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MiC~biOlOgy 1995), 141,2937-2943 Printed in Great Britain

Effects of cadmium and of YAP7 andCADllYAP2 genes on iron metabolism in theyeast Saccharomyces cerevisiaeEmmanuel Lesuisse and Pierre LabbeAuthor for correspondence: Emmanuel Lesuisse. Tel: +33 1 43 54 04 79. Fax: +33 1 44 27 57 16 .e-mail : elu@ccr.jussieu.fr

Laboratoire de Biochimiedes Porphyrines, lnstitut J.Monod, Tour 43,Universite Paris 7, 2 PlaceJussieu, 75251 Paris cedex05, France

Sacchammyces cerevisiaewas more resistant to cadmium when the growthmedium contained excess iron. Cadmium reduced the amount of iron taken upby cells during growth, and the cell ferrireductase activity was also stronglyinhibited. These effects depended on the YAP1and CADIIYAP2 gene dosage.The growth rate of cells in irondef cient conditions and their ferrireductaseactivity in the absence of added cadmium were also strongly affected by thedosage of YAP1 and CAD1IYAP2 genes. Our results suggest an indirectinfluence of these genes on iron metabolism, possibly via modification of thecell redox status.

Keywords: ron metabolism, cadmium toxicity, ferrireductase, Saccharomjces cerevisiae,YAP I I C A D I/ YAP2

INTRODUCTIONThe Saccharomyces cerevisiae proteins YAP1 and CAD1(yAP2) are transcription factors that are related to themammalian proto-oncoproteins c-jun (Moye-Rowley e tal., 1989; Bossier e t al., 1993; Wu e t al., 1993). Harshmane t al. (1988) showed that the transcription of genespreceded by the ARE (AP1 recognition clement) con-sensus sequence is activated by yAPl. Several phenotypesassociated with the disruption of Y A P 7 and C A D 7 /Y A P 2 have been described. For example, Ayap7 mutantsare more sensitive than their wild-type to cadmium andzinc (Wu e t al., 1993) and to oxidative agents (Schnell &Entian, 1991). Overexpression of Y A P 7 causes cells toacquire a pleiotropic drug-resistance phenotype and tobecome resistant to cadmium. This last phenotype is alsoobserved when C A D I is overexpressed, but Acad7mutants are not hypersensitive to this metal (Bossiere t al.,1993). Other data also suggest that the Y A P 7 andC A D 7 genes could be involved in the regulation of ironmetabolism. Overexpression of Y A P or C A D confersresistance to the iron/zinc chelator 1,lO phenanthroline(Bossier e t al., 1993;Schnell& Entian, 1991). Addition ofiron (but not zinc or copper) alleviates the growthinhibition of a Ayap7 mutant in alkaline medium (Schnell& Entian, 1991). Bossier etal. (1993) suggested that YAP1and CADl/yAP2 promote the production of a secreted

Abbreviations: ARE, AP1 rec ognition element; BPS, bathophenanthro-line disulfonic acid; DTNB, 5,5-dithio-bis(2-nitrobenzoate).

compound that competes with 1 lo phenanthroline forzinc or iron, but this proposition is unrealistic for iron,since S. cerevisiae produces no siderophore, and thepotential iron-chelating compounds excreted by this yeast[ 3-hydr0xy)anthranilate and Krebs cycle intermediates;Lesuisse e t al., 19921 form complexes with iron that arethermodynamically less stable (low Kf) than the iron-orthophenanthroline complex.Iron uptake by S.cerevisiae is a two-step process, involvingextracellular reduction of ferric complexes by an inducibleplasma-membrane-bound reductase (Lesuisse e t al., 1987;Dancis e t al., 1990), and transport of the free ferrous ionsby a high-affinity transport system (Eide e t al., 1992). Theferrireductase activity of S. cerevisiae depends on at leasttwo genes, F R E I (Dancis e t al., 1992) and F R E 2(Georgatsou & Alexandraki, 1994), both of which aretranscriptionally regulated by iron. Both genes also haveARE [(G) T G A C T C/A A]-resembling elements intheir promoter regions (see Discussion). The YAP1 andCAD1 proteins have cysteine-rich C-terminal regions,which make them good potential candidates for playing arole in metal-dependent regulation of transcription.Gounalaki & Thireos (1994) suggested that there couldbe direct or indirect interactions between YAP1 and theMAC1 protein, a transcription factor involved in the basaltranscription of F R E I (Jungmann e t al., 1993). Previouswork suggested that the ferrireductase activity of cellsdepends on their redox status (Lesuisse & Labbe, 1992).Cadmium tolerance itself probably depends on the redoxstatus of the cells, since fungi frequently neutralize this

