MOLECULAR AND CELLULAR BIOLOGY, JUlY 1990, p. 3551-3561 Vol. 10, No. 7 0270-7306/90/073551-11$02.00/0 Copyright C) 1990, American Society for Microbiology of the Yeast Mitochondrial Aconitase Gene (ACOI) and Evidence of a Synergistic Regulation of Expression by Glucose plus Glutamate SERGE P. GANGLOFF, DIDIER MARGUET, AND GUY J.-M. LAUQUIN* Institut de Biochimie Cellulaire et Neurochimie, Centre National de la Recherche Scientifique, 1, rue Camille Saint Saens, 33077 Bordeaux Cedex, France Received 27 November 1989/Accepted 3 April 1990

We have isolated genomic clones complementing the aconitase-deficient strain (glul-1) of Saccharomyces cerevisiae. Identification of the aconitase gene was established by enzymatic assays and molecular analyses. The corresponding mRNA has been characterized, and its direction of transcription has been determined. The complete nucleotide sequence revealed strong amino acid homologies with the sequences of some peptides isolated from the mammalian protein. Disruption of the gene by deletion-insertion led to glutamate auxotrophy. Expression of the aconitase gene was sensitive to glucose repression and was synergistically down regulated by glucose and glutamate.

Aconitase (citrate [isocitrate] hydro-lyase; EC 4.2.1.3), an tion. Recent work with Bacillus subtilis has demonstrated enzyme of the Krebs cycle (18) located mainly in the that the levels of aconitase are further reduced when a mitochondrial matrix, catalyzes the reversible isomerization source of glutamate is supplied with a rapidly metabolized of the tricarboxylic acids (TCA) citrate and isocitrate. cis- carbon source (34). By contrast, glutamate alone, or in Aconitate is formed as an intermediary product which nor- combination with a poor carbon source (e.g., citrate), does mally does not dissociate from the enzyme during the course not lead to reduction of the level of this enzyme (12). We of the reaction (32, 45). Like the other TCA cycle enzymes, report here the cloning of the S. cerevisiae gene coding for aconitase from Saccharomyces cerevisiae is encoded in the the mitochondrial aconitase by complementation of the nucleus and transported into the mitochondria (39). This glul-J mutation. We characterize its transcript and show enzyme is composed of a single polypeptide chain of Mr that gene disruption leads to glutamate auxotrophy and the about 80,000 (32, 42, 45). Aconitase has been purified from petite phenotype. Preliminary studies of ACOI gene expres- different organisms, and homologies in amino acid content of sion are also presented, and subcellular localization of the mammalian, Candida lipolytica, and S. cerevisiae species gene product is discussed. suggest a common evolutionary origin (42). Of considerable interest is the characterization of the enzyme as an Fe-S MATERIALS AND METHODS protein (36). As the enzymatic reaction involves only dehy- dration and hydration steps, the presence of an Fe-S cluster, Growth and transformation of S. cerevisiae. All experi- normally implicated in electron transfer, was unexpected. ments described were performed with S. cerevisiae GRF18 This cluster appears to be a 4Fe-4S center capable of (MATa leu2-3,112 his3-11,15 canl-100), DBY747 (MATa reversible rearrangement to yield a 3Fe-4S center; the labile leu2-3112 his3-AJ trpl-289 ura3-52) or its derivatives, and iron seems to be involved in the binding of the enzyme the aconitase-deficient strain GL153 (MATat leu2-3,112 his3- substrate (2, 6, 15). Aconitase provides the best-character- Al ura3-52 glul-1), obtained by sporulation of the cross ized example of a nonheme iron enzyme that does not between MO-48-C (glul-1) and DBY747. SG3-2, SG4-6, function as an agent of electron transport. In yeast cells, SG6-1, and SG7-1 are the glul-J strain GL153 transformed aconitase also takes part in the functioning of the glyoxylate with the complementing plasmids of the same name. YPD cycle, and extramitochondrial aconitase activity has been and SD media were prepared as described previously (41). found (5). This dual cell localization has also been described YPR and SR were the same as YPD and SD except that 2% for citrate synthase activity, for which two functional nu- glucose was replaced by 2% raffinose; similarly, SDG and clear genes have been characterized (17, 29). In S. cerevi- SRG were the same as SD and SR but with the addition of siae, the first enzymes of the TCA cycle, i.e., citrate 1% glutamate. All synthetic media were supplemented with synthase and aconitase, are required both for synthesis of the required auxotrophic markers at a final concentration of glutamate and for efficient utilization of nonfermentable 20 mg/liter for histidine and uracil, 30 mg/liter for leucine and energy sources (e.g., glycerol, lactate, or acetate) (17, 24). tryptophan, and 100 mg/liter for glutamate. Yeast transfor- An aconitase-deficient strain (glul-J) has been isolated (24); mation was carried out either by the spheroplast method its main phenotypic features are glutamate auxotrophy, described by Beggs (1) or by the lithium method reported by citrate accumulation when glucose is present as a carbon Ito et al. (14). source, and inability to grow on acetate, lactate, ethanol, Bacterial strains and cloning vectors. Subcloning was per- and glycerol. Studies (28, 38, 47) of the effects of glucose formed with the Bluescript vector (Stratagene) and the repression on the activities of the TCA cycle enzymes have shuttle vectors YEp24 (3) and pFL44 (a gift from F. La- shown that aconitase activity is subject to catabolite regula- croute). The pFL44 vector was derived by insertion into the pUC19 Alul sites 629 and 747, respectively, of the URA3 HindIII fragment with BglII linkers and of the 2,um replica- * Corresponding author. tion origin MstI fragment with ClaI linkers. The host Esch- 3551 3552 GANGLOFF ET AL. MOL. CELL. BIOL. erichia coli strain was TG-1 [A(lac-pro) supE thi hsdD5 (F' ized by a common restriction pattern within a 7.5-kbp traD6 proA+B+ laclIq lacZ AM15)]. Competent cells were region. To confirm that the complementation of the aconi- prepared according to Maniatis et al. (20). Transformants tase mutation (i.e., ability to grow without an exogenous were grown on LB plates supplemented with 100 pug of supply of glutamate) was directly related to the presence of ampicillin per ml. these plasmids, we reintroduced them into the glul-J mutant Construction of plasmid pSE-3 AACO and gene replace- strain. The wild-type phenotype (i.e., glutamate indepen- ment. The 0.65-kilobase-pair (kbp) SalI-EcoRI fragment dence) was restored in all cases and was accompanied by from pAT153 was replaced with the 3.3-kbp SalI-EcoRI loss of the petite phenotype. We also assayed the enzymatic aconitase-bearing fragment from pSG7-1. The inner 0.35-kbp activities of crude extracts of the different transformants, BglII fragment was then removed, and the 1.2-kbp URA3 together with activities of the parental strain controls, which marker was inserted. The 4.2-kbp newly generated SalIl- had been grown on SD medium supplemented with 0.01% EcoRI fragment was then introduced into pFL44, in which glutamate for strains SG3-2, SG4-6, SG6-1, SG7-1, and the BglII sites resulting from the URA3 deletion had been DBY747, enzyme activities were, respectively, 160, 160, filled by reaction with Klenow enzyme and religated to form 180, 160, and 70 nmol of cis-aconitate transformed per min plasmid pSE-3 AACO. Gene disruption was performed ac- per mg of protein; no activity was detectable for strain cording to the one-step method described by Rothstein (35). GL153. The transformed cells yielded aconitase activities up DNA sequence analysis. Sequencing of both strands of the to 2.5-fold that obtained with wild-type strain DBY747, EcoRI-SalI aconitase-bearing fragment subcloned in both suggesting the presence of the aconitase structural gene on a orientations in the Bluescript vector was carried out as multicopy plasmid. The mitochondrial localization of this described by Sanger et al. (37). Sequences of the ACOJ gene activity has been investigated by cell fractionation experi- were serially deleted from upstream or downstream first with ments. In the mitochondrial fraction, we recovered 70% of exonuclease III and then with exonuclease VII (49). The the total activity and obtained with the transformed mutant ends were made blunt with T4 DNA polymerase, and the at three- to fourfold-higher specific activity than with the vectors were religated. The single-stranded , overlap- wild-type strain. In addition, total DNA from wild-type ping on at least 100 bp, were isolated by published methods strain DBY747 was digested with 10 different restriction (22), and primer extension was performed with the M13 enzymes, blotted onto nitrocellulose, and probed with the universal 17-mer primer. Approximately 450 bp was read 2.6-kbp PvuII-EcoRI fragment (Fig. 1B). The results indicate from each deleted fragment. that there are no BamHI, ClaI, EcoRI, or PvuII assays. Aconitase activity was assayed by an sites within this region and that KpnI, PstI, and XbaI are adaptation of the method described by Fansler and Lowen- present once and BglII and EcoRV are present twice in this stein (7). Crude extracts were prepared from 100-ml cultures fragment. The deduced genomic pattern is identical to the harvested in the early log phase. Cells were suspended in one determined by restriction analysis of the cloned DNAs potassium phosphate buffer (20 mM, pH 7.4) supplemented and indicates that no recombination had occurred during the with 1 mM phenylmethylsulfonyl fluoride and broken with cloning steps. glass beads by vortexing. After 5 min of centrifugation at Messenger identification and subcloning experiments. To 3,000 x g, the assays were performed on the supernatant identify more precisely the region responsible for the recov- fractions. Specific activities are given as nanomoles of ery of aconitase activity, we restricted the yeast insert DNA cis-aconitate transformed per minute per milligram of pro- from plasmid pSG3-2 with BamHI and KpnI into three tein. Protein concentration was determined by the biuret fragments (1.5, 3.5, and 5.0 kbp) (Fig. 2A). After nick method (10). This regimen produced activity measurements translation, these fragments were used as probes in Northern that were reproducible within 15%. Assays were performed hybridization experiments with wild-type RNAs. Probe I in duplicate, and results were averaged. (1.5 kbp) showed no autoradiographic signal, whereas probe Southern blots, Northern (RNA) blots, probes, and in vitro II (3.5 kbp) identified a 2.6-kb messenger corresponding to transcription experiments. S. cerevisiae genomic DNA was the predicted size of the transcript for the 80-kilodalton prepared by the method of Sherman et al. (41), digested with aconitase protein (42). Probe III (5.0 kbp) detected the appropriate restriction enzymes, subjected to electrophore- 2.6-kb messenger plus an unidentified 1.6-kb transcript (Fig. sis through 1% agarose gels, and blotted onto nitrocellulose 2A). as described by Southern (44). Yeast total RNA was ex- On the basis of these results, three different DNA frag- tracted from early-log-phase cells (41), subjected to electro- ments from plasmid pSG7-1, PstI-PstI (5.0 kbp), SaIl- phoresis through formaldehyde-agarose gels, and blotted BamHI (5.6 kbp), and SalI-EcoRI (3.3 kbp), were subcloned onto nitrocellulose (44). Blots were hybridized with purified into the corresponding polylinker restriction sites of the nick-translated (30) or oligonucleotide-labeled (8) probes. In shuttle vector pFL44 to form plasmids pPP-1, pSB-2, and vitro transcription reactions were carried out as directed by pSE-3, respectively. After transformation of the glul-1 mu- the supplier (Stratagene). tant strain, the colonies were tested for glutamate prototro- phy (Fig. 1B). The minimal region necessary for complemen- RESULTS tation of the aconitase glul-J mutation was located within the 3.3-kbp SalI-EcoRI fragment of plasmid pSG7-1. Since Isolation of the gene for mitochondrial aconitase. We sep- the Sal site of this subclone was derived from the YEp24 arately transformed the aconitase-deficient strain GL153 vector, the minimal yeast DNA necessary for complement- with the three different pools of DNA from the YEp24 ing this aconitase mutation lies within the 3.0-kbp region genomic libraries constructed by Carlson and Botstein (3). proximal to the SalI restriction site of plasmid pSG7-1. This The uracil-independent transformants were plated onto glu- is in agreement with the 2.6-kb size of the mRNA detected tamate-lacking medium, and 11 independent clones were by Northern blot hybridization experiments. Moreover, the selected. The DNA from the transformed colonies was PstI-PstI construction indicates that the region from the PstI prepared, and four different plasmid species were isolated site to the Sall site is necessary for the recovery of aconitase (pSG3-2, -4-6, -6-1, and -7-1; Fig. 1A) which were character- activity. VOL. 10, 1990 YEAST ACONITASE GENE 3553

