using dithizone (20) prior to the addition of the remaining trace metals. This medium contained approximately 15 nM copper and 170 nM iron as determined by flameless atomic absorption spectrophotometry and is referred to as metal-free medium in the text. As noted, bathophenanthroline sulfonate (BPS, 50 µM) was included also to further limit the bioavailability of residual iron and copper. BPS strongly inhibits the uptake of both metals. Fe(III) and Cu(II) were added as the chlorides as indicated; BPS was omitted in these cases. The carbon source in this medium was glucose (2%, w/v).

Yeast Strains and Genotypes

The strains used were: DH5alpha (MATalpha lys2-801 leu2-3, 112 ura3-52 hisDelta200 trp1-1), DH5fs (DH5alpha mac1-1), DH5 up1 (DH5alpha MAC1), F113 (MATaMAC1 FRE1 ura3-52 ino1-1), and FDeltaFe (F113 fre1::URA3). The allele, mac1-1, is a frameshift mutation (a G deletion at nucleotide 127) while the MAC1 allele contains a single T-A transversion which results in an His-Gln substitution in Mac1p. Both alleles were introduced by homologous recombination at the MAC1 locus in DH5alpha to yield the respective mutant strains (15) . F113 and FDeltaFe (9, 10) were obtained from A. Dances (NIH). Two additional strains were used for the test of dominance of the MAC1 allele: S144 (MATamet3 leu2-1 MAC1, obtained from the Yeast Genetics Stock Center, Berkeley, CA) and BCup1a (MATaMAC1 S144). The latter strain resulted from an initial cross of the MAC1 allele into S144, selecting for the mutant genotype (and MATa), followed by two additional backcrosses into S144. Except as noted, all strains exhibited a genotype which included FRE1 CUP1 ACE1 as determined by Northern analysis of FRE1 and CUP1 mRNA. Copper-induced CUP1 transcription requires ACE1.

Methods

Cell Growth

Cell growth was monitored by turbidity at 660 nm. Liquid stocks were prepared using as inocula washed single colonies taken from YPD plates. These stocks were prepared in the copper-free, synthetic medium. Growth of experimental cultures was initiated at A = 0.05 or less; the cultures were allowed to grow to A = 0.8 to 1.2 (log phase, 1.4-2.0 times 10^7 cells/ml) prior to use.

Cu and Fe Uptake Protocols

Cells for uptake experiments were washed with uptake buffer (five times culture volume) and resuspended in the uptake buffer at the A of 1.0 (1.6 ± 0.1 times 10^7 cells/ml). This buffer contained 0.2 M MES and glucose (2%, w/v), pH 6.0. Citrate (20 mM) was included also as indicated. This buffer contained no detectable copper or iron. Following a 15-min equilibration in this buffer at 30 °C, CuNO(3) (10 µM) or FeCl(3) (2 µM) was added to initiate uptake. Uptake of copper was observed to be linear for at least 20 min and for iron for at least 40 min; initial (t = 15 s), nonspecific periplasmic binding of either metal was shown to be statistically insignificant. Based on these observations, the uptake data reported are based on a single 10 min (Cu) or 30 min (Fe) time point sampled in triplicate in all experiments. Uptake was followed by quenching samples (1 ml) in 5 ml of an ice-cold quench solution of 10 mM EDTA in 0.1 M Tris succinate (pH 6.0). These cell samples were collected by suction though nitrocellulose filters (0.45 µm, Millipore) which were presoaked in the quench solution; the retained cells were washed with a total of 20 ml of quench solution. The filters were allowed to air dry and then were counted in an LKB CompuGamma counter. Counts were corrected for background measured by counting blank filter, efficiency, and decay to yield nanograms of metal retained.

Northern Analysis

Total RNA was isolated using glass beads in the presence of 1% SDS and the standard mixture of chloroform/phenol(21) . Following two ethanol precipitations, the RNA was stored in H(2)O at -70 °C. Northern blots were obtained by capillary transfer from 1% agarose gels using Immobilon-N (Millipore). Filter strips were hybridized at 42 °C with P-labeled probe (10^7 counts/min) for 20 h; the final wash was carried out at 50-55 °C in the presence of 2% SDS in 2 times SSC. Probes were prepared by random priming (Boehringer) using as templates restriction fragments internal to the coding regions of ACT1 (actin, control) and FRE1 (iron reductase component). Autoradiographic films were quantitated by scanning laser densitometry using an LKB UltraScan instrument. Linearity of response was ensured by comparison of peak heights and areas with amount of total RNA applied or exposure time.

