Copyright 0 1996 by the Genetics Society of America

The Saccharomyces cwmkiae RTG2 Is a Regulator of Aconitase Expression Under Catabolite Repression Conditions

Christian Velot, Peter Haviernik’ and Guy J.-M. Lauquin Institut de Biochimie et Ginitique Cellulaires, Centre National de la Recherche Scientajique, University of Bordeaux 11, 33077 Bordeaux, France Manuscript received September 11, 1995 Accepted for publication July 6, 1996

ABSTRACT The ACOl gene, encoding mitochondrial aconitase of Saccharomyces cermisiae, is required both for oxidative metabolism and for glutamate prototrophy. This gene is subject to catabolite repression; the ACOl mRNAlevel is further reduced when glutamate is supplied with glucose. To further explore regulation of ACOl expression, we have screened for mutations that reduce expression of an ACOl-lac2 fusion borne on a multicopy vector. We identified a gene required for wild-type expression of ACOl only under catabolite repression conditions. Sequencing of the corresponding cloned gene revealed that it is identical to RTG2 previously cloned as a pivotal gene in controlling interorganelle retrograde communication. Cells containing either the original rtg2-2 mutation or a null rtg2 allele are not petite but show a residual growth on minimum glucose medium with ammonium sulfate as the sole nitrogen source. This growth defect is partially restored by supplying aspartate or threonine, and fullywith glutamate or proline supplement. Surprisingly, this phenotype is not observed on complete medium lacking either of these amino acids. In addition,a genetic analysis revealed an interaction between RTG2 and ASP5 (encoding aspartate amino transferase), thus supporting our hypothesis that RTG2 may be involved in the control of several anaplerotic pathways.

CONITASE (citrate [isocitratel hydro-lyase; EC chondrial aconitase is still not understood. A dual cell A 4.2.1.3) is an located mainly in the mito- location has also been described for citrate synthase chondrial matrix, where it is involved in theKrebs cycle activity, for which three functional , CITI, CIT2, (KREBS and LOWENSTEIN1960), catalyzing the revers- and CIT3, have been characterized (KIM et al. 1986; ible isomerization of the tricarboxylic acids (TCA) ci- ROSENKRANTZet al. 1986; BECAMet al. 1995). However, trate and isocitrate via ciJiaconitate (ROSEand O’CON- the extramitochondrial citrate synthase activity (CS2, NELL 1967; VILLAFRANCA and MILDVAN 1971).In encoded by CIT2),which operates as part of the glyoxy- Saccharomyces cermisiae, as in other eukaryotic cells, all late cycle, was clearly localized in peroxisomes (LEWIN the of the TCA cycle are encoded in the nu- et al. 1990). Interestingly, the effects of a CITI or ACOl cleus and are transported into the mitochondrial ma- disruption on citrate synthase or aconitase activity, re- trix. The mitochondrial aconitase of S. cereuisiae is en- spectively, are very different. In the latter case, both coded by the ACOl gene, previously cloned by mitochondrial and extramitochondrial aconitase activi- GANGLOFFet al. (1990) by complementation of the glul- ties are abolished ( GANGLOFF1990). On the contrary, 1 mutation (OGURet al. 1964). In yeast cells, aconitase in acitl strain, it has been found thatCIT2 transcription activity is also involved in the glyoxylate cycle, and the and CS2 activity are increased (LIAOet al. 1991) and extramitochondrial aconitase activity probably fulfills that this regulation (termed retrograde) depends on this role (DUNTZEet al. 1969),although it does not two genes, RTGl and RTG2 (LIAOand BUTOW1993). seem to be located in peroxisomes where the glyoxylate The role of the TCA cycle is twofold. First, it is oxida- cycle is thought to take place in yeasts (KAWAMOTO et tive, generating NADH, that drives mitochondrial respi- al. 1977; WALES et al. 1980; MCCAMMONet al. 1990) as ration and hence the synthesisof ATP. Second, the in plants and other fungi (ZIMMERMANand NEUPERT Krebs cycle provides the carbon skeletons used in many 1980; TOLBERT1981; WANNER and THEIMER1982; TREL anabolic pathways, including a-ketoglutarate, the main EASE 1984; DE BELLISet al. 1990; REYNOLDS and SMITH precursor of glutamate. Thus, in S. cermisiae, citrate syn- 1995). Moreover, the genetic origin of this extramito- thase and aconitase are required both for glutamate prototrophy andfor efficient utilization of nonfer- Corresponding author; Guy Lauquin, Laboratoire de Physiologie Mo- mentable carbon sources (OGURet al. 1964; OGURet al. Ikculaire et Cellulaire, IBGC/CNFS, l,rue Camille Saint Saens, 33077 1965; KIM et al. 1986; GANCLOFFet al. 1990), implying Bordeauxcedex, France. E-mail: [email protected] that aconitase, like citrate synthase, will be highly regu- ‘Present address; Department of Biochemistry, Faculty of Sciences, Comenius University, Mlynska dolina CH-I 842-15 Bratislava, Slo- lated. It has been reported for the bacterium Bacillus vakia. subtilis that levels of citrate synthase and aconitase are