1 kb v -.1211-Z-11 z I pSG3-2

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L/ ,' .-' ' g.. ." /. r, -1 el -, z -/- -..r-z, ., Z .., Z 11- -, " " -, -., .., ,, Z ", -.., '-' -/-1, ., -e., al -/ / 4 ., ,, Z _7 --, 7.1 .7 ". pSG06-1

if S v X E II L E C V PXR KL L R E: C B IYJEX K E XEi 11 I 11 11 III 11I 1 1 1 11 1111 III ACOC Chromosonial locus ({DBY747)

S VP E E B I'l1-1 I-' n ~B L C E R m- H K P V X E I pSG7-1 _f 1T I I 1 I

23. g 9.4 >" _ 6.6 PPP-I 4.4 -- gm II ITIDl

2.3 am 2.( - >- pS1B-2 I 1 ILIZ LL]

pSE-3 AbilitL lo I kb conmplemeiit the glu -I mnUtaLioln

FIG. 1. Restriction map and subcloning analysis of the ACOI gene. (A) Restriction map of the genomic area around the aconitase locus of wild-type strain DBY747. Hatched boxes over the map indicates the extent of DNA inserts of the Yep24 plasmids encoding aconitase activity; the hatched box below the map represents the DNA fragment used to probe the Southern blot. (B) Southern analysis of genomic DNA. Wild-type DNA (1 ,ug) was cut with restriction enzymes, and the resulting DNA fragments were hybridized at high stringency to the 32P-labeled PvuII-EcoRI fragment on a Southern blot. Arrows on the left indicate the DNA size standards of lane m. Sizes are given in kilobases. (C) Localization of ACOJ within the genomic insert of pSG7-1. pPP1, pSB-2, and pSE-3 harboring various DNA fragments from pSG7-1 were used to transform the mutant strain, and each construction was tested for glutamate prototrophy (indicated as + or -). Restriction site abbreviations: B, BamHI; E, EcoRI; H, HindIII; K, KnpI; L, BglII; P, PstI; R, EcoRV; S, Sall; V, PvuII; X, XbaI; C, ClaI.