Analytical Methods

Atomic absorption analyses were performed on a Perkin Elmer model 1100B spectrophotometer equipped with a model 700 graphite furnace and an AS-70 auto-sampler. Total cell protein was determined on all cell samples analyzed for Cu or Fe accumulation or metal reductase activities (22) while protein in soluble extracts was determined by BCA (bicinchoninic acid) assay (Pierce) using bovine serum albumin as standard(23) . Reductase activities in whole cells were determined as described (8) using the citrate-containing uptake buffer in all cases. Reduction of the tetrazolium salts dimethylthiazolyldiphenyltetrazolium bromide, iodophenylnitrophenylphenyltetrazolium chloride, and triphenyltetrazolium chloride (TTC), respectively, was followed spectrophotometrically as described(24) . Reduction of paraquat was followed by coupling the resulting paraquat free radical to the reduction of ferricytochrome c (followed at 550 nm) via the superoxide radical. A plate assay for the MAC1 allele was developed which took advantage of the enhanced reductase activity associated with this allele. In this screen, a TTC-containing overlay (25) was poured on fresh colonies grown on YPD plates which contained CuCl(2) (50 µM). Cells carrying the MAC1 allele were indicated by their fast (2-4 min) pink-red color development. In the presence of the copper, wild type colonies remained white. mac1-1-carrying strains were screened by their inability to grow on YPG (glycerol) plates unless CuCl(2) (50 µM) was included(15) . Statistical analyses were performed using the INSTAT program from GraphPAD (San Diego, CA).


RESULTS

The FRE1-dependent Reductase Is Nonspecific and Requires the MAC1 Gene Product

Table 1gives the rates of reduction of a variety of one-electron acceptors by wild type cells. The absolute rates of reduction of these substrates span a range of 10^5-fold; aside from Fe(III), Cu(II) is the best substrate of those tested. Also shown are the relative activities (as a percent of wild type) of fre1Delta-, mac1-1-,and MAC1-containing strains toward these several substrates. In most cases, a given mutation similarly affects the reduction of all substrates. For example, reduction of nearly all substrates is strongly inhibited in fre1Delta and mac1-1-containing strains, while in all cases the reduction is elevated in the gain-of-function MAC1 mutant. This pattern suggests that most of this reductase activity, which exhibits little specificity, is due to Fre1p. This conclusion is based on the fact that the FRE1 transcript is absent in the two former strains (9, 10, 15) but elevated in the latter one(15) . However, there are two significant additional features about these data. First, there is an apparent inverse correlation between the rate of reduction of a substrate and the fold increase reduction in a MAC1 strain. Second is that the rate of Cu(II) reduction is uniquely less sensitive to deletion of FRE1. Nonetheless, these results are generally consistent with a model in which the predominant reductase activity in the plasma membrane is due to the Fre1p-containing complex which, in turn, is regulated by Mac1p.



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 fre1Delta-containing (FDeltaFe) strains and a MAC1-containing mutant. The YPD plates on which the spores were grown were overlaid with TTC. The elevated reductase activity in MAC1FRE1 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) times MAC1 cross show the expected 2:2 segregation of this phenotype (left plate, Fig. 1). In contrast, the spores from the fre1Delta (FDeltaFe) times MAC1 cross show a characteristic tetratype segregation in which non-parental ditypes (e.g., fre1Delta 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(4) 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 fre1Delta allele. Strain DF5 up1 (MAC1FRE1) was crossed with strains F113 (wild type, MAC1FRE1) and FDeltaFE (MAC1 fre1Delta), the two diploids sporulated and the resulting tetrads dissected. The spores here were grown on YPD plates containing 50 µM CuSO(4) which were subsequently overlaid with a TTC-containing agar solution at 30 °C (see ``Experimental Procedures''). The presence of the MAC1FRE1 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 MAC1fre1Delta genotype remains white due to the suppression of MAC1 by fre1Delta.



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(4) 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 (MATalpha MAC1) was crossed with strains BCup1a (MATa MAC1) and wild type S144 (MATa MAC1), while S144 was crossed with parental wild type strain DF5alpha (MATalpha 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(4) indicated. The photograph was taken following 48 h of growth at 30 °C.



Copper, in Addition to Iron, Represses FRE1 Transcription

As described extensively, transcription from FRE1 is strongly repressed by iron in the medium(9, 10, 15) . This is evident in Northern blots probed for FRE1 mRNA (Fig. 3, lane 2). However, copper addition to the medium also strongly represses FRE1 transcription (lane 3). This copper-dependent repression was not due to contaminating iron; extracted medium made 40 µM in CuCl(2) was shown by flameless atomic absorption spectrophotometry to contain residual iron only ([Fe] leq 170 nM, see ``Experimental Procedures''). Iron added together with copper is not synergistic (lane 4) indicating that at the metal ion concentrations used (40 µM of either metal) iron or copper alone can lead to strong transcriptional repression of FRE1. Literature data suggest that higher metal concentrations (e.g., 200 µM iron) would be even more repressing, however(9, 13) . The results here are consistent with a model in which both metals work though the same or overlapping regulatory elements. These results suggest also that the reductase activity of Fre1p toward Cu(II) (Table 1) is not gratuitous, but rather is associated with the normal metabolism of this metal ion. Note that in MAC1-containing strains FRE1 mRNA levels are only weakly repressed by iron or copper(15) .