Genetics 144: 893-903 (November, 1996) 894 C. Vklot, P. Haviernik and G. J.-M. Lauquin downregulated by glucose alone (catabolite regulation) 8-kbp fragment (3A). YCp50-3A1 and YCp50-3A2 were ob- and synergistically by glucose and a glutamate source tained by deletion of the 4.8-kbp SalI fragment and the 4.8- kbp EcoRI fragment, respectively, from the 3A insert (see Fig- (HANSONand COX 1967). ROSENKRANTZet al. (1985) ure 3A). The 1.7-kbp BglII-EcoRI, 3.4kbp SalI-KpnI, 4.4kbp have shown that, in the case of aconitase, this control BglII-KpnI subclone fragmentsfrom 3A were inserted between is exercised at the transcriptional level. In S. cerevisiae, the corresponding sites of the centromeric vector pRS316 a similar control pathway operates for citrate synthase (SIKORSKIand HIETER 1989) to generate pRS3A3, pRS-3A4 (KIM et al. 1986) and for aconitase (GANGLOFFet al. and pRS3A5, respectively (see Figure 3A). To construct pRS- Artg2 in which the RTGZ gene is disrupted by the 7W’I 1990). Moreover, GANGLOFFet al. (1990) found a se- marker, the 750-bp EcoRI-SalI fragment from the 3A5 sub- quence in the ACOl promoter corresponding to the clone was replaced by the 800-bp EcoRI-SalI TKI’Z-bearing inverted upstream activating sequence UAS2 UP1 fragment from YDp-W (BERBENet al. 1991). (GUARENTEet al. 1984; FORSBURGand GUARENTE 1988), Genetic manipulations: Yeast transformations were carried which has been shown to be the HAF’2/3/4/5 - out either by the spheroplast method described byBE:(;(;s (1978) and modified by BURGERSand PEKCIVAI.(1987), or by responsive site of the CYCl gene (OLESENet al. 1987; the lithium method reportedby IT(> rt al. (1983) and modified FORSBURGand GUARENTE1989; MCNABB et al. 1995). by GIETZ et al. (1992). Crosses, diploid selection, sporulation Accordingly, GANGLOFF(1990) showed that expression and tetrad analysis were carried out by standard procedures of the ACOl gene is under the control of both HAP2 (SHERMANPt al. 1986). Colony hybridization was performed and HAP3 genes (HAP4 and HAP5 were unknown at as previously described (HANAHAN andMESEISON 1980). that time). Southern analysis and DNA sequencing: S. ccrpuisiae DNA was prepared by the method of SHERMANet al. (1986), di- To study further the regulation of aconitase expres- gested with appropriate restriction enzymes, subjected to elec- sion, we sought mutants affected in ACOl expression trophoresis through 1% agarose gels and blotted onto nitro- by using lac2 reporter constructs to mimic the regula- cellulose as described by SOUTHERN(1975). Blots were tion of ACOI. We have identified and cloned a nuclear hybridized with purified random priming probes (FEINHERG and VOGELSTEIN1984). Sequencing was carried out using di- gene required for a normal level of ACOl expression deoxy chain termination (SANGERet al. 1977). TheSa&-EcoRI under catabolite repression conditions and found it to fragment from 3A5 insert was sequenced directly on plasmids be identical to RTG2 (LIAOand BUTOW1993). Further- pRS-3A3 and pRS-3A4 (see Figure 3A) with the universal 20- more, we performed a genetic study on the relation- mer reverse and SK primers. ships between genes RTG2 and ASP5 (encoding aspar- Enzyme assays: Crude extracts were prepared from 100 ml cultures harvested in the early log phase. Cellswere sus- tate aminotransferase), andfound rtg2 to partially pended in potassium phosphate buffer (20 mM, pH 7.4) sup- suppress the asp5 mutation. plemented with 1 mM phenylmethylsulfonyl fluoride, and bro- ken with glass beads (0.5 mm diameter) by vortexing. “hole cell extracts were obtained following successive centrifuga- MATERIALS AND METHODS tions of 5 min at 1200 X gand 20 min at 30,000 X g. Protein Strains and media: The S. cereuisiae strains used in this study concentration was determined by the bicinchoninic acid-us- are presented in Table 1. All of the strains listed are isogenic to ing method described by SMITHet al. (1985). the reference strain S288C, except STX32-1OA and SN/Z-50. Aconitase activity was assayed by an adaptation of the Standard rich (YPD), synthetic complete (SC), synthetic method described by FANSLERand LOWENSTEIN(1969). Spe- minimal (SD) presporulation (SP1) and minimal sporulation cific activities are given as nanomoles of cicaconitate trans- (AcK) media were described previously (SHERMANet al. 1986). formed per minute permilligram of protein. Assays were per- Other media were SD + X (like SD but including nutrient(s) formedat 22“ induplicate or triplicate and results were X), SC -Y (like SC but lacking nutrient(s) Y) and X-Gal averaged. This regimen produced activity measurements that medium. X-Gal plates contained 70 mM potassium phosphate were reproducible within 20%. (pH 7.0),0.17% yeast nitrogen base without amino acid and Quantitative /?-galactosidase assays were carried out either ammonium sulfate, 0.5% (NH4)$S04, 2% glucose, 40 pg/ on colonies grown on SC -Ura plates and suspended in 1 ml ml X-Gal, 20 pg/ml uracil, 60 pg/ml leucine and 20 pg/ml Z buffer so that ODeoowas about 2 units, or on yeast strains histidine. grown overnight in the appropriate selective synthetic me- Plasmids: The ACOl-lacZ fusion contains a 794bp EcoRI- dium, diluted in fresh selective medium, and grown to log EcoRV fragment of the 5‘ region ofACOl plus sequences phase (ODti,,,)= 1). After cell permeabilization as previously encoding the first 49 amino acids fused in frame to the lacZ described (GUARENTE 1983),/?-galactosidase assays with the gene of Escherichia coli. The multicopy version was directly colorimetricsubstrate o-nitrophenyl-/?-u-galactopyranoside obtained by inserting the 794bp EcoRI-EcoRV fragment from were performed as described by MILLER(1972). Actit‘Tities are pSE-31 (GANGLOFFet al. 1990) between the EcoRI and SmaI given as Miller units. Assays were carried out in duplicate or sites ofYEp358 (MYERS et al. 1986). The ACOl-lacZ fusion was triplicate and averaged, and were reproducible within 20%. also placed in an integrative vector bearing the HIS3 marker Isolation of ACOl expression mutants: Strains FYlA and and derived from pAT153 (TWIGG and SHERRAT1980). This NlB bearing pYACOl’Z were mutagenized with ethyl meth- last plasmid was used to integrate the ACOl-lacZfusion at the ane sulfonate (FINK1970) to -50% survival and plated on ACOl chromosomal locus. Southern analysis was performed 0.45 pm nitrocellulose filters laid onto SC pura plates. After to confirm that the integration event had occurred and that 48 hr of growth, filters were transferred to minimal glucose the ACOl chromosomal copy was intact. Such strains are des- plates containing the /?-galactosidase chromogenic substrate ignated “/Z” (see Table 1). (X-Gal plates). About 20,000 colonies were screened (10,000 The yeast genomic library (CEN BANK,ATCC 37415) from each strain) and 227 pale blue or white colonies were (ROSEel al. 1987) was constructed in the centromeric vector identified. Of these 227 colonies, only eight strains consis- YCp50. YCp50-3A is an isolate from this library containing an tently displayed reduced levels of /?-galactosidaseactivity. Only Aconitase Regulation in S. cermisiae 895