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metal by binding it to glutathione or glutathione poly-mers. In this context, it could be significant that AjapImutants are hypersensitive to both oxidative stress and tocadmium. Similarly, the loss-of-function mutants ofMAC7 are deficient in ferrireductase activity and hyper-sensitive to H,O, and cadmium. The last phenotype isblocked by adding excess iron (Jungmann e t a/ . , 1993).The present paper describes the links between YAP7 andC A D / YAP2 genes, iron metabolism and cadmiumtolerance in S. cereuisiae.

METHODSYeast strains and plasmids. Strains SEY6210 (wild-type),SMlO (A jap l ) , JG 1 (Acadl) a nd Y A W 6 4 (A3apl Acadl), and theplasmids YEp351-YAP1 and pAW18 used for overexpressionin the wild-type strain of YAPl and C A D I , respectively, werekindly provided by Dr W. S. Moye-Rowley, Department ofPhysiology and Biophysics, University of Iowa, IA, USA. Thegenotypes of these strains and descriptions of th e plasmids havebeen previously published (Wu e t a/., 1993). The plasmidscarrying the FREI-lacZ fusion were a gift from D r R. Labbe-Bois, Laboratoire de Biochimie des Porphyrines, Institut J.Monod, Paris, France. A D N A fragment containing 800 bp ofth e F R E l promoter and the first 15 bp of the co ding sequencewas cloned in the multicopy vectors YEp357 and YEp367(Myers eta/., 1986).

RESULTSEffect of YAP genes on iron-limited grow thSeveral reports (Schnell& Entian, 1991;Wu e t a!., 1993;Bossier e t a/ . , 1993) have stated that overexpression ofYAP1or CADIIYAP2genes makes cells more resistantto the iron chelator 1 , l O phenanthroline on agar plates.We confirmed this observation and tested the resistance ofthe cells to other hydrophobic and hydrophilic ironchelators, including bathophenanthroline, bathophen-anthroline disulfonic acid (BPS), ferrozine, 2,2 bipyridyl,8-hydroxyquinolein and 8-hydroxyquinolein disulfonicacid. All these molecules had nearly the same effect on thegrowth of all strains (wild-type, Aya pl, Acad l, Ayap IAcadI,YAP overexpressed, C A D overexpressed) on agarplates : for example, both 2,2-bipyridyl and 8-hydroxy-quinolein had a slight inhibitory effect at 150 pM and fullyinhibited the growth of all strains at 250 pM; BPS had astrong inhibitory effect on the growth of all strains at1 mM (data not shown). Schnell & Entian (1991) pre-viously reported that BPS did not lead to a YAPI-dependent resistance phenotype and attributed this to thehydrophilic nature of the chelator, which is not believedto cross the cell membrane. But this seems unlikely, sincethe hydrophobic chelators 2,2 bipyridyl and 8-hydroxy-quinolein had the same inhibitory effect on the growth ofall the strains.

Cell growth, reductase assays and iron uptake. Cells weregrown in liquid YNB-glucose medium (Yeast Nitrogen Base,Difco, 0.67 %, glucose 2 % ,w/ v) in aerobic conditions (20 mlmedium in 100 ml Erlenmeyer flask in a gyro-shaker at200 r.p.m.), T he cu lture media were treated w ith 8-hydroxy-quinolein (Nicholas, 1957) prior to use to remove iron;ZnSO, (1 pM) was added to the m edium after hydroxyquinoleintreatment. Residual iron in th e treated media was estimated byinductively coupled plasma atom ic emission spectroscopy. F orthe reductase assays, the cells were pre-cultured for 40 h at30 OC, then washed twice with E D TA (2% , w/v ) and twicewith distilled water. T he cells were then inoculated to OD,,, 0.2(Kontron model Uvicon 860) into fresh culture medium, plusbathophenanthroline sulfonate (BPS; 200 pM), or differentconcentrations of iron [as Fe(II1)-citrate].Th e cells were grow n for 8 h at 30 OC. Aliquots were w ithdrawnfrom the cultures every 2 h, filtered and washed with water o nnitrocellulose filters. The ferrireductase activity of the cells wasmeasured with Fe(II1)-citrate (360 pM ) as substrate (Lesuisseet a/., 1987). Under these experimental conditions, the cellferrireductase activity was maximum after 4-6 h of gr ow th andthen decreased. Only maximal values of ferrireductase activitiesare shown in the Results.For iron uptake experiments, iron was added as [6SFe(III)]-citrate (0.1-1 pM) to either liquid YNB-glucose medium orsolid YP G o r YNB-glucose agar media. Th e cells were washedon th e filters wi th 20 ml Na,S,O, (1 %, w/v) and 20 ml ED TA(2 %, w/v , in 50 mM citrate, pH 6.5).Thiols assays. Total low-molecular-mass thiols and surfacesulfhydryl groups were assayed with DTNB as previouslydescribed (Lesuisse & Labbe, 1992).

