The Yeast Fre1p/Fre2p Cupric Reductases Facilitate Copper Uptake and Are Regulated by the Copper-modulated Mac1p Activator

Abstract

Fre1p and Fre2p are ferric reductases which account for the total plasma membrane associated activity, a prerequisite for iron uptake, in Saccharomyces cerevisiae.The two genes are transcriptionally induced by iron depletion. In this communication, we provide evidence that Fre2p has also cupric reductase activity, as has been previously shown for Fre1p (Hassett, R., and Kosman, D.J. (1995) J. Biol. Chem. 270, 128–134). Both Fre1p and Fre2p enzymes are functionally significant for copper uptake, as monitored by the accumulation of the copper-regulatedCUP1 and CTR1 mRNAs in fre1Δ,fre2Δ, and fre1Δfre2Δ mutant strains. However, only Fre1p activity is induced by copper depletion, even in the presence of iron. This differential copper-dependent regulation of Fre1p and Fre2p is exerted at the transcriptional level of the two genes. We have shown that Mac1p, known to affect the basal levels of FRE1 gene expression (Jungmann, J., Reins, H.-A., Lee, J., Romeo, A., Hassett, R., Kosman, D., and Jentsch, S. (1993)EMBO J. 12, 5051–5056), accounts for both the copper-dependent induction of FRE1 and down-regulation of FRE2 gene. Finally, Mac1p transcriptional activation function is itself modulated by the availability of copper. Fre1p and Fre2p are ferric reductases which account for the total plasma membrane associated activity, a prerequisite for iron uptake, in Saccharomyces cerevisiae.The two genes are transcriptionally induced by iron depletion. In this communication, we provide evidence that Fre2p has also cupric reductase activity, as has been previously shown for Fre1p (Hassett, R., and Kosman, D.J. (1995) J. Biol. Chem. 270, 128–134). Both Fre1p and Fre2p enzymes are functionally significant for copper uptake, as monitored by the accumulation of the copper-regulatedCUP1 and CTR1 mRNAs in fre1Δ,fre2Δ, and fre1Δfre2Δ mutant strains. However, only Fre1p activity is induced by copper depletion, even in the presence of iron. This differential copper-dependent regulation of Fre1p and Fre2p is exerted at the transcriptional level of the two genes. We have shown that Mac1p, known to affect the basal levels of FRE1 gene expression (Jungmann, J., Reins, H.-A., Lee, J., Romeo, A., Hassett, R., Kosman, D., and Jentsch, S. (1993)EMBO J. 12, 5051–5056), accounts for both the copper-dependent induction of FRE1 and down-regulation of FRE2 gene. Finally, Mac1p transcriptional activation function is itself modulated by the availability of copper. Iron and copper are elements that have many similar chemical properties. Both are transition metals with several oxidation states, have close atomic and ionic radii numbers, and have very similar electronegativity. They are relatively homogeneously distributed on the planet and have thus been integrated in many biochemical reactions during evolution. Both are essential for life in almost every species (bacteria, fungi, plants, mammals) (1Theil, E., Raymond, N. K., Bioinorganic Chemistry, Bertini, I., Gray, H. B., Cippard, S. I., Valentine, J. S., 1994, University Science Books, Mill Valley, CA.Google Scholar, 2Crichton R.R. Charloteaux-Wauters M. Eur. J. Biochem. 1987; 164: 485-506Google Scholar). Excess quantities of iron and copper exert similar amount-dependent cytotoxicity, both favoring the formation of hydroxyl radicals which are disastrous to the cell (3Halliwell B. Cutteridge M.C. Methods Enzymol. 1990; 186: 1-85Google Scholar). Iron presents a particular problem for its uptake, since in the oxygen-containing atmosphere it is practically all in the form of insoluble ferric hydroxides. Organisms have therefore developed complex mechanisms of high fidelity and precision to achieve an appropriate homeostasis of these two metals. In Saccharomyces cerevisiae, two proteins of the plasma cell membrane, Fre1p and Fre2p, reduce Fe(III) to Fe(II) in the proximal vicinity of the cell. The expression of both corresponding genes is regulated by the environmental iron concentration by a negative feedback mechanism which takes place at the transcriptional level (4Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Google Scholar,5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar). The coupled function of the cell surface Fet3p multicopper oxidase, which catalyzes the conversion of Fe(II) to Fe(III) extracellularly (6De Silva D.M. Askwith C.C. Eide D. Kaplan J. J. Biol. Chem. 1995; 270: 1098-1101Google Scholar), and the recently reported Ftr1p permease (7Strearman R. Yuan D.S. Yamaguchi-Iwai Y. Klausner R.D. Dancis A. Science. 1996; 271: 1552-1557Google Scholar) are also required for high affinity iron uptake. Thus, a link between iron and copper metabolism was first noted by the isolation of the FET3 gene in a scheme aiming to clone the ferrous transporter (8Askwith C. Eide D. Ho A.V. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Google Scholar). Fet3p requires copper to function, and therefore high affinity iron uptake requires copper (8Askwith C. Eide D. Ho A.V. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Google Scholar, 9Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Google Scholar). Mutations either in the high affinity copper transporter gene CTR1 or in the CCC2 gene, encoding a member of the family of P-type ATPases proposed to transport cytosolic copper into the lumen of a secretory organelle, results in iron deficiency in the cell (9Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Google Scholar, 10Yan D.S. Stearman R. Dancis A. Dunn T. Beeler T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2632-2636Google Scholar). A similar mechanism has been postulated in mammals for the release of newly absorbed iron from intestine to blood involving the plasma glycoprotein ceruloplasmin, a copper-binding protein with ferrous oxidase activity (11Chang A. Fink G.R. Curr. Biol. 1994; 4: 532-533Google Scholar, 12Kaplan J. O'Halloran T.V. Science. 1996; 271: 1510-1512Google Scholar). Finally, the iron regulated transcription of the yeast genes FRE1,FRE2, FET3, CCC2, FTR1, andFTH1 (FTR1 homologue of unknown function) is affected by the Aft1p transcriptional activator which recognizes a specific consensus sequence on their promoters (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Google Scholar, 14Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Google Scholar). Moreover, expression of FRE1 and CTR1 mRNAs depends on the nuclear protein Mac1p, which is involved in iron and copper utilization (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar). 1E. Georgatsou, L. A. Mavrogiannis, G. S. Fragiadakis, and D. Alexandraki, unpublished observations. We have previously shown that although Fre1p and Fre2p have seemingly redundant functions in S. cerevisiae, the two genes are up-regulated by the absence of extracellular iron in a kinetically different way, implying that they are subject to distinct regulation (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar). Since there is evidence that copper reduction might be a component of copper uptake (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar) and that Fre1p is also a copper-repressible cupric reductase (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar, 16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar, 17Lesuisse E. Labbe P. Plant Physiol. 1992; 100: 769-777Google Scholar), in this study we have investigated the participation of FRE1 and FRE2 gene products in copper metabolism. Our data clearly point to a role of both activities in copper uptake, although the two genes are differentially affected by the function of Mac1p under conditions of copper (and iron) depletion. Mac1p transactivating function is itself modulated by the availability of copper, being higher in its absence. The yeast strains used in this study are all derivatives of the S288C strain. The wild type and fre1Δ, fre2Δ, andfre1Δfre2Δ strains have been previously described (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar). For the MAC1 gene disruption in the yeast genome, a 480-base pair StyI fragment (codons 41–201) was replaced by the 1.1-kilobase HindIII fragment of theURA3 gene on a MAC1 (−100 to 963)/pBluescript recombinant phagemid. The resulted insertion fragment was excised and used to transform the desired ura3–52 strains (18Guthrie C., and Fink, G. (eds) (1991) Methods in Enzymology Vol. 194, Academic Press, New YorkGoogle Scholar). Uracil prototrophy was used for the selection of transformants. Transformants able to grow on glycerol only in the presence of 50 μmcopper (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar) were further confirmed for the MAC1 gene deletion by DNA blot hybridization. The yeast strain used for transcriptional induction and repression assays of the LexA-Mac1p fusion proteins to lacZ reporters was L9FT5, a derivative of FY105 (MATα,his3Δ::LexAopHIS3 leu2::PET56 ura3–52 trp1Δ63). The growth media used were SD (2% glucose, 0.67% yeast nitrogen base; Difco) or SD supplemented either with 100 μmbathophenanthroline disulfonic acid-Na2 salt (BPS) (Fe(II) and Cu(II) chelator) or with 100 μm bathocuproine disulfonic acid-Na2 salt (BCS) (Cu(I) chelator) (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar, 19Landers J.W. Zak B. Am. J. Clin. Pathol. 