TABLE 1

Yeast strains used

Strain Genotype Source FYlA MATa ura3-52 leu2Al Laboratory collection FYI B MATa ura3-52 leu2Al his3A200 Laboratory collection 1B-1 MATa ura3-52 bu2Al his3A200 rtg2-2 This study FYlB/Z MATa ura3-52 leu2Al his3A200 ACOl-lacZHIS3 This study" lB-l/Z MATa ura3-52 leu2Al his3A200 rtg2-2 ACOl-lacZHIS3 This study" FY23 MATa ura3-52 leu2Al trpIA63 B. DUJON (Institut Pasteur) FY23 1 MATa/MATa ura3-52/ura3-52 leu2Al/bu2Al his3A200/HIS3 trplA63/TRPl rtg2-2/RTG2 This study' FY231-00 MATa/MATa ura3-52/ura3-52 leu2Al/leu2Al his3A200/HIS3 trplA63/TRPl rtg2-2/rtg2-2 This study' FY231-10 MATa/MATa ura3-52/ura3-52 leu2Al/leu201 his3A200/HIS3 trplA63/TRPl rtg2-2/RTG2 This study" FY231-11 MATa/MATa ura3-52/ura3-52 leu2Al/leu2Al his3A200/HIS3 trplA63/TRPl This study' RTG2/ RTG2 FYc5 MATa ura3-52 trplA63 his3A200 Laboratory collection FYC5/Z MATa ura3-52 trplA63 his3A200 ACOl-lacZHIS3 This study" FYC5-Vl MATa ura3-52 trplA63 his3A200 rtg2::TRPl This study FYc5/z-v1 MATa ura3-52 trplA63 his3A200 ACOl-lacZHIS3 rtg2::TWl This study" STX32-10A MATa ura3 his3 arg8 asp5 Berkeley collection SFY/Z-50a MATa ura3-52 trplA63 his3 asp5 ACOl-lacZHIS3 This study"*/ SFY/Z-50a MATa ura3-52 trplA63 his3 asp5 ACOl-lacZHIS3 This study",' Strains bearing the ACO1-lac2 fusion integrated at the ACOl chromosomal locus. ' Formed by mating 1B-1 and FY23. 'Formed by mating two rtg2-2 segregants from FY231. " Formed by mating a rtg2-2 segregant and a wild segregant fromFY231. Formed by mating two wild segregants from FY231. [Segregants from STX32-10A X FYC5/Z. five of these (all bearingthe mating type a)appeared to carry type and mutant strains bearing the integrated fusion: chromosomal lesions; these were subjectedto preliminary ge- the results are presented in Table 2. To confirm that netic and phenotypic analysis. expression of ACOl was affected, aconitase activity was assayed in whole-cell extractsfrom FYlB and 1B-1 RESULTS strains cured of any ACO1-lacZ fusion and grown in Isolation of amutant with altered expression of YPD medium. Theresults obtained corroboratedthose ACO1: To further explore ACOl gene expression regu- observed with the reporter gene, although the decrease lation, we screenedfor mutations that would affect in activity for aconitase was lower than for P-galactosi- transacting regulatory and reduce expression of an ACOI-lac2 fusion borne on a 2p URA3 vector. TABLE 2 Mutant clonesgiving rise to pale blueor white colonies ACOl-lacZ fusion and aconitase expression on minimal glucose XGal plates were isolated (see MA- in wild FYlB and mutant 1B1 strains TERIALSAND METHODS). Eight mutants with reduced or abrogated P-galactosidase expression were further ana- Strain lyzed; three exhibited a plasmid-linked phenotype and FY1 B 1B-1 were presumed to be ACOI-lac2 structural gene mu- Enzyme tants. The remaining five mutants derived from FYlB activity"GlucoseLactateGlucoseLactate bearing the mating type a) and were named (ie., 1B- &galactosidase' 42 153 3 177 1, 1B-3, 1B-24, 1B-35 and 1B-45. Preliminarygenetic Aconitase"18 t- 4 110 t- 21 6 t- 3 135 +- 25 and phenotypic analysis revealed that 1B-3 and 1B-24 bore mutations not affecting ACOl-lacZ expression at "Average values determined on three independent experi- ments. all but probably the numberof copies of the 2pm-based P-galactosidase activitiesare given as Miller units and were plasmid. Moreover, 1B-35 and 1B-45 proved to be pet measured as describedin MATERIALS AND METHODS after mutants with complex phenotypes for which a strategy growth in SC -His medium containing either 2% glucose for gene cloningwas difficult to set up. So we decided (catabolite repression conditions) or 2% lactate (derepres- to furtherstudy the mutant 1B-1, the only one left with sion conditions). "Aconitase activitiesare given as nmolof cis-aconitate trans- the expected phenotype. formed per min per mg of protein and were determined on Phenotypiccharacterization: The levels ofexpres- crude extracts of the strains grown to early log phase either sion of the ACO1-lacZ fusion were examined in wild- on YPD (glucose) or on YPL (lactate). 896