Determination of transcription direction of the ACOI mes- plasmid to form pSE-3 AACO (see Materials and Methods). senger. To determine the orientation of the putative aconi- The resulting 3.4-kbp PvuII-EcoRI linear fragment in which tase transcript, in vitro transcription experiments were per- URA3 is inserted in the opposite transcriptional direction formed. The 3.3-kbp SalI-EcoRI fragment from pSG7-1 was was used to transform strain DBY747 to uracil prototrophy subcloned into the corresponding sites of the Bluescript+KS (Fig. 3a). Transformed cells were then tested on synthetic polylinker, whereas the 2.6-kbp PvuII-EcoRI was inserted medium lacking glutamate. All of the uracil prototrophs into the Smal-EcoRI sites, thus yielding the reverse orien- failed to grow when plated on glutamate-lacking medium, tation. Transcription was performed from the T7 promoter, and no aconitase activity was detectable in crude extracts and the labeled RNA probes were hybridized with wild-type made from cultures of these colonies, indicating that the RNAs (Fig. 2B). The 2.6-kb autoradiographic signal was construction had inactivated the ACOI gene. In addition, all obtained with the latter construction only, indicating that of the transformants were found unable to grow on glycerol transcription proceeds from the EcoRI to the PvuII site (Fig. medium and exhibited a petite phenotype when tested with 2B). Similar experiments carried out with single-stranded the triphenyltetrazolium chloride technique (43). To confirm probes (13) derived from the 5.0-kbp BamHI-KpnI fragment that the transformants carried the disrupted acol:: URA3 cloned in M13mpl8 and -mpl9 vectors suggest that the allele, we performed Southern analyses of genomic DNA 1.6-kb unidentified transcript is transcribed in the same digested with EcoRI and KpnI enzymes from untransformed direction as the ACOJ gene (data not shown). and transformed cells (Fig. 3b). The 1.2-kbp intragenic Disruption of the ACOI gene and genetic linkage analysis. If EcoRV fragment spanning the BglII deletion was used as a the ACOJ gene is essential for cell viability in the absence of probe. As expected, since there is one KpnI restriction site exogenous glutamate, its disruption in haploid cells should and no EcoRI site within the probe, the deletion-insertion lead to lethality in growth medium lacking glutamate. We event increased the size of the EcoRI and KpnI fragments inserted the 1.2-kbp Bglll URA3 selectable marker from the spanning the BglII sites by about 0.8 kbp, leaving unchanged pFL44 vector, in which the inner PstI site had been removed the size of the second KpnI fragment detected by the probe. by mutagenesis (F. Lacroute, personal communication), into To exclude the possibility that ACOJ is an extragenic the 0.35-kbp BglII-deleted aconitase gene borne on pSE-3 suppressor of the glul-l aconitase mutation, we crossed the 3554 GANGLOFF ET AL. MOL. CELL. BIOL.

A B

1 11 111 1 11

1i -< 2.6 k b :. i. 4 1. k

2.6 kb->- Olga

B I E V K E F' B E K T7 B SE V S X I I I t- I I 111

II A4 C0 T7 Vi 2 R E S X LI i I I I II tco FIG. 2. Northern analysis of the ACOI locus. (A) Transcription map of the ACO1 region. Total RNA (8 sLg) from wild-type strain DBY747 was subjected to electrophoresis, blotted onto nitrocellulose, and hybridized with 32P-labeled DNA probes. The map represents 10 kbp of insert DNA from plasmid pSG3-2. The fragments obtained by KpnI-BamHI restriction, called I, II, and III, were used to probe the RNAs. The open bar represent the presumed coding sequence of the aconitase gene. (B) Determination of transcription direction of the aconitase messenger. In vitro transcription experiments were performed from the T7 promoter; the resulting labeled RNAs were hybridized on total wild-type RNAs. Lanes I, RNA probe obtained from the SalI-EcoRI fragment of pSE-3 inserted into the corresponding sites of the Bluescript polylinker; II, RNA probe obtained from the PvuII-EcoRI fragment of pSE-3 subcloned into the SmaI-EcoRI sites of the Bluescript polylinker. The arrow adjacent to the T7 promoter indicates the direction of transcription of the RNAs used to probe the Northern blot. The thick arrows below the constructions indicate the deduced direction of transcription of the aconitase gene. T7, T7 promoter; (V), the SmaI site of the Bluescript polylinker eliminated by ligation with the PvuII end of the insert DNA. The open bar shows the inserted aconitase DNA. Sizes of the various RNAs detected were determined by comparison with a lambda scale (not shown). Restriction site abbreviations are as for Fig. 1. glul-J mutant strain GL153 (MATot leu2-3,112 his3-AJ ura3- polylinker sites, respectively. We crossed the resultant 52 glul-J) with the newly generated strain AACO1 (MATa integrated GL153 strain (MATot leu2-3,112 his3-AJ ura3-52 ura3-52 leu2-3,112 his3-AJ trpl-289 acol::URA3). The re- gluJ-J: :pBS-ESU) with the glul-J mutant strain GL152 sulting diploid cells (Ura+ Trp+) did not grow on synthetic (MATa leu2-3,112 his3-AJ ura3-52 glul-J trpl-289) and se- medium lacking glutamate. Because of poor germination lected the diploid cells on the basis of size. Random spore capacity of the haploid spores, random spore analysis was analysis was performed on 100 haploid spores, and 52 Glu+ carried out. We analyzed 75 Trp+ colonies obtained after and 53 Trp- spores were scored; among the 52 Glu+ spores, ether treatment of the sporulated diploids and found that all 27 were Trp+. This result is indicative of a 2+:2- segrega- of them were Glu-, 38 were Ura+, and 37 were Ura-. We tion pattern for glutamate and tryptophan auxotrophies, also verified that the analyzed clones were haploid by confirming that we did not clone an extragenic suppressor of crossing them with tester strains. Thus, we can infer from the glul-J mutation. These results indicate that the cloned that result that spores probably segregated 0+ :4- for gluta- ACOJ DNA is the GLUJ DNA. mate auxotrophy and 2+:2- for uracil auxotrophy, indicat- Sequence analysis of the ACOJ DNA. The EcoRI-SalI ing that the cloned ACOJ DNA is genetically linked to the aconitase-bearing fragment on the Bluescript vector was GLUI DNA. To confirm this linkage, we integrated the entirely sequenced in both orientations as described in ACOJ DNA at the glul-J locus of strain GL153 by transfor- Materials and Methods; one single large open reading frame mation with the ClaI-linearized plasmid pBS-ESU (data not (ORF) was found on this DNA fragment. The nucleotide and shown) and observed that a single copy of this DNA is derived polypeptide sequences are shown in Fig. 4. The capable of reversing the mutant phenotype. Plasmid pBS- ORF extends for 2,337 bp and can be translated into 779 ESU is a Bluescript vector in which the URA3 BglII frag- amino acid residues (Mr 85,685). The first 24 residues of the ment from pFL44 and the EcoRI-SalI ACOJ-bearing frag- protein encoded by the ORF include an abundance of ment have been inserted into the BamHI and EcoRI-SalI hydrophobic residues, five basic and five hydroxylated (Ser VOL. 10, 1990 YEAST ACONITASE GENE 3555

a .) EIZ2 R probe b) E m K 1 2 1 2 E V- PRKLLR E E p I I B11 II1LF I DBY747 ,':I 3.04