Figure 3: Repression of FRE1 transcription by iron and copper. Total RNA was isolated from log-phase cultures of strain DF5alpha grown in metal-free, defined media containing the additions as noted in the figure ([FeCl(3)], [CuCl(2)] = 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.



Fe and Cu Uptake Is Inhibited by Citrate

The assay for metal reductase activity uses BPS [Fe(II)] and bathocuproine disulfonic acid [Cu(I)] as the ``reporter'' of metal reduction. Coupling metal reduction to binding by a chelator can strongly perturb the reduction reaction, however(26) . Since these two phenantholine derivatives chelate the reduced ions so strongly, they effectively raise the oxidation-reduction potentials of Fe(III) and Cu(II) to a point at which any reducing equivalent(s) can support the reduction of these higher valency metal ions. A chelating agent like citrate or lactate is needed at relatively high concentration (20 mM) to counteract the cell-free blank rate of Cu(II) or Fe(III) reduction driven by the phenantholine reagent.

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, fre1Delta, 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.



Metal Reduction and Uptake: Repression by Iron and Inhibition by Pt(II)

First, the data in Table 2and Table 3confirm that the repression by iron of FRE1 transcription strongly suppresses Fe(III) reduction (Table 2) and Fe uptake (Table 3) as has been reported. As discussed below, the new results presented in these tables show that the effect of iron (and copper) on Cu(II) reduction and uptake is quantitatively different. Several contrasting results in these two tables strongly suggest the hypothesis that although Fre1p can support Cu(II) uptake, yeast also has an additional, Cu(II)-specific reductase. These results are as follows.



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 fre1Delta 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 versusFe 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).

Uptake of Cu in the Presence of Ascorbate: Transport Is Not Regulated by Copper in the Medium Nor Is It Inhibited by Pt(II)

Addition of ascorbate to uptake buffer containing Fe(III) makes Fe uptake independent of the Fre1p reductase(9, 13) . This observation is consistent with the model of iron uptake in which the first step is Fe(III) reduction since ascorbate alone readily reduces Fe(III) to Fe(II). Cu(II) uptake is similarly Fre1p reductase-independent (at least) in the presence of ascorbate as indicated by the fact that Pt(II) does not inhibit ascorbate-supported Cu uptake (compare the last three columns, Table 4). Like Fe(III), Cu(II) is readily reduced by ascorbate also. This suggests a mechanistic analogy to the Pt(II) inhibition of Fe(III) but not Fe(II) uptake, namely, that Cu(II) uptake also proceeds though an intermediate which is formed by a reductase or reductases, in a manner that can be partially but not completely inhibited by Pt(II). Another significant observation is that the rates of Cu accumulation in wild type, mac1-1, and MAC1 strains are similar when ascorbate is present in the uptake buffer (compare first to third columns, Table 4). Since in the presence of ascorbate Cu uptake is reasonably reductase-independent, Mac1p must have a limited role in regulation of the activity of the copper transporter, e.g., the CTR1 gene product. In contrast, Mac1p appears to be (partially) necessary for wild type expression of the putative Cu(II)-reductase. This inference is shown by the rates of Cu uptake in the presence of Pt(II) for wild type and the mac1-1-containing strains (0.5 ± 0.03 versus 0.1 ± 0.01 nmol Cu/min/mg protein, respectively, Table 4). With either strain, only Fre1p-independent activity would remain active in the presence of Pt(II); the loss of 80% of this activity in the mutant strain under these conditions indicates the role for Mac1p in expression of the putative Cu(II)-reductase activity. The data in Table 4show also that copper in the growth medium represses Cu uptake in the absence of ascorbate by only 50%; in contrast, copper (40 µM) represses Fe uptake by >80% (data not shown) presumably due to copper-repression of FRE1 transcription (Fig. 3). This difference is consistent with the presence of a Cu(II)-specific reductase which, in comparison to FRE1, is less strongly regulated by copper.




DISCUSSION

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 times 10^3 s, while at Fe(II) it is 3 times 10^6 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 10^8 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(2)-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, fre1Delta 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(2)CX(3)HX(4)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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46787. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 716-829-2842; Fax: 716-829-2725; camkos{at}ubvms.cc.buffalo.edu.

(^1)
The abbreviations used are: MES, 4-morpholinepropanesulfonic acid; BPS, bathophenanthroline disulfonic acid; TTC, triphenyltetrazolium chloride.


ACKNOWLEDGEMENTS

We thank Annette Romeo for assistance in the Northern analysis of FRE1 transcription, Dr. Cecile Pickart for her several critical readings of this manuscript, and Dr. Andrew Dancis for providing strains F113 and FDeltaFe. This work was made possible by our collaboration with Drs. Joern Jungmann and Stefan Jentsch which is gratefully acknowledged.


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