Fyc5 FyC5-Vl TABLE 3

1 s1 AGO1 expression decrease phenotype segregates !as a single nuclear locus Aconitase activity" 2 Sl+E Tetrad

3 Sl+T Scgrcgant" 1 2 3 4 5 6

1 4 14 23 8 30 13 4 Sl+D h 12 27 6 28 24 30 C 27 33 17 (5 8 8 d 30 9 5 33 8 24 5 Sl+D+T I'alucs are nmol of ci.wconitate transformed/min per mg of protein. "Scgrcgants of the diploid strain resulting from ;I 6 Sl+P W231 cross lmween 1B-1 antl FY23 for which aconitase activities (in the same units) were respectivclp (5 and 28. "Aconitase activities wcre assayed on crude extracts of the 7 SC-E segregants grown on WD to early log phase.

amino acids and adenine)as well as proline (forreasons a SC-PT explained in DISCCSSIOS) for the ability to restore nor- mal growth on this minimalmedium. The results

9 SC-D-T-E showed that either aspartate or threonine could par- tially restore growth without being additive, and that FIc;l'w I .-Gro\v111 phrnor~psol. str;Iins hearing either eitherglutamate or prolinecould fully restore the the original Hg2-2 mutation (IRI) or the r/g2 null allele growth of the 1R1 mutant to wild-type levels (Figure (FYCS-\'I), and additional growth requirements. IVild-type 1 ). Surprisingly, no growth defect was ohsewed on syn- and mutant strains were grown overnight in liquid YPD cul- thetic complete mediumlacking either one orall of the ture, washed twice in sterile water and resuspended m 2 X three aminoacids (aspartate, threonine and glutamate) 10" cells/ml in water. For each strain, 3 p1 drops (IO' cells) were laid onto different synthetic minimal (SD) and complete (Figure 1). Since a strain harboring theghI-1 mutation (SC) glucose media. SI medium is SD plus uracil, histidine, or a null allele of ACOI is unable to grow on SC -Cln leucine and tryptophan (1): same as (1) plus glutamate (2); medium, these results suggest that there is a reduced same as (1) plus thrconine (3); same as (1) plus aspartate but significant ACOl expression in the 1R1 mutantcells (4); same as (1) plus aspartate and threonine (5);same as that is reflected in the low aconitase activity detcctcd (1) plus proline (6); SC medium lacking glutamate (7); SC medium lacking aspartate and threonine (8); SC medium in this mlltant strain (Table 2). lacking aspartate, threonine and glutamate (I)). Genetic characterization: Todetermine whether these phenotypes were due to a single genetic lesion, dase (Table 2). The same experiments were carried out 1R1 was crossed with the wild-type strain FY23, generat- in the absenceof catabolite repression by replacing the ing the FY231 diploid strain. Segregants were analyzed 2% glucose by 2% lactate, a nonfermentable carbon after spornlation and tetrad dissection. Aconitase activi- source. Results show that no repression phenotype is ties were determined on crude extracts of segregants observed in theseconditions (Table 2), i.~.,the 1Rl (from six tetrads dissected) grown to early log phase strain does not exhibit any decrease in aconitase or P- on YPD culture medium, and thesame segregants were galactosidase activities compared to the wild-type strain. also tested fortheir abilit!r to grow normally on SD Since an aconitase-deficient strain is characterized by medium supplemented with uracil,histidine, leucine a petite phenotype, the mutant strain 1B-1 was plated and trvptophan (SD + Ura + His + Leu + Trp). The onto various rich media supplemented with a nonfer- results showed that both phenotypes segregated 2':2- mentable carbon source, i.P., either 2% lactate (YPL), and cosegregated in all six tetrads (Table 3 and Figure 2% glycerol, 2% potassium acetate or 3% ethanol. 2A), suggesting that a single mutation caused these phe- Strain 1B-1 proved to be able to grow fully on these notypes. To confirmthese results, the study was ex- different media. tended to tetrads resulting from the cross between 1R However, the1R1 mutant grew vel? poorly com- 1/Z and FY(:.5/7, (see Table l), where each segregant pared with wild-type strain FYlB on synthetic minimal bore the integratedACOI-IurZfusion and could be sub- glucose medium supplemented with uracil, leucine and jected directly to P-galactosidase assays. In each of the histidine (SD + Ura + Leu + His). To understand the 18 tetradsanalyzed, two segregants displayed low P- origin of this growth defect, we tested each of the other galactosidase activity and grew poorly on SD + Ura + nutrients presentin the SC medium (i.~.,other essential His + Leu + Trp medium, thereby confirming that a Aconitase Regulation in S. rmwisinc 89 7