S V PR KL LR E 9.4 I ---I -IL pSE-3 AACOI UTRA3 4.4 ->C E V PR KL LR E E p I1 _11F 2.3 URA 3 DBY747 AACOI - 0

FIG. 3. Disruption of wild-type aconitase gene by deletion-insertion. (a) Restriction maps of the aconitase gene in the disrupted and wild-type strains. The middle drawing represents the plasmid bearing the deleted-inserted aconitase gene whose PvuII-EcoRI linear fragment was used to transform the wild-type strain (see text). Open bars represent the aconitase gene; the hatched box represents the deleted portion; the direction of transcription of the inserted URA3 gene is shown by a black arrow within the box. The probe is the EcoRV fragment represented above the map of the wild-type strain. Enzyme restriction sites are as for Fig. 1. (b) Southern hybridization of chromosomal DNA from deletion mutant and wild-type strains. Mutant (lanes 1) and wild-type (lanes 2) DNAs were restricted by EcoRI (E) or KpnI (K). The probe is the labeled EcoRV fragment (see above). The indicated scale was generated by restricting lambda DNA with Hindlll and probing with 32P-labeled lambda DNA (lane m). and Thr) amino acids, and a lack of acidic residues, a To investigate whether the same situation occurs in S. common feature of mitochondrial presequences (46). There- cerevisiae, cultures were grown on different media. Cultures fore, this amino-terminal region is likely responsible for the grown to early log phase were divided into two parts, one to mitochondrial targeting of the ACOJ product. be assayed for aconitase activity and the other to be assayed The codon usage in ACOJ was compiled, and the codon for extraction of total RNA, which was hybridized with the bias index according to Sharp and Li (40) was calculated to 1.25-kbp EcoRV intragenic probe. be 0.43, which indicates a moderately expressed gene. We Preliminary experiments performed on cells grown on analyzed the 5' noncoding region and found a putative raffinose, a fermentable carbon source that does not cause TATA box (TATATAAT; positions -141 to -134; under- catabolite repression to nearly the same extent as glucose lined in Fig. 4). Interestingly, we also found at positions (50), displayed degrees of aconitase activity and transcript -291 to -284 a sequence corresponding to the inverted similar to those obtained with a nonfermentable carbon upstream activation sequence UAS2 UP1 (9), which has source (e.g., glycerol), indicating that raffinose does not been shown to be a HAP2-HAP3-responsive site of the result in the repression of aconitase (data not shown). On the CYCI gene (25). contrary, the results (Table 1) indicated that aconitase activ- Comparison of the deduced amino acid sequence with ity was down regulated about threefold by the presence of cysteinyl-tryptic peptides from beef heart aconitase. Former glucose as the sole carbon source in either rich or synthetic studies focused on isolation of the reactive cysteine of beef medium, as compared with the activity obtained with raffi- heart aconitase (26) have led to the determination of the nose as the sole carbon source (catabolite regulation). Most amino acid sequence of seven cysteine-containing oligopep- of the mitochondrial enzymes are subject to a similar regu- tides; no similarities between these sequences and that lation (28, 38, 47). When glutamate was added to the culture reported for the phenacyl bromide-reactive cysteine-con- (SDG and SRG media), the activity was further reduced taining peptide isolated from pig heart aconitase (11) have twofold in the presence of glucose but not significantly with been reported. We compared our deduced amino acid se- raffinose. These data suggest that glutamate severely de- quence and those determined earlier and also found no creases aconitase activity only when sufficient amounts of apparent similarity with the pig heart aconitase peptide glucose are present in the medium. To define more accu- sequence, whereas all seven oligopeptides from beef heart rately the level at which this regulation occurs, we analyzed aconitase were found to be present in the mitochondrial the relative amounts of aconitase RNA present in the cells aconitase of S. cerevisiae, with similarities ranging from grown on the various media. Both catabolite regulation and about 50 to nearly 100% (summarized in Fig. 5). These amino synergistic repression by glucose and glutamate occurred acid sequence comparisons together with the results pre- principally at the mRNA accumulation level (Fig. 6). Indeed, sented above provide concrete evidence that the cloned the relative abundance of the transcripts as characterized by ACOJ DNA corresponds to the mitochondrial aconitase Northern blot experiments was closely related to the enzyme structural gene. activities found for the same cultures. Regulation of aconitase expression. Recent studies on B. subtilis (34) have raised the problem of the regulation of DISCUSSION aconitase expression. In this organism, aconitase is down regulated at the transcriptional level by glucose alone (ca- In this report, we describe the isolation and identification tabolite regulation) or synergistically by glucose and gluta- of a nuclear gene encoding the S. cerevisiae mitochondrial mate. aconitase. Several key enzymes of intermediary metabolism gaattccaaaggctcacaaatggcattccgtactgatatacttcgcactttacatatgcttatataaaaat -577

gccagtttcccatcgtcaattacgcagaggtagccttcgtttattttctctctttttgtatatcattgtatt -505

aatcataatccattgtattttactttgtcttatctggctttaatgaatgacgccggtcacacgcgggtgcct -433

gattctcgattgtgccaagccatttgggcacggtgtcaaattacctaaaaaatggccgagagccgcaaaagg -361

gaggtccgcggggccgggcaataccctttgtttttcgagcatttcggcgccgaaatcggaaaggtcctgacc -289

aatcaatagagaaattagtgcatacgagaaaaatttgaacccttcagttgttctccgcaggcgactttaacc -217

atcaaacctccaatcgctgccggttttcggaaaggcaagcacaaaaagggaggacaaggaaaatcttttgtt -145

atatatataatcttccggttttagaggttaattaggaggagtatgttgcttaattccgttgtcccttgttct -73

gttcactctttcttagttattacatagtagaacgaaggtaataaatactatcattattactatagatataca -1

ATG CTG TCT GCA CGT TCT GCC ATC AAG AGA CCC ATT GTT CGT GGT CTT GCG ACA 54 Met Leu Ser Ala Arg Ser Ala Ile Lys Arg Pro Ile Val Arg Gly Leu Ala Thr 18

GTC TOC AAC TTG ACT AGA GAT TCA AAA GTC AAC CAA AAC TTA TTA GAA GAT OAT 108 Val Ser Asn Leu Thr Arg Asp Ser Lys Val Asn Gln Asn Leu Leu Glu Asp His 36

TCT TT ATT AAC TAC AAG CAG AAT GTG GAA ACG CTG GAT ATC GTA AGA AAA AGA 162 Ser Phe Ile Asn Tyr Lys Gln Asn Val Glu Thr Leu Asp Ile Val Arg Lys Arg 54

TTA AAC AGG CCAQTT ACC TAC GCG GAA AAG ATT TTG TAC GGT OCA TTG GAT GAC 216 Leu Asn Arg Pro Phe Thr Tyr Ala Glu Lys Ile Leu Tyr Gly His Leu Asp Asp 72

CCT OAT GGT CAA GAT ATT OAG AGA GGT GTT TCA TAC CTA AAA TTA AGA CCA GAT 270 Pro His Gly Gln Asp Ile Gln Arg Gly Val Ser Tyr Leu Lys Leu Arg Pro Asp 90

CGT GTT GCC TGT CAA GAT GCT ACT GCT CAA ATG GCT ATT TTA MA TTT ATG TCC 324 Arg Val Ala Cys Gln Asp Ala Thr Ala Gln Met Ala Ile Leu Gln Phe Met Ser 108

GCT GOT TTA CCA CAG GTT GCT AAG CCA GTC ACT GTC QCA TGT GAC OAT TTG ATT 378 Ala Gly Leu Pro Gln Val Ala Lys Pro Val Thr Val His Cys Asp His Leu Ile 126

CAM GCA CAM GTT GGT GGT GAA AAA GAT TTG AAG AGA GCT ATA GAT CTA AAC AAG 432 Gln Ala Gln Val Gly Gly Glu Lys Asp Leu Lys Arg Ala Ile Asp Leu Asn Lys 144

GAA GTT TAT GAT TTC TTG GCC TCT GCC ACT GCG AAA TAT AAC ATG GGT TTC TGG 486 Glu Val Tyr Asp Phe Leu Ala Ser Ala Thr Ala Lys Tyr Asn Met Gly Phe Trp 162