10 30 50 70Time (h)Fig. 1. Growth curves of different yeast strains under iron-deficient (upper pa nel) or iron-su ff cient (lower panel) conditions.Cells were in oculated o OD,, 0.01 in iron-deficien t (YNB-glucose+100 pM BPS, uppe r p anel) o r iron-suff cient (YNB-glucose,lower panel) media. 0 , Wild-type; 0 , AyapI; A, Acadl; I,AyaplAcadl;0 , YAPl overexpressed;A, AD1 overexpressed.

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Table 1. Effects of cadmium and iron on the grow th of different yeast strainsCells were patched on YPG-agar medium containing different amounts of cadmium and iron. The grow th of the colonies w as estimatedafter 1 week at 30 OC. + + +,Normal growth; + , poor growth; -, no g rowth.

~~~ ~

[Cd2+](pM).. 180 240 300 360 420 480 180 240 300 360 420 480[Fej'] (pM) ... 10 10 10 10 10 10 103 103 103 103 103 103Wild-type + + + + - - - + + + + + + + + + + + + + + + +Acad 1 + + + + - - + + + + + + + + + + + + + +YaP 1Ayap 1Acad 1YEp351-YAPI + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

- - + + + + + + + + +- -+-

- - - - - - - - - - -+ +PAW18 + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +

Table2. iron uptake by cells grow n on agar platesThe agar medium (YPG) contained 1 pM 55Fe(III)-c itrate. After 1 week of growth at 30 OC, th ecellular paste was suspended in water and cells were washed ont o a filter with E D TA and dithionite.Iron uptake was determined by liquid scintillation counting of the solubilized samples.

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Strain Iron uptake [pmol (mg wet wt cells)-']No addition Cd2+ 60pM) Cd2+120 pM) BP S (100 pM)

Wild-type 22.5 7.5 3.8AYaP 1 23.2 4.5 3.8Acad 1 17.3 6.7 3.0Ayap 1Acad 1 19.5 4 5 3.0YEp351-Y A P 1 21.0 7.5 3.8PAW18 20.2 7.5 4 8

3.74.53.72.65.73.7

The situation was different when the cells were grown inliquid medium (YNB-glucose). The Ayap 7 mutant thenhad the highest growth rate and the YAP7-overexpress-ing strain had the lowest growth rate when the mediumwas supplemented with 100 pM BPS (Fig. 1).Thus, over-expression of YAP7 did not favour the growth of cells iniron-deficient conditions as was suggested by Bossier e t a/.(1993). It resulted rather in an increased inhibition ofgrowth on iron-deficient liquid medium (Fig. 1). Thiseffect could not be related to differences in the ability ofthe strains to take up iron from the iron-deficient medium,as shown by the following experiment : wild-type, Ayap7and YAP 7-overexpressing strains were grown on eitheriron-sufficient (YNB-glucose) or iron-deficient medium(YNB-glucose +100 pM BPS) to which 0.25 pM 55Fe asFe(II1)-citrate) was added. The amount of iron taken upby the cells was measured after 24 h. N o significantdifferences were observed between the strains whengrown on either iron-sufficient medium [the three strainsaccumulated 15-20 pmol iron (mg wet wt cells)-'] oriron-deficient medium [the three strains then accumulated4-6 pmol iron (mg wet wt cells)-']. Curiously, the growthdefect in iron-deficient conditions of the YAP 7-over-expressing strain was alleviated by adding the oxidativeagent menadione disulfonate (25-100 pM) to the medium.For example, after 4 d of growth in iron-deficient

conditions (YNB-glucose medium +100 pM BPS, in-oculated to ODsooO-OS), that strain reached ODsoo0.8(versus 0.3 for the control culture) when the medium wassupplemented with 25 pM menadione disulfonate. In thesame conditions, the wild-type reached OD,,, 1.7 (versus1.3 for the control culture). Again, this effect did not resultfrom a difference in the amount of iron accumulated bythe cells, since menadione disulfonate had no significanteffect on the total iron taken up by the cells (data notshown).Cadmium toxicity and iron metabolismWu & Moye-Rowley (1994) showed that transcriptionalactivation of GSH7 - he first gene of glutathione bio-synthesis- mediated by YAP1 is essential for normalcadmium tolerance in S. cerevisiae. However other factorsseem involved in cadmium resistance/toxicity. As shownin Table 1, the cadmium resistance of the cells increasedwhen the growth medium contained excess iron [Fe(III)-citrate]. This effect was specific to iron since it was notobserved when excess copper, or citrate alone, was addedto the medium (data not shown). The effect of irondepended on the copy-number of YAP7 and CAD7genes. For example, it was less pronounced in the Ajap7Acad7 mutant (Table 1).Analysis of the iron content of