4.3-khp I.G-oRV-I.:roRV fragment(Figure SR). The de- ducedgenomic pattern was identicalto that deter- a) Aconitase activity mined by restriction analysis of the cloned insert SA, HW HM HT and indicatedthat no recombination eventhad oc- I 1Xf3 4f2 17f3 ht curred during the cloning steps. Moreover, the size of fragments generated by digestion with I.:coRI and Hin- dIII restriction enzymes corresponded to those ex- pected from the EcoRI/HindIII physical map of the yeast genome (OL.SOSP! nl. 1986; RII.ESP! 01. 1995). FIGL'KE2.-Genctic chalacterization. (A) Poor growth phe- Disruption of the RTG2 gene and characterizationof notype on synthetic minimal glucose media segrcptcs as a single the Artg2 strain: Subcloning experiments and comple- nuclear locus. Segregants analyzed in Table 3 (letters from a to mentation tests revealed that a 0.7-kbp I:'coRI-Snfl frag- d refer to segregmts and n1unhel-s from 1 to 6 rcfcr to tetlads) were subjected to a growth test as described in the legend of ment within the SA.? subclone was probably essential Pipre 1, on SD meclirlm supplemented with tu-,cc.il, histidine. for complementation of the 1R1 mutant (Figure JA). leucine and tnpophan. Comparison benwen Table 3 and Fig- To perform the disruption of the corresponding gene, we 2A show that the nvo phenotypes cosegregate. (B) Domi- we replaced this fragment by the 0.8-khp I.;coRI-Snfl nance/recessiveness analysis. Homozygous wild R7'(;2/R7'(;2 7'Wl selectable marker to form pRSAr/g2 (see M~~TERI- FY23I-I1 (HW), heterozygous R7T,2/$$2FY231-10 (HT) and homozygous mutant r/g2-2/r/g2-2 IT23 1-00 (HM) diploids were AI.SASD METHODS).After checking thatthis new plasmid formed by mating segregants from FY231. (a) flg2-2 mutation was unable to complement the @-galactosidaseactivity is recessive for the reduced aconitase expression phenotype. decrease and residual growth phenotypes in mutant The mlues shown here represent nmol of ricaconitate fonned strains 1R1/Z and 151, respectively (Figure SA), the per min per mg of protein ? SE of three independent experi- newly generated 4.5-kbp KjmI-S/wI rfg2:: TWI-bearing ments (b) ttg2-2mutation is recessive for residual growth phene hve. These diploids were subjected to a growth test as described linear fragment from pRSArfg2 was used to transform in the legend of Figure 1 on SD medium supplemented with strains FYC5 and FYC.5/7, to tryptophan prototrophy. uracil and leucine. We confirmed that the tryptophan-independent trans- formantscarried the disrupted allele by performing single mutation was responsible for both phenotypes. Southern analysis of genomic DNA from both untrans- For reasons that will become clear below, the mutation formed and transformed cells. The resulting r/g2dis- was termed rfg2-2. rupted strains were termed FYC5-VI and FYC5/Z-V1 To determine whether the7-4.72-2 mutation was reces- (see Table 1). sive, homozygous mutant rfg2-2/r/g2-2 (FY231-00), het- To characterize these disrupted strains, we measured erozygous RTG2/rfg2-2 (FY2S 1-1 0) and homozygous 0-galactosidase activity in the yeast strains FYC.5/7, and wild H7%2/RTG2 (FY2Sl-11) diploids were formed by FYC.i/Z-VI, as well as aconitase activity on crude ex- matingsegregants from FY231 (seeTable 1). These tracts of FYC.5 and FYC5-VI grown to early log phase diploids were tested for their ability to grow normally on glucose (YPD) and lactate (YPL) media (Table 4). on SD + Ura + Leu medium, and aconitase activities As expected, both enzyme activities were decreased in were assayed on crude extracts of each of them grown the disrupted strains but this phenotype was obsened to early log phase on YPD medium. Results (Figure 2R) only under catabolite repression conditions as seen for indicated that the rfg2-2 mutation was clearly recessive the strain bearing the original rtg2-2 mutation. How- for both phenotypes. ever, we noted that the aconitase activity decrease ap Cloning R7E2 andchromosomal location: Twelve peared to be more severe in the disrupted strain since Ura' transformants of the 1R1 mutant able to grow no activity was detectable. Moreover, growth tests per- normally on SD + His + Leu medium were selected formed on different synthetic minimalor complete glu- after transformation with a YCp50-based library (ROSE cose media showed thatthe FYC5-VI strain behaved PI nl. 1987). Complementation in all 12 transformants like mutant 1R1 (Figure 1). Finally, FYC5-VI, like the proved to be conferred by the same plasmid, YCp50- 1B-lstrain, was able to grow fully on rich medium sup SA.YCp50-3A restoredaconitase activity to wild-type plemented with various nonfermentable carbonsources levels (Figure SA). After subcloning experiments (see (i.P., lactate, glycerol, potassium acetate, or ethanol) MATERIXLSAND METHODS), the minimal complementing (data not shown). region proved to be a 4.4kbp I3gflI-KpnI fragment (suh However, to exclude thepossibility that subclone3A5 clone 3A5, Figure SA). This fragment maps to the left bore an extragenic suppressor of the rfg2-2 mutation, arm of VII (behveen the HXK2 and HAP2 we crossed FYC5/7,-Vl with the r!g2-2 mutant strain 15 loci) by hybridizationto A clones ATCC 70008 and 1 /Z.The resulting diploidwas sporulated andsubjected 7071 3. to tetrad analysis. 0-galactosidase activity was assayed in In addition, total DNA from wild-type strain M1B the segregants from 18 tetrads that were likewise tested was digested with eight different restriction enzymes, for their ability to grow on SD + Ura + His + Leu + blotted onto nitrocellulose paper and probed with the Trp medium. The segregationpattern was 0':4- for 898 (3. \'Plot, P. Haviernikand G. J.-M. Iauquin IAl V VE Bg S Se E VNK K YCpSO-3A // EV \\a\I I , I1 \\\,~.,.\\\\\\\\\\,\,.~~~,,,~~~,,~~.~~I \ // HC ' Hp CSh Hp H \ \ \ \ HP rn E VHHpP K N XDbrn