AAG CCA GGT TCC GGT ATC ATT QC CAA ATT GTT CTG GAA AAC TAC GCT TTC CCA 540 Lys Pro Gly Ser Gly Ile Ile His Gln Ile Val Leu Glu Asn Tyr Ala Phe Pro 180

GGT GCT TTG ATC ATT GGT ACT GAC TCC OAT AOA CCA AAT GCT GGT GGT TTA GGT 594 Gly Ala Leu Ile Ile Gly Thr Asp Ser His Thr Pro Asn Ala Gly Gly Leu Gly 198

CAA TTG GCT ATT GOT GTT GGT GGT GCT GAT GOC GTT GAT GTT ATG GCA GGT CGT 648 Gln Leu Ala Ile Gly Val Gly Gly Ala Asp Ala Val Asp Val Met Ala Gly Arg 216

CCA TGG GAA TTG AAG GCT CCA AAG ATC TTA GGT GTT AAG TTG ACT GOT AAG ATG 702 Pro Trp Glu Leu Lys Ala Pro Lys Ile Leu Gly Val Lys Leu Thr Gly Lys Met 234

AAC GGT TGG ACT TCT CCA AAG GAT ATT ATT TTG AAA TTG GCT GGT ATC AOA ACT 756 Asn Gly Trp Thr Ser Pro Lys Asp Ile Ile Leu Lys Leu Ala Gly Ile Thr Thr 252

GTC AAA GGT GGT ACT GGT AAA ATT GTT GAA TAT TTT GGT GAT GOT GTT GAC ACT 810 Val Lys Gly Gly Thr Gly Lys Ile Val Glu Tyr Phe Gly Asp Gly Val Asp Thr 270

TTC TOC GCT ACT GOT ATG GGT ACC ATT TGT AAT ATG GGT GOT GAA ATC GOT GCT 864 Phe Ser Ala Thr Gly Met Gly Thr Ile Cys Asn Met Gly Ala Glu Ile Gly Ala 288 ACC AOA TCT GTT TTC CCA TTC AAC AAA TCT ATG ATT GAA TAT TTG GAA GCA ACT 918 [EIThr Ser Val Phe Pro Phe Asn Lys Ser Met Ile Glu Tyr Leu Glu Ala Thr 306 GGT CGT GGT AAG ATC GCT GAC ITT GCT AAA TTA TAC OC AAG GAT CTA TTA TCT 972 Gly Arg Gly Lys IlelAla Asp Phe Ala Lys Leu Tyr His Lys Asp Leu Leu Ser 324 GCT GAT AAG GAT GCT GAA TAC GAT GAG GTC GTC GAA ATT GAC TTG AAC ACT CTG 1026 Ala Asp Lys Asp Ala Glu Tyr Asp Glu Val Val Glu Ile Asp Leu Asn Thr Leu 342 FIG. 4. Nucleotide and deduced amino acid sequences of the ACOI gene with its 5'- and 3'-flanking regions (GenBank accession number M33131). The noncoding strand and translation of the largest ORF are presented. The nucleotide sequence is numbered from nucleotide 1 of the presumed initiation codon. A potentially active TATA box and the inverted upstream activation sequence UAS2 UP1 are underlined (-145 to -134 and -291 to -284, respectively). The boxed amino acid sequences represent the oligopeptides corresponding to those sequenced by Plank and Howard (26). Oligopeptide numbering given in the text is depicted in Fig. 5. 3556 GAA CCA TAC ATC AT GGG CCA m ACC CCC GAT TTG GCT ACT CCA GTT TCT AAG 1080 Glu Pro Tyr Ile Asn Gly Pro Phe Thr Pro Asp Leu Ala Thr Pro Val Ser Lysi 360 ATG AAG GAA GTT GCT GTT GCT AAT AAC TGG CCA TTG GAT GTC AGA GTC GGT TTG 1134 Met Lys Glu Val Ala Val|Ala Asn Asn Trp Pro Leu Asp Val Arg|Val Gly Leu | 378

ATC GGT TCT TGT ACC AAT TCC TCT TAT GAA GAT ATG TCT CGT TCA GCA TCC ATT 1188 Ile Gly Ser Cys Thr Asn Ser Ser Tyr Glu Asp Met Ser Arg Ser Ala Ser Ile 396

GTC AAG GAC GCT GCT GCT CAT GGT TTG AAA TCC AAG ACC ATr TTC ACT GTT ACT 1242 Val Lys Asp Ala Ala Ala His Gly Leu Lys Ser Lys Thr Ile Phe Thr Val Thr 414

CCA GGT TCT GAA CAA ATC AGA GCC ACT ATT GAA CGT GAT GGC CAA TTA GAA ACC 1296 Pro Gly Ser Glu Gln Ile Arg Ala Thr Ile Glu Arg Asp Gly Gln Leu Glu Thr 432

TTC AAA GAA TTT GGT GGT ATC GTT TTG GCA AAC GCC TGT GGC CCA TGT ATT GGT 1350 Phe Lys Glu Phe Gly Gly Ile Val Leu Ala Asn Ala Cys Gly Pro Cys Ile Gly 450

CAA TGG GAT CGT AGA GAT ATC AAG AAA GGT GAC AAG AAT ACT ATT GTT TCC TCT 1404 Gln Trp Asp Arg Arg Asp Ile Lys Lys Gly Asp Lys Asn Thr Ile Val Ser Ser 468

TAC AAC AGA AAT TTC ACT TCT AGA AAT GAT GGT AAC CCA CAA ACT CAT GCT TmT 1458 Tyr Asn Arg Asn Phe Thr Ser Arg Asn Asp Gly Asn Pro Gln Thr His Ala Phe 486

GTT GCA TCT CCA GAA TTA GTA ACT GCG TTC GCC ATT GCG GGT GAT TTG AGA TTC 1512 Val Ala Ser Pro Glu Leu Val Thr Ala Phe Ala Ile Ala Gly Asp Leu Arg Phe 504

AAC CCT CTA ACA GAC AAA TTA AAG GAC AAG GAT GGT AAT GAG TTC ATG TTG AAA 1566 Asn Pro Leu Thr Asp Lys Leu Lys Asp Lys Asp Gly Asn Glu Phe Met Leu Lys 522

CCA CCA CAT GGT CGA TGG TmT GCC TCG AAA GAG GTT ATG ATG CTG GTG AGA ACA 1620 Pro Pro His Gly Arg Trp Phe Ala Ser Lys Glu Val Met Met Leu Val Arg Thr 540

CTT ACC AAG CTC CAC CTG CAG ACC GTA GCC ACC GTT GAA GTT AAA GTT TCT CCA 1674 Leu Thr Lys Leu His Leu Gln Thr Val Ala Thr Val Glu Val Lys Val Ser Pro 558

ACT TCA GAC CGT CTA CAA CTG TTG AAA CCA TTC AAA CCT TGG GAT GGT AAG GAT 1728 Thr Ser Asp Arg Leu Gln Leu Leu Lys Pro Phe Lys Pro Trp Asp Gly Lys Asp 576

GCT AAA GAC ATG CCA ATC TTG ATT AAG GCC GTC GGT AAG ACA ACT ACT GAT CAT 1782 Ala Lys Asp Met Pro Ile Leu Ile Lys Ala Val Gly Lys Thr Thr Thr Asp His 594

ATT TCT ATG GCT GGT CCA TGG TTG AAA TAC AGA GGT CAT TTA GAA AAC ATT TCT 1836 Ile Ser Met Ala Gly Pro Trp Leu Lys|Tyr Arg Gly His Leu Glu Asn Ile Ser 612