1958; 29: 590-592Google Scholar,20Eide D. Davis-Kaplan S. Jordan I. Sipe D. Kaplan J. J. Biol. Chem. 1992; 267: 20774-20781Google Scholar). For all experiments described, cells were grown to saturation in SD medium, subsequently resuspended in the medium, and grown for They were to the desired or at a concentration of were from every and for copper and iron activities fusion were from the a fragment lacZ gene for the of the gene, and a of the gene. The and of FRE1 and FRE2 by were in of the described in fusion to the The MAC1 gene was from a yeast by the of a The sequence of the is For the of the LexA-Mac1p fusion the sequence from to was by and into of a derivative of the and by an and encoding the nuclear the from and the D. 1994; Scholar). The used for the are and The used for the and assays were in which of the the lacZ gene transcription D. 1994; Scholar, R. R. E. D. D. J. G. J. A. and (eds) in and New YorkGoogle Scholar), and in which two activation and of the gene the lacZ gene transcription D. 1995; Scholar, J. M. Cell. 1992; Scholar). The of the used as in the blot are and of these with was by DNA R. R. E. D. D. J. G. J. A. and (eds) in and New YorkGoogle Scholar). The described were by the of and of total and yeast DNA were and to R. R. E. D. D. J. G. J. A. and (eds) in and New YorkGoogle Scholar). the a DNA fragment the of the FRE1 gene to was into the of the The recombinant was by at the on gene and a homogeneously was by the in the presence of D. 1994; Scholar). the a fragment was which the of the of the A was with this to this which an For the FRE2 DNA fragment the of the FRE2 gene to was into the of the described for the The recombinant was by at the on gene and a was by the as for the the FRE2 were the of the the FRE2 is used for A was with this to a of the For the a HindIII DNA fragment the of the from to was into the HindIII of the described for the The recombinant was by at and a was by transcription with the of were with of total in a and The was in of a and and with A at for by with in at for The was at for and on a reduction activity was as described previously (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar). the conditions were used for the copper reduction that and was used of and For the an of was used to the complex at The used for at was activity assays R. R. E. D. D. J. G. J. A. and (eds) in and New YorkGoogle Scholar) were of growth in the desired has been shown previously that Fre1p ferric reductase is also cupric reductase induced by copper (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar). In this we have Fre2p ferric reductase for cupric activity in a strain (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar). We have first used iron conditions known to Fre2p (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar) to of the For we have wild in to the conditions described under The results in that Fre2p as as Fre1p have cupric reductase and Fre2p induction under copper and iron reductase reductase of reductase activities were in of the strain at different of growth in and The by a of from experiments at and of growth activities of the two reductases were are are as or Fe(II) in a of reductase activities were in of the strain at different of growth in and The by a of from experiments at and of growth activities of the two reductases were are are as or Fe(II) Since we previously that the two enzymes kinetically distinct induction by iron (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar), we have the strains under conditions of copper during of Our results that Fre1p was induced under copper at similar levels as by iron depletion. In Fre2p was modulated by copper depletion. Fre2p activity was induced even in the presence of the to the of of the Cu(II) in the by the We have the results only in by from its with the of copper in activity in strain which was affected by and for of the activity in We have similar ferric activity in this fre1Δfre2Δ strain (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar) Fre2p induction in this was between and in and relatively during the growth of the cells We that to the proposed of (8Askwith C. Eide D. Ho A.V. Bernard P.S. Li L. Davis-Kaplan S. Sipe D.M. Kaplan J. Cell. 1994; 76: 403-410Google Scholar, 9Dancis A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Google Scholar) of copper also iron depletion, which in induced Fre2p results that both Fre1p and Fre2p are cupric they the of copper reduction is for copper uptake. that Fre1p and Fre2p are for copper uptake, this be in a fre1Δfre2Δ strain. We have monitored copper into the cell and its utilization by the expression of two different copper and The regulation of these genes by copper distinct encoding a is induced by the transcription copper concentration in the cell M. R. A. P. J. S. Proc. Natl. Acad. Sci. U. S. A. Scholar, D.J. Mol. Cell. Biol. Scholar), encoding the copper transporter is induced by copper and is affected by the