!3.1"I 9.44 6.5-1 4.3-1

2.- 2.0-1

Ability to lkb complement both 0.5-1 - phenotypes of the rrg2-2 mutation

F1c;rxe 3,"Restriction map and suhcloning analysis of the genomic area around the RK2locus. (A) Restriction map of the genomic clone ahle to complement the rfg2-2 mutation and suhcloning experiments. Hatched boxes indicateDNA insert (SA) or suhclones (SA1 -3A5), and thin lines indicate vectorDNA. Gray box helow the map of insert 3A represents the DNA fragment used to probe the Southern blot. Plasmid YCp5O-SA as well as plasmids hearing various DNA fragments from the insert were used to transform mutant strains (IB-l/Z and IB-1) and tested for their ability to complement the 0-galactosidase decrease phenotype (in 16-1/7, strain) and poor growth phenotype (in 1B-1 strain). Each construction was either ahle (indicated as +) or unable (indicated as -) to complement hoth phenotypes. (R) Southern analysis of genomic DNA. Wild-type DNA (from stlain NIB) was cllt with different restriction enzymes (Dh means a douhle EmRI + Hind111 digestion), and the resultingDNA fragments were hyhridized at high stringency to the '"P-laheled LmRV-lhR\T fragment on a Southern hlot. Arrows on the left indicate the DNA size (in khp) stanclards of lanes m (A DNA cut with HincllII). Restriction site ahhreviations: R, BnmHI; C;, Chi; E, EmRI; H, HintlIII; Hp, IfprI; I(, KpI; N, Ncol; P, l'd; S, Snn; Se, SpI; Sh, Sphl; V, hRV; X, XhnI. both phenotypes in all tetrads, thus confirming thatwe encodesaspartate aminotransferase, which catalyzes had not cloned an extragenic suppressor and conse- transaminationfrom glutamate onto oxaloacetate to quently that FYC5/Z-V1 (and FYC.?-Vl too) did bear a form a-ketoglutarate and aspartate. An asp5 mutant is null allele (r(g2::TRPI) of the RTG2 gene. therefore auxotrophic for aspartate;it was observed that Sequence analysis of the 3A5 subclone DNA M'e de- this auxotrophy is best scored on media lacking both termined the sequence of this 3A.5 region as described aspartate and threonine (JONES and FINK1982). Conse- in MATERIALS AND METHODS. Approximately 500-base quently, owing to the aspartate, threonine, and gluta- readings were obtained, and computer searchesof avail- mate requirements of the ~/g2mutants, we looked for abledatabases from EMRL+GenRank revealed that interactions between the r(g2 and nsj15 mutations. For these sequences belonged to theRTG2 gene, previously this purpose, the 1R1/7, strain was mated to strainSFY/ cloned by LIAOand BUTOM'(1993). This gene is pre- Z-50a (see Table 1). After sporulation of the resulting sumed to be a pivotal gene in controlling retrograde diploid strain, the spores of 11 complete tetrads were communication between mitochondriaand the nucleus characterized by determiningtheir mating type and (i.p., the fact that expression of some nuclear genes is their ability to grow on SC media lacking either uracil sensitive to the functionalstate of mitochondria). R7%2 (SC -U), histidine (SC -H), leucine (SC -L), trypto- encodes a protein of 394 amino acids of unknown fhc- phan (SC -W), or both aspartate and threonine (SC tion and was shown to affect the mRNA retrograde ex- -D -T). As expected, thesegregation pattern, foreach pression of the CIT2 gene, which encodes the peroxi- tetrad, was Y':2", 0+:4- on SC -U medium, 4+:0- on somal isoform of citratesynthase (CS2) (LIAOand SC -H medium, and 2+:2- on SC -L, SC -W, and SC BVTOM'1993). -D -T media. However, some spores grew slowly on Genetic relationships between rtg2 and asp5 ASP5 SC -D -T, so thatthe segregationpattern became Aconitasein Regulation S. cermisiae 899

TABLE 4 rtg2::TRPl mutation (indicated by d). Thus, this result Artg2 strain is characterized by an ACOl transcription suggests that in the presenceof the rtg2 null allele, the decrease under catabolite repression conditions asp5 strain is able to synthesize enough aspartate to prevent the accumulation of AIR in the purine biosyn- Aconitase thetic pathway, but not enough to support growth on activity& 0-galactosidase SC media lacking aspartate. Consequently, this result Strain activity" (SC-His') WD' WL confirms the partial suppression of asp5 by the rtg2-2 allele, initially observed on SC -D -T medium. Similar FYC5/Z 44 results for pigmentation were later seen in the cross FYC5/Z-V1 8 FYc5328 28 involving the original rtg2-2 and asp5 mutations. To FYC5-Vl 0 371 know if this suppressor effect was reciprocal, we mea- suredthe 0-galactosidase activity in each segregant "Average values given as Miller units and determined on from six of the 12 tetrads shown in Figure 4. No evi- three independent experiments. "Values shown here represent nmol of czFaconitate formed dence for other than2':2- segregation was seen for p- per min per mg of protein and were determined from two galactosidase expression (for all six tetrads), indicating independent experiments undercatabolite repression condi- that this partial metabolic suppression was probably tions (WD) and from only one underderepression conditions "unidirectional" (data not shown). WL). 'All cultures were performed to early log phase. DISCUSSION