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I I100 1000 10000010 Cadmium concentration (nM )Fig. 2. Effect of cadmium concentration in the grow th m ediumon the ferrireductase activity. Cells from 40 h pre-cultures weregrown in liquid medium (previously treated with 8-hydroxyquinolein) containing different concentrations ofCdSO,. The ferrireductase activity of the cells was measuredafter 5 h of growth..,ild-type; 0 , Ayapl; A, Acadl; 0 ,YAP1 overexpressed; 0 , CAD1 overexpressed. Values, ex-pressed as percentage of maximal activity (in the absence ofadded cadmium), are means of 3-5 experiments.cells grown on agar plates with or without added cadmiumshowed that iron uptake decreased in the presence ofcadmium (Table 2). At a cadmium concentration of120pM , the iron content of the cells was roughly similarto that of cells grown in the presence of 100pM of the ironchelator BP S (Table 2). When the cells were grown in_ . . . *. . . .the extracellular cadmium concentration reached 1 pM(not shown). At low extracellular cadmium concen-trations (1-10 pM), only the wild-type strain and theAyap7, Acadl and Ayap7 Acad7 mutants showed a significantdecrease of their intracellular iron pool. The iron contentsof these cells in the presence of 10 pM cadmium were 65 YO(wild-type), 50 % (AyapI), 80 % (Acad7) and 15YO AyaplAcadl) of that found in the absence of cadmium, whileYAP 7- and CAD7-overexpressing strains were notaffected.Further investigations showed that cadmium had a strongrepressive effect on the ferrireductase activity of the cells(Fig. 2). When added during the reductase assays, thismetal showed no direct inhibitory effect on the ferri-reductase activity of the cells (not shown). The inhibitoryeffect of cadmium was observed when Cd2+was presentduring growth : the ferrireductase activity of the wild-type grown in iron-limited conditions ( < 0.1 pM Fe) wasdecreased by 50YOwhen 1pM cadmium was added to thegrowth medium (Fig. 2). Again, the effect of cadmiumdepended on the YAP7 and CAD7 gene activities:overexpression of YAP7 resulted in a steeper inhibition

1

1

2 3Fig. 3- Effect of growth medium iron concentration onferrireductase activity of different yeast strains. Cultures weregrown as described in Methods in YNB-glucose medium(treated with 8-hydroxyquinolein) supplemented with either200 pM BPS (0 , ron-deficien t conditions) or 0.1-200 p M Fe(ll1)-citrate (0,.1 pM; , 2 pM;.,0 pM;.,00 pM). Cells weregrow n for 5 h, harvested, washed, and their ferrireductaseactivity was measured. 1, Wild-type; 2, Ayapl; 3, Acadl. Data aremeans+sE of fou r experiments.curve, while overexpression of CA D lead to the oppositeeffect (Fig. 2). Disruption of YAP7 had little effect, whiledisruption of CAD7 caused the cell ferrireductase to be10-fold more sensitive to the effect of cadmium (50%inhibition at 0.1 pM ) than the wild-type (Fig. 2).

Effect of YAP genes on ferrireductase activityThe ferrireductase activity of the cells in the absence ofadded cadmium was also affected by the YAP7 andCADI gene contents. The effects of YAP7 and CAD7on the cell ferrireductase depended greatly on theexperimental conditions (pre-culture and culture ; ata notshown), and varied according to the iron content of thegrowth medium (Fig. 3) . The ferrireductase activity of thewild-type was maximum when cells were grown in iron-deficient conditions (200 pM BPS), and then droppedwhen iron was added. However, the decrease in ferri-reductase activity was not strictly proportional to theconcentration of iron added (Fig. 3) . The greatest decreaseoccurred at 0.1 pM iron and there was a reproducible smallpeak of activity at 2-20 pM iron. The Ayap7 strain showedmaximal repression at an iron concentration of 2 pM, anda well-marked peak of activity at 20 pM iron (Fig. 3). Theferrireductase activity of the Acadl strain remained almostfully induced when iron was 0.1 pM , and decreasedsharply around 2 pM (Fig. 3) . Two genes - F R E (Dancisetal., 1992) and F R E Z (Georgatsou & Alexandraki, 1994)-have so far been identified as playing a direct role in cellferrireductase activity. Both genes probably encode plas-ma membrane structural components of the reductasesystem(s). To see whether FRE7 could be transcrip-tionally regulated by YAP1 and/or CAD1, he strains usedin this study were transformed by a multicopy plasmidbearing the lacZ coding sequence fused to the FRE7promoter. /3-Galactosidase activity was determined in