AAT AAC TAT ATG ATT GGT GCT ATT AAT GCT GAA AAC AAG AAG GCT AAC TGT GTT 1890 Asn Asn Tyr Met Ile Gly Ala Ile Mn Ala Glu Asn Lys Lys Ala Asn Cys Val 630

AAA AAT GTA TAT ACT GGT GAA TAC AAA GGT GTT CCA GAC ACT GCT AGA GAT TAC 1944 Lys Asn Val Tyr Thr Gly Glu Tyr Lys Gly Val Pro Asp Thr Ala Arg Asp Tyr 648

AGA GAC CAA GGT ATC AAG TGG GIT GTT ATT GGT GAT GAA AAC TTT GGT GAA GGT 1998 Arg Asp Gln Gly Ile Lys Trp Val Val Ile Gly Asp Glu Asn Phe Gly Glu Gly 666

TCC TCT CGT GAA CAC GCT GCT TTG GAA CCA AGA TTC TTG GGC GGT TTC GCT ATC 2052 Ser Ser Arg Glu His Ala Ala Leu Glu Pro Arg Phe Leu Gly Gly Phe Ala Ile 684 ATC ACA AAG TCT TTC GCT CGT ATC CAT GAA ACT AAC TTG AAA AAA CAA GGT CTA 2106 Ile Thr Lys Ser Phe Ala Arg Ile His Glu Thr AMn Leu Lys Lys Gln Gly Leu 702

TTG CCA TTG AAC TTC AAG AAC CCA GCT GAC TAT GAC AAG ATC AAC CCT GAT GAC 2160 Leu Pro Leu Asn Phe Lys AMn Pro Ala Asp Tyr Asp Lys Ile Asn Pro Asp Asp 720

AGA ATC GAT ATT CTG GGT CTA GCT GAA TTG GCT CCA GGT AAG CCT GTA ACA ATG 2214 Arg Ile Asp Ile Leu Gly Leu Ala Giu Leu Ala Pro Gly Lys Pro Val Thr Met 738

AGA GTT CAT CCA AAG AAT GGT AAG CCA TGG GAT GCT GTG TTG ACC CAT ACT TTC 2268 Arg Val His Pro Lys Asn Gly Lys Pro Trp Asp Ala Val Leu Thr His Thr Phe 756

AAC GAT GAG CAA ATT GAA TGG TTC AAA TAT GGT TCT GCC TTA AAT AAA ATT AAG 2322 Asn Asp Glu Gln Ile Glu T Phe Lys Tyr Gly Ser Ala Leu Asn Lys Ile Lys 774

GCC GAT GAG AAG AAA taatgaaaacattgttataatcttttaaaggttattatttattttgtcttc 2388 Ala Asp Glu Lys Lys 779

tgtacacgtacccttgtttatcttttctgccttaaatttaatgacgttcggctggagaagtcaagactatga 2460

aatatatctogtaatttatgatc 2483

3557 3558 GANGLOFF ET AL. MOL. CELL. BIOL.

114 VAKPVTVHCDHLIQAQVGGEK ** * * ****** ** **** VAVPSTIHCDHLIEAQLGGEK peptide 5

255 GGTGKIVEYFGDGVDTFSATGMGTICNMGAEIGAT **** **** * *** * *** ************ GGTGAIVEYHGPGVDSISCTGMATICNMGAEIGAT peptide 9

312 ADFAKL..YHKDLLSADKDAEYDEVVEIDLNTLEPYINGPFTPDLATPVSKMKEVAV ** * * ** * * ** ** * * * ********** ** ** ADIANLADEFKDXLVPDSGCHYDQLIEINLSELYPHINGpFTpDLAAHpv. .AEVGS peptide 10 376 VGLIGSCTNSSYEDMSR *************** * VGLIGSCTNSSYEDMGR peptide 3

435 EFGGIVLANACGPCIGQWDR ************** *** DVGGIVLANACGPCIGEWDR peptide 7

589 TTTDHISMAGPWLK ****** ****** CTTDHISAAGPWLK PePtide 4

729 ELAPGKPVTMRVHPKNGKPWDAVLTHTFNDEQIEW ***** * ** * ** * *** DFAPGKPLTCIIKHPNGTQETILLNHTPNETXIEW peptide 8 FIG. 5. Comparison of the ACOJ deduced amino acid sequence with sequences of cysteinyl-tryptic peptides from beef heart aconitase. The different amino acid sequences from S. cerevisiae are presented in the standard one-letter code and are aligned with the corresponding oligopeptides from beef heart aconitase by the Smith and Waterman program (43a). Breaks in the sequences are shown as periods and have been introduced to maximize identities. Homologies between the sequences are indicated by stars. The number above each pair of compared sequences indicates the position of the first amino acid in the S. cerevisiae peptide sequence. are known to have both cytosolic and mitochondrial iso- Plank (26) for the beef enzyme, the cysteines equivalent to forms. This is the case for citrate synthase (17, 29, 33), residues 199, 250, 305, 383, 565, and 714, found in peptides malate dehydrogenase (21), fumarase (48), and aconitase (5), 1, 9, 10, 2, 4, and 6/8, respectively, do not exist within the which are present principally in the mitochondria and to a yeast sequence. Conversely, cysteines 94 and 629 of the lesser extent in the cytoplasm, where they are implicated in yeast aconitase are not present in the beef enzyme. Recent the glyoxylate cycle. results by Robbins and Stout (31) indicated that the 4Fe The ACOJ gene has been isolated from a genomic library cluster of beef heart aconitase has three thiols ligands, in both orientations on YEp24 plasmids. The presence of the namely, cysteines 358, 421, and 424. Thus, possibly the gene is indicated by the following results: (i) all of the cloned equivalent cysteines in the yeast enzyme, i.e., 382, 445, and genomic DNAs share a common restriction pattern within 448, respectively, are also the putative thiols ligands for the 7.5 kbp and reverse the glutamate auxotrophy mutant phe- 4Fe-4S cluster. In contrast, Plank et al. (27) have recently notype; (ii) aconitase enzyme activities assayed on crude suggested the possibility of a four-thiol ligand system; they extracts of transformed cells were four times higher than propose that the cysteine 383 in peptide 2 could be the fourth those obtained on wild-type cells; (iii) and the nucleotide ligand. As there is no equivalent cysteine in the yeast sequence revealed amino acid homologies of up to 100% aconitase, the yeast 4Fe-4S cluster cannot involve a fourth with the seven cysteinyl-tryptic peptides from beef heart thiol ligand, and the same might be true for the beef aconitase characterized by Plank and Howard (26). aconitase. These authors have also suggested that the linear As previously noted for the beef enzyme (26), there is no 3Fe-4S cluster of purple aconitase uses both cysteines in clustering of cysteines. There are only 7 cysteine residues in peptide 7 and both cysteines in peptide 9 as ligands. The the yeast enzyme but 11 in the beef heart aconitase. More yeast enzyme cannot provide two cysteines within the specifically, according to the nomenclature of Howard and equivalent peptide 9, and thus it would be interesting to VOL. 10, 1990 YEAST ACONITASE GENE 3559