3+:1- for eight of the 11 tetrads after several days at In this paper, we present the isolation and identifica- 28". To test the hypothesis that rtg2-2 was suppressing tion in S. cereuisiae of a nuclear gene required to main- the asp5 mutation and to pursue the study further, the tain, under catabolite repression conditions, awild-type genetic analysis was repeated but theoriginal rtg2-2 mu- level of expression of the ACOl gene, encoding mito- tation was replaced by the rtg2::TRPl null mutation, chondrial aconitase. A mutant was isolated, after EMS so that rtg2 segregants could be identified directly by mutagenesis, displaying a second phenotype associated tryptophanprototrophy. The diploid strain resulting to the aconitase activity decrease: a partial growth defect from a cross between FYCS/Z-Vl and SFY/Z-50a (see on synthetic minimal glucose medium that was partially Table 1) was sporulated and subjected to tetrad analysis. and fully overcome by aspartate and glutamate, respec- Growth tests (on the above-mentioned media) were car- tively. Sequence analysis revealed thatthe gene we ried out for segregants from 12 complete tetrads. Re- cloned by complementation of the growth defect was sults showed that these tetrads were all tetratype, but identical to RTG2, previously cloned, along with RTGI, that the 2+:2- segregation pattern on SC -D -T me- by LIAOand BUTOW(1993) as genes required for CZT2 diumremained 2+:2- even after severaldays at 28". retrograde regulation. These tetrads were kept on a WD plate at 4". After RTG genes andretrograde communication: RTGI several weeks, we observed that for each tetrad, three encodes a proteinof 177 amino acids resembling mem- spores were whiteand onewas pink like the asp5 parent bers of the bHLH family of transcription factors (LIAO (Figure 4). The pink/red coloration is well-known for and BUTOW 1993). RTG2 encodesa protein of 394 ade2 and adel strains and is due to the accumulation amino acids of unknown function and was recently in these mutants of a red pigment that apparently de- shown to be related to bacterial phosphatases and to rives from phosphoribosylaminoimidazole (also termed contain an ATPase domain of the Hsp7O/actin/sugar AIR), a substrate of the enzyme encoded by ADE2. AIR kinase superfamily (KOONIN1994). The term "retro- is carboxylated by this enzyme to form phosphoribosyl- grade communication" describes the fact that the mito- aminoimidazolecarboxylate (CAIR), which in turn is a chondrial state can influence expression of specific nu- substrate, with aspartate and ATP, of the enzyme en- clear genes, like CIT2, which encodes peroxisomal coded by ADEI, leading to phosphoribosylsuccinocar- citrate synthase (CS2) that operatesas part of the glyox- boxamide aminoimidazole (SAICAR) synthesis (JONES ylate cycle (ROSENKRANTZet al. 1986; LEWINet al. 1990). and FINK1982). Thus, the pink colorationis not unex- The CZT2 transcription is elevated six- to 30-fold in a pected for cells deficient in aspartate, since this defi- po petitecompared with isochromosomal respiratory ciency could result in reduced conversion of CAIR to competent (p') cells and this effect can be mimicked SAICAR, thereby resulting in an adel phenocopy by the absence of afunctional CITI geneencoding (JONES and FINK 1982).The single pink spore clone of mitochondrial citrate synthase (CSI) (LIAO et al. 1991). each tetrad shown in Figure 4 bears only the asp5 muta- Recently, CHELSTOWSKAand BUTOW (1995) showed tion (indicated by a). Therefore, the segregant bearing that RTG genes are also requiredfor expression of both rtg2 :: TRPI and asp5 mutations (indicated by 6) is genes encoding peroxisomal proteins. systematically white, just like the wild-type RTG2 ASP5 RTG2 regulates ACOl expression: On the one hand, spore(indicated by c), and thatbearing the single the finding thatRTG2 also affects ACOl expression sup- 900 C. Velot, P. Haviernik and G. J.-M. Lauquin

FIGURE4.-Evidence of a partial metabolic suppression of asp5 by the rtg2 null allele. Strain FYC5/ZV1 (rtg2::TWI) was mated to strain SFY/Z-50a (asp5) and the resulting diploid was sporulated and subjected to tetrad analysis. Twelve tetrads were kept at 4” on YF’D plate, and the pink coloration appeared progressively over several weeks. Numbers from 1 to 12 refer to tetrads, and letters from a to d refer to segregants as follows: single asp5 mutant (a); double rtg2::TWl, asp5 mutant (b);wild- type RTG2, ASP5 segregant (c); single rtg2::TWl mutant (d).

ports the suggestion of LIAO and BUTOW(1993) that on synthetic minimal media, together with the recent RTGl and RTG2 act at a critical juncture between the finding that pyruvate carboxylase activityis reduced to glyoxylate and TCAcycles. On the other hand, this 50% in Artgmutants (SMALLet al. 1995), suggests that result indicates that RTG2 might not necessarily be a these mutants may be depleted for oxaloacetate. Thus specific actor of the retrograde regulation. Indeed, in it seems that RTG2 may also affect expression of en- contrast to the “CITI-CIT2system” where the CIT2 zymes that catalyze reactions leading to oxaloacetate transcription and CS2 activity are stimulated when mito- synthesis or oxaloacetate consumption, such as aspar- chondrial function is altered, an acol strain does not tate aminotransferase, encoded by ASH. This hypothe- displayany aconitase activity(GANGLOFF 1990) al- sis is supported by the partial suppression effect be- though an extramitochondrial one has been found in tween rtg2 and asp5 mutations, which gives evidence of wild-type cells (DUNTZEet al. 1969). Extramitochondrial direct or indirect interactions between the correspond- aconitase, whose origin is still not understood, is sup ing genes. posed to be involved in the glyoxylate cycle. Moreover, Finally, it appears that RTG2 acts at the juncturebe- we measured P-galactosidase activity inisogenic p+ and tween the Krebscycle andthe anaplerotic pathways po strains transformed with a plasmid bearing theAC01- leading to TUintermediate synthesis. lacZ fusion and no significant difference was observed Auxotrophies and nutrient requirementsof rtg2 mu- (data not shown). Theabsence of aconitase activity in tants: It is currently accepted that the glutamate re- mol cells indicates that not only the TCA but also the quirement phenotype for aconitase-deficient cells is glyoxylate cycle is altered in the mutant as it is for cells due to an a-ketoglutarate depletion (OGURet al. 1964), bearing a double disruption of CZTl and cIT2. Conse- but its understanding needs that of nitrogen metabo- quently, it appears that ACOl is required for a func- lism. The nitrogen source used in synthetic minimal tional glyoxylatecycle, either directly or indirectly. and complete media is usually ammonium sulfate, and Thus, it seems that RTG2 is related to genes required utilization of ammonia occurs exclusively by its incorpo- for expression of a fully functional glyoxylatecycle rationinto glutamate and glutamine (MAGMANE rather thanto genes whose expression is elevated when 1992), the main donors of cellular nitrogen (REITZER mitochondrial function is impaired. However, it is likely and MAGMANIK 1987). This incorporation requires a that the role of RTG2 is not restricted to the controlof key metabolite that is a-ketoglutarate (MITCHELLand the glyoxylatecycle. Indeed,the fact that aspartate MAGASANIK 1983; MITCHELL 1985; WIAMEet al. 1985; could partially restore the growth defect of an rtg2 strain FOLCHet al. 1989; MILLER and MAGMANIK 1990; MAGA- Aconitasein Regulation S. cereuisiae 90 1