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Table 3. Ferrireductase activity, surface thiols and intracellular low-molecular-mass thiolsassociated with cells of different strainsCells were gro wn for 5 h in YNB -glucose medium (treated with 8-hydroxyquinolein) supplementedwith either 200 pM BPS (- Fe) or 01 pM Fe(II1)-citrate (+Fe). T he cell ferrireductase activity wasmeasured as described in M ethods. T he thiol groups (intracellular and surface) were assayed withDT NB after incubation of the washed cells for 3 min in citrate buffer (50 mM, pH 65) containing 5%glucose. Values are expressed in nmol (mg wet wt cells)-'.

.... .............................. . .......... ................. ............. ......................................... . ....... ................... ..... ..... ................................................ . ...... ..................

Strain Growth Ferrireductase Surface thiols Intracellular thiolscondition activity (nmol (nmol mg-') (nmol mg-l)

min-' mg-')Wild-type - e 3.8Wild-type +Fe 0.5A!aP 1 - e 5.3A a P 1 +Fe 2.2Acad 1 - e 6.5Acadl +Fe 7.0

0.80-20.90 51.21.3

3.52.72 61.64 74.7

each strain grown under the conditions described above.The results showed that YAP1 and CAD1 had no directinfluence on F R E1 transcription. /l-Galactosidase activitywas induced by iron-deprivation (+BPS;1500-2000 U /l-galactosidase) in all strains tested and was stronglyrepressed when iron (0-1-200pM) was added to theculture medium (100-300 U, data not shown).An indirect influence of YAPICAD genes on ferrire-ductase activity could theoretically be exerted via modu-lation of the cell redox status. Therefore, we measured theintracellular low-molecular-mass thiols and the surfacesulfhydryl groups in different strains grown in differentconditions (Table 3). The level of reduced thiols waspreviously shown to be increased in iron-deprived cells(Lesuisse & Labbe, 1992). As shown in Table 3, this wasagain observed here. Interestingly, we found a constantrelationship between the ferrireductase activity of the cells-whatever the strain - and the amount of surface sulf-hydryl groups accessible to the membrane-impermeantEllman's reagent (DTNB) (Table 3). For example, theAcad7 strain, which showed unrepressed ferrireductaseactivity when grown with 0.1 JAM ron, also showed thehighest level of reduced surface thiols and of intracellularlow-molecular-mass thiols under this growth condition(Table 3). The Ayap7 strain which is expected to have alower glutathione pool than the wild-type (Wu & Moye-Rowley, 1994) showed indeed a lower content of in-tracellular reduced thiols, but was unaffected in its surfacethiol pool and similarly unaffected in its ferrireductaseactivity (Table 3).

DISCUSSIONAn excess of iron in the growth medium makes the cellsmore resistant to cadmium; reciprocally, cadmium in-hibits ferrireductase activity and iron uptake. Thus, whilecadmium tolerance is mainly a function of the YAP7 andCAD7 gene activity- probably via modulation by these

genes of glutathione synthesis (see Wu & Moye-Rowley,1994)- he toxicity of cadmium could at least partly belinked to the defects in iron metabolism induced by thismetal.The dosage of YAP1 and CADI/YAPZ genes has amarked effect on the ability of cells to grow in iron-deficient medium, on ferrireductase activity and on therate of ferrireductase repression by cadmium. On YPG-agar medium, YAP-overexpressing cells were moreresistant than wild-type cells to 1 lo-phenanthroline, butnot to other iron chelators. We believe that the resistanceto 1 lo-phenanthroline is relevant to a detoxificationprocess, which could be less effective with other chelatorslike 2,2'-bipyridyl or 8-hydroxyquinolein. The growthof YAPI-overexpressing cells on iron-deficient liquidmedium was strongly inhibited. Possibly, the YAP -overexpressing cells grown in such conditions could sufferfrom an imbalance of their redox status, more than froma lack of iron sensu stricto, since overexpression of YAP1is known to result in an increase of the intracellularglutathione pool (Grey & Brendel, 1994). The growth ofcells in iron-deficient conditions also results in a significantincrease in the intracellular and surface reduced thiolpools (Lesuisse & Labbe, 1992). According to thishypothesis, menadione would alleviate the growth defectby shifting the cell redox balance towards a more oxidizedstate. It should be pointed out that identical conditions ofgrowth cannot be reproduced on agar plates, since agarmedium cannot be treated by 8-hydroxyquinolein.The YAP gene products could theoretically act onferrireductase activity a t several levels. The FRE7 andFREZ genes contain ARE-resembling elements in theirpromoter regions and could then be candidates fortranscriptional regulation by YAP1 and/or CAD1. TheFR E7 gene contains the sequence TTTTTGCTCAYC atpositions - 67 and - 84 from ATG. Dancis e t a/. (1992)showed that this repeated sequence could constitute acandidate binding site for an iron-responsive regulatory

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protein. The FRE2 gene also contains ARE-resemblingelements at positions -306 (TGGCTCA) and -489(GTGACTC) from ATG. The influence of Y A P geneson ferrireductase activity does not seem to be exerted viatranscriptional regulation of FRE 7 . However, YAP1and/or CAD1 could regulate transcription of other genesencoding other structural components and/or regulatorycomponents of the reductase system(s) (FRE2 or somestill-unidentified component). YAP1 and /or CAD1 couldalso influence the ferrireductase activity of the cellsindirectly, by acting on genes involved in the post-transcriptional regulation of reductase activity. Y A P ,for example, appears to play an important role in theoxidative stress response of S. erevastae (Kuge & Jones,1994). Since the ferrireductase activity of the cells alsoseems to be related to their redox status (Lesuisse &Labbe, 1992), YAP1 (and/or CAD1) may have an indirecteffect on ferrireductase activity at several levels, such asglutathione synthesis (Wu & Moye-Rowley, 1994), gluta-thione reductase and/or glucose-6-phosphate dehydro-genase activities (Schnell e t al., 1992). The products ofY A P 7 and/or C A D I genes could also act on thereductase activity via the RAS/cAMP pathway. Previouswork (Lesuisse e t a/., 1991) suggested that a CAMP-mediated activation of protein kinase A could be neededto fully derepress ferrireductase activity. Gounalaki &Thireos (1994) showed that there is a link betweentranscriptional activation by YAP1 of TPS2 (encodingtrehalose phosphate phosphatase) and the activity ofCAMP-regulated protein kinase. It is worth noting thatFREl shows significant homologies with one componentof the neutrophil NADPH oxidase system (Dancis e t al.,1992), the activity of which is partly regulated byphosphorylation/dephosphorylation. Nothing is pre-sently known about the post-transcriptional regulation ofthe yeast ferrireductase system(s). Recent studies suggestthe existence of complex inter-connections between themetabolisms of iron and copper (Dancis e t al., 1994) andthe metabolic pathways involved in the response tooxidative stress (see for example the phenotype of maclmutants; Jungmann e t al., 1993). We believe that cellferrireductase activity is regulated at several levels in-volving not only the iron and copper contents of the cellsbut also the cell redox status -which in turn is related tothe iron/copper status- s a function of the growth phaseand of the extracellular redox conditions. The adaptativerole of Y A P 1 and C A D I I Y A P 2genes in relation to ironmetabolism remains to be defined in that complicatedbackground.

ACKNOWLEDGEMENTSWe thank D r W. S. Moye-Rowley for the yeast strains andplasmids and Dr 0.Parkes for checking the manuscript.

REFERENCESBossier, P., Fernandes, L., Rocha, D. & Rodrigues-Pousada, C.(1993). Overexpression of Y A P 2 , coding for a new YA P protein,an d Y A P in Saccbaromyces cerevisiae alleviates growth inhibitioncaused by 1 lo-phenanthroline. J Biol Cbem 268, 23640-23645.

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.............................................. ................................... ..................... . ...... .... ... ... .................. ....Received 11 May 1995; revised 27 June 1995; accepted 24 July 1995.

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