A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; Scholar). shown in high levels were cells grown in which that the mutant cells copper uptake the wild type of copper to a concentration of μm levels in both that the also the fre1Δfre2Δ significant was in the accumulation between the two gene to levels of copper gene A. Haile D. Yuan D.S. Klausner R.D. J. Biol. Chem. 1994; Scholar). under Fe(II) and Cu(II) conditions both reductases were in the accumulation of both CTR1 and mRNAs were between fre1Δfre2Δ and wild type strain. higher concentration was to CTR1 levels in the mutant This that the two reductases were clearly required for copper uptake under conditions of by the two under conditions the between the two strains almost since copper was exerted the of The might a higher concentration in the presence of reductases the transporter to with the the of of the reductases for copper uptake, we have the and under conditions at which the and wild type cells shown the of the repression in the strains clearly that both reductases in copper uptake. The between and wild type cells that Fre1p the almost as as both However, a between and fre1Δfre2Δ strains concentration was required to in the was to the Fre2p participation in copper uptake. The accumulation in either between the presence or absence of Fre2p, clearly to the of The differential induction of the two reductases in copper cells to the levels and FRE2 mRNAs under these an A we were able to and an mRNAs in total of a wild type strain. A that FRE1 under copper and FRE2 was at very at all as described for the induction of the corresponding In experiments FRE1 mRNAs both the induction for the corresponding reductase activities (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar) of activities by or FRE2 promoters in wild type cells grown under copper and iron clearly that was able to both and expression of only expression The only known nuclear protein in both copper and iron basal expression of gene is Mac1p (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar). mutant cells from copper iron since of copper or iron their (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar). Mac1p a to the copper-dependent transcription (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google Scholar). We have the of FRE1 genes to copper in a strain during of The results shown in A was in cells grown in and induced copper depletion. In FRE1 basal levels were in the cells with the wild type levels and at very levels in the iron of the both FRE1 mRNAs were However, by to the induction in wild type we a of the FRE1 induction to in the in to a of the FRE2 accumulation to A and activity levels by the in a strain grown under copper or iron conditions that the differential accumulation of FRE1 and FRE2 mRNAs resulted from the transcriptional of the two genes these results to the accumulation we that at of growth the assays were the accumulation of was in the strain with the wild type strain by the FRE1 and by the FRE2 The results of reductase assays on and were in with the induction of their under copper Mac1p function has a negative role on FRE2 gene it gene. In the absence of Mac1p under copper might for both since FRE2 and levels are higher in the as in the shown in Mac1p to be involved in the differential expression of genes under iron are further under The copper-dependent regulation genes affected by Mac1p function to the copper of Mac1p protein we have the Mac1p sequence to the for its to transcription of a to the D. 1994; Scholar, R. R. E. D. D. J. G. J. A. and (eds) in and New YorkGoogle Scholar). shown in Mac1p protein was of transactivating the in cells grown in This activity was cells were from copper, which that the Mac1p protein was at these Mac1p levels were under conditions results a of copper on Mac1p its transcriptional activation of the LexA-Mac1p fusion for repression function on a D. 1995; Scholar, J. M. Cell. 1992; Scholar) on the expression of this that Mac1p have This is for the for its different role on the expression of the genes under Iron and copper are metals and have been The mechanisms by which to their and are being J. O'Halloran T.V. Science. 1996; 271: 1510-1512Google Scholar). The enzymes Fre1p and Fre2p are at the of the that extracellular to iron communication, to nuclear that gene as as at the of a being regulated by iron to its into the cell. a has been (13Yamaguchi-Iwai Y. Dancis A. Klausner R.D. EMBO J. 1995; 14: 1231-1239Google Scholar, 14Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Google Scholar), the two reductase genes are differentially induced E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google and this In this study we have the participation of the ferric reductases Fre1p and Fre2p in copper and we have elements of their have the plasma membrane activity E. Labbe P. Plant Physiol. 