SANIK 1992). Therefore, it was theoretically possible to grow fully on rich media with glycerol, lactate, ethanol restore growth of the rtg2 mutants on minimal media or acetate as carbon source, in contrast to strains bear- by supplying a nitrogen source ableto give rise to gluta- ing the glul-1 mutation or anacol null allele, which do mate without passing througha-ketoglutarate. Since not grow at all on these rich media. However, LUO and proline is degraded directly to glutamate within mito- BUTOW (1993) showed that an rtg2 null mutant does chondria in a two steps (COOPER 1982),we expected not grow on minimal acetate/ammonia medium even that proline, when added to 0.1% w/v, would enable when supplemented with glutamate. We have con- rtg2mutants to grow at wild-type rate onminimal media. firmed this observation andnoted that this carbon This result was obtained and confirms that the pool of source utilization defect is specific for acetate and etha- a-ketoglutarate is limited in rtg2 mutants. Since proline nol (not seen with lactate or glycerol) and observed is absent from the SC medium, the normal growth of only on synthetic minimal media (and not on SC me- the rtg2 mutants observed on SC medium lacking gluta- dia) (data not shown). mate is probably due to the cumulative effect of several The inability of cells to grow on acetate is diagnostic nutrients, among which could be arginine, since it can of some defect in the TCA cycle (KIM et al. 1986; Mc- be degraded into proline (MIDDELHOVEN1964). How- ALISTER-HENNand THOMPSON1987; WSPAL et al. 1989; ever, we showed that arginine alone fails to restore the CUPPand MCALISTER-HENN1991). For rtg2 mutants, growth defect probably because its degradation pathway this phenotype, like the growth defect phenotype, is requires a-ketoglutarate as the amino group acceptor observed only on minimal media. This means that, in in the conversion of ornithine to glutamate-y-semialde- complete media, several nutrients contribute to restor- hyde catalyzed by ornithine transaminase (MIDDELHO- ing a normal pool of some Krebs cycle intermediates VEN 1964). (such as a-ketoglutarate and probably oxaloacetate) Aspartate was able to partially restore the growth of and thus to the recovery of a fully functional TCA cycle. rtg2 mutants on minimal medium, while LIAOand BU- When cells are shifted from a complete to a minimal TOW (1993) indicated that, like glutamate, it fully re- medium, the absence of some metabolites necessarily stores the growth of the null rtg2 mutant. This differ- requires some anabolic pathways, and the fact that rtg2 ence was unexpected and could be due to the presence mutants behave differently on these two types of media of differentauxotrophic markers, ie., to adifferent indicates that at least some of these pathways are con- composition of the minimal media. trolled (directly or indirectly) by RTG2. This is consis- In addition, we have shown that the partial restora- tent with the suggestion of SMALLet al. (1995) that tion of the growth observed with aspartate is also ob- phenotypes for the Artgmutant cells could result from tained with threonine but without additive effect, indi- the cumulative effect of many small changes in the en- cating that thepathway by which the restoration occurs zyme composition of the cell. is the same for aspartate and threonine. This is not In any case, the elucidation of the specific role of surprising because aspartate is the precursor of threo- the RTG genes requires the identification of new target nine (and methionine) via homoserine. Moreover, it genes aswell as the physiological conditions under has been observed that asp5 mutants can be fed by a which the RTG genes operate for each one of these combination of threonine and methionine (JONES and targets. Furthermore, it will be of great interestto deter- FINK1982) and that the aspartate auxotrophy of the mine whetherRTG2is an actor of the synergistic regula- asp5 mutants is best scored onmedia lacking both aspar- tion by glucose and glutamate of the ACOl gene. tate and threonine. Aspartokinase, the first enzyme of This investigation was supported in part by research grants from the common pathway for synthesis of threonine and the Centre National de la Recherche Scientifique, the University of methionine from aspartate, is end-product inhibited by Bordeaux I1 and the Commission of the European Communities (Ac- threonine (but notby methionine) and repressed fairly tion for Cooperation in Science and Technology with Central and strongly (four- to sevenfold) by addition of threonine, Eastern European Countries, ERB35/OPL933615). C.V. was “alloca- but onlyweakly (30%) by addition of methionine taire” of the French Ministere de la Recherche et deI’Enseignement Superieur. (JONES and FINK 1982). Therefore, threoninecould re- store the growth of the rtg2 mutants not by leading to LITERATURE CITED aspartate synthesis but by preventing the consumption of thedecreased pool of aspartate. Altogether, this BkM, A,“., Y.JIA, P. P. SI.ONIMMSKIand C. J. 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