TABLE 1. Aconitase activity determined on crude extracts of different yeast strains grown on various culture media cn0 0 e Enzyme activity' (nmol of cis-aconitate Medium transformed/min per mg of protein) DBY747 GRF18 SD 68 55 SDG 25 20 SR 180 160 SRG 180 160 YPD 140 140 YPR 290 270 ACO_ - ' Average values determined on at least three independent experiments. ACT _ Activity in strain GL153 and the AACO mutant strain was not detectable. W know whether yeast aconitase also exists as a stable purple aconitase. Our comparisons are similar to those drawn by Plank and Howard (26) regarding the similarities to other Fe-S proteins (thioredoxins or ferredoxins). Contrary to FIG. 6. Regulation of expression of the aconitase gene detected their findings, however, there is no cysteine residue in our by Northern blot hybridization. Wild-type strain DBY747 was their grown on SD (lane SD), SR (lane SR), SDG (lane SDG), and SRG peptide sequence corresponding to oligopeptide 4, (lane SRG). RNAs isolated from these cultures (8 ,ug) were sub- which they had implicated as containing the single cysteine jected to electrophoresis, blotted onto nitrocellulose, and hybridized that is found at or near the and which can be with a 32P-labeled EcoRV aconitase fragment. Actin DNA was used modified by a variety of sulfhydryl reagents. The absence of to probe the various amounts of RNA present in each lane. ACO, this cysteine residue in S. cerevisiae would seem to support Aconitase transcript; ACT, actin messenger. the conclusions of Kennedy et al. (16), who suggested that this cysteine residue may interfere in substrate binding to the active site rather than being directly involved in the catalytic process. a related gene, the product of which is at a concentration too The ACOJ gene encodes the mitochondrial form in cells low to be detected by our biochemical assay. This product grown on a nonfermentable carbon source, and its disruption would not contain the necessary information to be targeted leads to both glutamate auxotrophy and petite phenotype. to the mitochondria and thus would be implicated only in the Therefore, if a second gene encoding cytosolic aconitase glyoxylate cycle. Further work is needed to understand the activity exists, it cannot compensate for the mitochondrial mechanism of aconitase partition between intra- and extra- isoform defect. Furthermore, no aconitase activity is detect- mitochondrial compartments. Amino acid sequence analysis able in crude extracts of disrupted cells, and Southern ofthe N-terminal part of both forms of aconitase would be of analysis did not reveal any homologous sequences at high considerable help in this direction. stringency of hybridization within the yeast genome; thus, In this study, we also explored the regulation of aconitase any second gene would have diverged quite early from its expression. This enzyme is required for growth on nonfer- mitochondrial counterpart. mentable carbon sources and for biosynthesis of glutamate. Taken together, these observations suggest the presence It has been reported for the bacterium B. subtilis that levels of a single-copy gene encoding both cytoplasmic and mito- of aconitase are regulated by the combination of a rapidly chondrial aconitase. Such a mechanism has been reported metabolized carbon source (e.g., glucose) and a source of recently for the TCA enzyme fumarase (48). glutamate (34); a similar control pathway operates for the Differential sorting of products encoded by a single gene citrate synthase of S. cerevisiae (17). have already been described for invertase (3), some tRNA We have shown by studying the mRNA steady-state levels synthetases (4, 23), and more recently fumarase (48). All of and enzymatic activities of cells grown in various conditions these enzymes are synthesized as signal sequence-bearing that the pattern of regulation of aconitase in S. cerevisiae is precursors whether they are targeted to the mitochondria or superimposable on that observed for B. subtilis aconitase secreted into the periplasmic space. It has been shown that and S. cerevisiae citrate synthase; moreover, it would ap- this regulation is related to the presence of two transcription pear in these three cases that primary control is exercised at start sites and two initiation codons, one upstream and one the level of transcription. Furthermore, recent results have downstream of the signal sequence. In the case of the shown that expression of the aconitase gene is under the mitochondrial aconitase of S. cerevisiae, no second transla- control of both HAP2 and HAP3 genes (unpublished data). tion start is found in the vicinity of the first ATG; the closest More accurate information about specific regulation will be one would be Met-101, and therefore another kind of mech- provided by the study of mutant strains affected in the anism accounting for the dual cell localization must be expression of both aconitase and citrate synthase. considered. The observation that the strain carrying the disrupted acol allele displays no aconitase activity might be explained by the existence of a single-copy gene whose ACKNOWLEDGMENTS product is not localized solely within the mitochondria S. P. Gangloff is "allocataire" of the French Ministere de la because of a low efficiency of the targeting signal. Thus, the Recherche et de l'Enseignement Superieur. This work was sup- aconitase enzyme fraction that does not enter the mitochon- ported in part by research grants from the University of Bordeaux dria could operate in the glyoxylate cycle. Another explana- II, from la Fondation pour la Recherche Medicale, and from the tion for the results presented above may be the presence of Centre National de la Recherche Scientifique. 3560 GANGLOFF ET AL. MOL. CELL. BIOL.