1992; 100: 769-777Google Scholar) and Fre1p (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar) as being by copper. We have that Fre2p is a cupric as is only Fre1p is induced in copper for of the plasma membrane The activity, which is modulated by copper, to the by described by Hassett and Kosman (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar). This reductase is by gene. data We have further shown that the distinct of the two cupric reductases to copper is from the differential transcriptional regulation of the two genes. FRE1 gene was transcriptionally FRE2 gene to to copper depletion. we have shown that Mac1p nuclear protein or to this differential of the two reductase genes. FRE1 gene basal and induced expression were on its in with the that Mac1p is involved in the of the copper to the FRE1 gene. In gene basal and expression were only in the absence of for this which to be in the a previously shown to transcription of a gene (4Dancis A. Roman D.G. Anderson G.J. Hinnebusch A.G. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3869-3873Google Scholar), distinct from the Aft1p which be for the Mac1p as by Yamaguchi-Iwai Y. Stearman R. Dancis A. Klausner R.D. EMBO J. 1996; 15: 3377-3384Google Scholar). are also on the FRE2 (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar) and A. Yuan D.S. Haile D. Askwith C. Eide D. Moehle C. Kaplan J. Klausner R.D. Cell. 1994; 76: 393-402Google Scholar) promoters in different CTR1 gene expression is also on the presence of Mac1p (15Jungmann J. Reins H.-A. Lee J. Romeo A. Hassett R. Kosman D. Jentsch S. EMBO J. 1993; 12: 5051-5056Google on the FRE2 are these to the between and transcription of FRE2 gene is at from the first and the is at these were affected by Mac1p, they might different on the different the expression of FRE1 Mac1p in of the formation of the transcriptional complex on the R. M. Scholar). This is also in with results that Mac1p a role in the different induction of the two reductase genes by the of the We that Mac1p FRE1 expression and of Aft1p of results in This scheme is in with on the role of Mac1p was able to a gene to its the Mac1p repression function on a gene an that Mac1p function to the cupric reductases was the that its function was modulated by the availability of copper, being in its absence. The role of Mac1p be by the study of its as as of the elements of the transcriptional in which it Our the role of the cupric reductases on the uptake of copper. the copper reduction is for its uptake, the from a is since Cu(II) are in a form in as to copper the cell as Cu(II) or was in have shown a in Fe(III) and Cu(II) activities in to of these metals from the 1993; Scholar, J. Plant Mol. Biol. 1992; Scholar). be either by the of a role of reductases in the of the plasma membrane which affect and E. Labbe P. Plant Physiol. 1992; 100: 769-777Google Scholar, J. Plant Mol. Biol. 1992; Scholar) or a specific role in copper uptake which into account environmental availability of Cu(II) which form very with environmental Hassett and Kosman (16Hassett R. Kosman D.J. J. Biol. Chem. 1995; 270: 128-134Google Scholar) evidence for copper in S. of copper uptake was the was in the uptake that is for the and function in uptake S. Kosman D.J. D.J. 1996; Scholar). We have evidence for the of reduction for copper uptake by the CTR1 and mRNAs as reporters to and utilization of copper in the cell. We have that activities higher copper as shown by the quantities in the fre1Δfre2Δ strain with wild type This activity was an prerequisite for copper uptake, to the of Cu(II) or to by the plasma membrane activity Our results provide evidence that Fre1p also Fre2p have a to the uptake of copper we used a its form This might be very for the since there are many of Cu(II) in the in The of the two ferric reductases in copper and iron metabolism distinct and since Fre1p and Fre2p to or to the it that ferric reductases have to also in copper metabolism. Since iron the cells in conditions of copper induction of the ferric reductases by iron be of copper was the copper is Fre1p is first able to of copper into the and for copper to be Fre2p to its being induced of iron A of Fre2p was both under copper and in the strain. Our results clearly that reduction of copper its into the We that Fre1p and Fre2p, although for copper uptake, are and in that under In the involving two genes in (5Georgatsou E. Alexandraki D. Mol. Cell. Biol. 1994; 14: 3065-3073Google Scholar) in close for iron and copper with clearly is a very in mechanisms that have in to is to as by the sequence of the S. with the Fre1p and Fre2p and are involved in specific membrane reductase activity We and for and for with and strain for and for