ADDENDUM IN PROOF 20. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, In our discussion on subcellular localization of aconitase Cold Spring Harbor, N.Y. we have overlooked previous work on shared gene products 21. McAlister-Henn, L., and L. M. Thompson. 1987. Isolation and concerning the TRMI and MOD5 genes (J.-M. Li, A. K. expression of the gene encoding yeast mitochondrial malate Hopper, and N. C. Martin, J. Cell Biol. 109:1411-1419, 1989; dehydrogenase. J. Bacteriol. 169:5157-5166. D. Najarian, M. E. Dihanich, N. C. Martin, and A. K. 22. Messing, J. 1983. New M13 vectors for cloning. Methods Hopper, Mol. Cell. Biol. 7:185-191, 1987). A porcine aconi- Enzymol. 101:20-78. 23. Natsoulis, G., F. Hilger, and G. R. Fink. 1986. The HTSl gene tase cDNA was recently cloned and sequenced (L. Zheng, encodes both the cytoplasmic and mitochondrial histidine- P. C. Andrews, M. A. Hermodson, J. E. Dixon, and H. tRNA synthetases of S. cerevisiae. Cell 46:235-243. Zalkin, J. Biol. Chem. 265:2814-2821, 1990). Comparison of 24. Ogur, M., L. Coker, and S. Ogur. 1964. Glutamate auxotrophs the deduced amino acid sequence with that of yeast indi- in Saccharomyces. I. The biochemical lesion in the glt-l mu- cated 70% similarity between both sequences. tants. Biochem. Biophys. Res. Commun. 14:193-197. 25. Olesen, J., S. Hahn, and L. Guarente. 1987. Yeast HAP2 and LITERATURE CITED HAP3 activators both bind to the CYCI upstream activation 1. Beggs, J. D. 1978. Transformation of yeast by a replicating site, UAS2, in an interdependent manner. Cell 51:953-961. hybrid plasmid. Nature (London) 275:104-109. 26. Plank, D. W., and J. B. Howard. 1988. Identification of the 2. Beinert, H. 1986. Iron-sulfur clusters: agents of electron transfer reactive sulfhydryl and sequences of cysteinyl-tryptic peptides and storage, and direct participants in enzymic reactions. Bio- from beef heart aconitase. J. Biol. Chem. 263:8184-8189. chem. Soc. Trans. 14:527-533. 27. Plank, D. W., M. C. Kennedy, H. Beinert, and J. B. Howard. 3. Carlson, M., and D. Botstein. 1982. Two differentially regulated 1989. Cysteine labeling studies of beef heart aconitase contain- mRNAs with different 5' ends encode secreted and intracellular ing a 4 Fe, a cubane, or a linear 3 Fe cluster. J. Biol. Chem. forms of yeast invertase. Cell 28:145-154. 264:20385-20393. 4. Chatton, B., P. Walter, J. P. Ebel, F. Lacroute, and F. Fasiolo. 28. Polakis, E. S., and W. Bartley. 1965. Changes in the enzyme 1988. The yeast VAS1 gene encodes both mitochondrial and activities of Saccharomyces cerevisiae during aerobic growth cytoplasmic valyl-tRNA synthetases. J. Biol. Chem. 263:52-57. on different carbon sources. Biochem. J. 97:284-297. 5. Duntze, W., D. Neumann, J. M. Gancedo, W. Atzpodien, and H. 29. Rickey, T. M., and A. S. Lewin. 1986. Extramitochondrial Holzer. 1969. Studies on the regulation and localization of the citrate synthase activity in baker's yeast. Mol. Cell. Biol. glyoxylate cycle enzymes in Saccharomyces cerevisiae. Eur. J. 6:488-493. Biochem. 10:83-89. 30. Rigby, P. W. J., M. Dieckmann, C. Rgodes, and P. Berg. 1977. 6. Emptage, M. H., T. A. Kent, M. C. Kennedy, H. Beinert, and E. Labeling deoxyribonucleic acid to high specific activity in vitro Munck. 1983. Mossbauer and EPR studies of activated aconi- by nick translation with DNA polymerase I. J. Mol. Biol. tase: development of a localized valence state at a subsite of the 113:237-251. (4Fe-4S) cluster on binding of citrate. Proc. Natl. Acad. Sci. 31. Robbins, A. H., and C. D. Stout. 1989. Structure of activated USA 80:4674-4678. aconitase: formation of the [4Fe-4S] cluster in the crystal. Proc. 7. Fansler, B., and J. M. Lowenstein. 1969. Aconitase from pig Natl. Acad. Sci. USA 86:3639-3643. heart. Methods Enzymol. 13:26-31. 32. Rose, I. A., and E. L. O'Connel. 1967. Mechanism of aconitase 8. Feinberg, A. P., and B. Vogelstein. 1984. A technique for action. J. Biol. Chem. 242:1870-1879. radiolabeling DNA restriction fragments to high 33. Rosenkrantz, M. S., T. Alam, K.-S. Kim, B. J. Clark, P. A. specific activity. Anal. Biochem. 137:266-267. and L. P. Guarente. 1986. Mitochondrial and nonmito- 9. Forsburg, S. L., and Mutational of Srere, L. Guarente. 1988. analysis chondrial citrate synthase in Saccharomyces cerevisiae are upstream activation sequence 2 of the CYCI gene of Saccharo- encoded by distinct homologous genes. Mol. Cell. Biol. 6: myces cerevisiae: a HAP2-HAP3-responsive site. Mol. Cell. 4509-4515. Biol. 8:647-654. 34. Rosenkrantz, M. S., D. W. Dingman, and A. L. Sonenshein. 10. Gornall, A. G., C. J. Bardawill, and M. M. David. 1949. citB is Determination of serum proteins by means of the biuret reac- 1985. Bacillus subtilis gene regulated synergistically by tion. J. Biol. Chem. 177:751-766. glucose and glutamine. J. Bacteriol. 164:155-164. 11. Hahm, K.-S., 0. Gawron, and D. Piszkiewicz. 1981. Amino acid 35. Rothstein, R. 1983. One-step gene disruption in yeasts. Methods sequence of a peptide containing an essential cysteine residue of Enzymol. 101:202-211. pig heart aconitase. Biochim. Biophys. Acta 667:457-461. 36. Ruzicka, F. J., and H. Beinert. 1978. The soluble "high poten- 12. Hanson, R. S., and D. P. Cox. 1967. Effect of different nutri- tial" type iron-sulfur protein from mitochondria is aconitase. J. tional conditions on the synthesis of tricarboxylic acid cycle Biol. Chem. 253:2514-2517. enzymes. J. Bacteriol. 93:1777-1787. 37. Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequenc- 13. Hu, N.-T., and J. Messing. 1982. The making of strand-specific ing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. M13 probes. Gene 17:271-277. USA 74:5463-5467. 14. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transfor- 38. Satrustegui, J., and A. Machado. 1977. The synthesis of yeast mation of intact yeast cells treated with alkali cations. J. matrix mitochondrial enzymes is regulated by different levels of Bacteriol. 153:163-168. mitochondrial function. Arch. Biochem. Biophys. 184:355-363. 15. Kennedy, C., R. Rauner, and 0. Gawron. 1972. On pig heart 39. Schatz, G., and T. L. Mason. 1974. The biosynthesis of mito- aconitase. Biochem. Biophys. Res. Commun. 47:740-745. chondrial proteins. Annu. Rev. Biochem. 43:51-87. 16. Kennedy, M., G. Spoto, M. Emptage, and H. Beinert. 1988. The 40. Sharp, P. M., and W.-H. Li. 1987. The codon adaptation active site sulfhydryl of aconitase is not required for catalytic index-a measure of directional synonymous codon usage bias, activity. J. Biol. Chem. 263:8190-8193. and its potential applications. Nucleic Acids Res. 15:1281-1295. 17. Kim, K. S., M. S. Rosenkrantz, and L. Guarente. 1986. Saccha- 41. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in romyces cerevisiae contains two functional citrate synthase yeast genetics. Cold Spring Harbor Laboratory, Cold Spring genes. Mol. Cell. Biol. 6:1936-1942. Harbor, N.Y. 18. Krebs, H. A., and J. M. Lowenstein. 1960. The tricarboxylic acid 42. Sholze, H. 1983. Studies on aconitase species from Saccharo- cycle, p. 129-303. In D. M. Greenberg (ed.), Metabolic path- myces cerevisiae, porcine and bovine heart, obtained by a ways. Academic Press, Inc., New York. modified isolation method. Biochim. Biophys. Acta 746:133- 19. Laemmli, U. K. 1970. Cleavage of structural proteins during the 137. assembly of the head of bacteriophage T4. Nature (London) 43. Slonimski, P. P., G. Perrodin, and J. H. Croft. 1968. Ethidium 227:680-685. bromide induced mutation of yeast mitochondria: complete VOL. 10, 1990 YEAST ACONITASE GENE 3561

transformation of cells into respiratory deficient non chromo- glucose repression and anaerobiosis on the activities and sub- somal "petites." Biochem. Biophys. Res. Commun. 30:232- cellular distribution of tricarboxylic acid cycle and associated 239. enzymes in Saccharomyces carlsbergensis. J. Gen. Microbiol. 43a.Smith, T. F., and M. S. Waterman. 1981. Comparative biose- 116:93-98. quence metrics. J. Mol. Evol. 18:36-46. 48. Wu, M., and A. Tzagoloff. 1987. Mitochondrial and cytoplasmic 44. Southern, E. M. 1975. Detection of specific sequences among fumarases in Saccharomyces cerevisiae are encoded by a single DNA fragments separated by gel electrophoresis. J. Mol. Biol. nuclear gene FUMI. J. Biol. Chem. 262:12275-12282. 98:503-517. 49. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved 45. Villafranca, J. J., and A. S. Mildvan. 1971. The mechanism of M13 phage cloning vectors and host strains: nucleotide se- aconitase action. J. Biol. Chem. 246:772-779. quences of the M13 mpl8 and pUC19 vectors. Gene 33:103-119. 46. von Heijne, G. 1986. Mitochondrial targeting sequences may 50. Zitomer, R. S., D. L. Montgomery, D. L. Nichols, and B. D. Hall. form amphiphilic helixes. EMBO J. 5:1335-1342. 1979. Transcriptional regulation of the yeast cytochrome c gene. 47. Wales, D. S., T. G. Cartledge, and D. Lloyd. 1980. Effects of Proc. Natl. Acad. Sci. USA 76:3627-3631.