Eur. J. Biochem. 204,983-990 (1992)
The ICL1 gene from Saccharomyces cerevisiae Ernestina FERNANDEZ, Fernando MORENO and Rosaura RODICIO Departamento de Biologia Funcional, Area de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Oviedo, Spain, (Received August 2, 1991)
EJB 91 1039
The glyoxylate cycle is essential for the utilization of Cz compounds by the yeast Saccharomyces cerevisiae. Withm this cycle, isocitrate lyase catalyzes one of the key reactions. We obtained mutants lacking detectable isocitrate lyase activity, screening for their inability to grow on ethanol. Genetic and biochemical analysis suggested that they carried a defect in the structural gene, ICLl . The mutants were used for the isolation of this gene and it was located on a 3.1-kb BglII-SphI DNA fragment. We then constructed a deletion-substitution mutant in the haploid yeast genome. It did not have any isocitrate lyase activity and lacked the ability to grow on ethanol as the sole carbon source. Both strands of a DNA fragment carrying the gene and its flanking regions were sequenced. An open reading frame of 1671 bp was detected, encoding a protein of 557 amino acids with a calculated molecular mass of 62515 Da. The deduced amino acid sequence shows extensive similarities to genes encoding isocitrate lyases from various organisms. Two putative CAMP-dependent protein-kinase phosphorylation sites may explain the susceptibility of the enzyme to carbon catabolite inactivation. Saccharornyces cerevisiae, like many other prokaryotic and eukaryotic microorganisms, can utilize ethanol or acetate as the sole carbon source. Growth on these substrates requires the glyoxylate pathway as an anaplerotic sequence for replenishing C, compounds to the tricarboxylic acid cycle. One of the key enzymes of the glyoxylate cycle is isocitrate lyase (see [l] for a recent review). It has been purified and biochemically characterized from many organisms, such as Escherichia coli [2,3], Neurospora crassa , Candida tropicalis , Phycomyces blakesleeanus  and S. cerevisiae . In S . cerevisiae, the enzyme was described as a multimer composed of four identical subunits, with a molecular mass of 75 kDa each. Its synthesis can be induced by growth on ethanol . In contrast to the enzyme of E. coli , Candidu lipoliticu [lo] and Aspergillus nidulans [ll], it is not induced by growth on acetate . Moreover, when glucose is added to cells adapted to growth on ethanol, the formation of new isocitrate lyase is repressed  and inactivation of the enzyme already present occurs . The latter is probably achieved via phosphorylation of specific amino acid residues . In addition to the biochemistry, the genetic regulation of the synthesis of isocitrate lyase has been studied in many organisms. Mutants affected in both structural and regulatory genes have been described for E. coli , A . nidulans [ll], C. lipolytica 116, 171, N . crussa  and Coprinus cinereus . More recently, the cloning of the structural genes has been Correspondence tu R . Rodicio, Departamento de Biologia Funcional, Area de Bioquimica y Biologia Molecular, Facultad de Medicina, E-33006 Oviedo, Spain Abbreviations. lCLl, gene encoding isocitrate lyase; Y EPD, YEPE, YEPP, SCD, SCE, SCP, growth media defined in Materials and Methods. Enzyme. lsocitrate glyoxylate-lyase (EC 126.96.36.199). Note. The novel nucleotide sequence data published here have been submitted to the EMBL sequence data bank and are available under accession number X61271.
reported for E. coli , C. tropicalis , A . nidulans 1221, and C. cinereus . A S. cerevisiae mutant defective in isocitrate lyase activity was described by Ciriacy . It was obtained in a combined screening for mutants defective in the intermediary carbon metabolism and mutants with altered carbon catabolite derepression. However, no detailed analysis of this icll mutant was provided. In the same screening and in previous work 1251, regulatory mutants with pleiotropic defects in derepression of isocitrate lyase and other enzymes necessary for the utilization of non-fermentable carbon sources have been described. In this paper we report on the isolation and characterization of mutants defective in isocitrate lyase activity. Genetic and biochemical analyses showed that they were defective in the structural gene. The mutants were used for cloning of the wild-type gene by functional complementation. The complete nucleotide sequence of the ICLl gene was determined, providing the basis for further studies on the transcriptional and post-translational regulation of the enzyme.
MATERIALS AND METHODS Strains and plasmids The S. cerevisiue strains 10.7-llA (MATa leu1 MAL3 suc33) and YGTG-1A (MATE trpl rnalO) described earlier 1261 were used in the isolation of mutants defective in isocitrate lyase and in further genetic analysis. Strains H44-3D (MATE Ieu2-3,112; kindly provided by J. Heinisch, Dusseldorf) and AMW-13C (MATu trpl (fs) ura3(fs) leu2-3,112 his3-I 1.15 can1 cir', where fs designates frameshift mutations; a gift from M. Whiteway Montreal, Canada) were used for deletion studies. Single-stranded DNA derived from recombinant M13mp18 and M13mp19 , was isolated from the E. coli strain XL1-Blue . For all other purposes, HBlOl was used as a host strain .
984 Plasmids containing the ZCLl gene were isolated from a yeast genomic library contructed by Nasmyth and Tatchell  in the multicopy vector YEpl3.
and incubated again with Staphylococcus protein-A-peroxidase - conjugate. After washing again, specific bands were detected by staining with 4-chloro-1-naphthol disolved in methanol.
Media, growth conditions and enzymatic analysis
The standard rich medium consisted of 1% yeast extract, 2 % peptone and was supplemented with either 2% glucose (YEPD), 3% ethanol (YEPE), or 3% pyruvate (YEPP). Synthetic medium contains 0.67% Difco yeast nitrogen base, supplemented with amino acids, adenine and uracil, as required. As carbon sources, either 20/0 glucose (SCD), 3% ethanol (SCE), or 3 % pyruvate (SCP) were added. Isocitrate lyase was assayed as described by Dixon and Kornberg . For preparations of crude extracts, cells were grown on SCD and then transferred to SCE for 8 h to allow for induction of isocitrate lyase. Leucine was omitted in the case of transformants. The protein concentrations were determined according to Lowry et al.  using bovine serum albumin as standard. Specific activities are expressed as nmol substrate consumed . min-l . mg protein-' in crude extracts. Selection of mutants defective in isocitrate lyase
Strain 10.7-11A was grown in 5 ml YEPD to a density of about lo8 cells/ml. Cells were treated with 4% ethylmethanesulfonate for 1 h at 30°C to give a survival rate of about 10%. After incubation for 7 h in 5 ml YEPD, cells were spread onto synthetic medium containing 3% pyruvate as the sole carbon source to select against mutants with a general defect in the utilization of non-fermentable carbon sources. They were then replica plated onto synthetic medium with ethanol as the sole carbon source. Colonies not growing on this medium were used for further analyses. Nucleic acid preparations, hybridization experiments and sequencing
Competent E . coli cells were prepared according to Hanahan . Amplified plasmid DNA was isolated by the alkaline lysis method of Birnboim and Doly , with minor modifications. If necessary, plasmids were further purified on a cesium chloride/ethidium bromide gradient as described by Clewell . Restriction fragments were isolated from agarose gels by the electroelution procedure of Smith . Yeast cells were transformed by the lithium acetate method according to Ito et al. . Chromosomal and plasmid DNA preparations from yeast were performed as described by Hoffman and Winston . Total RNA was prepared from yeast cells according to Sherman et al.  and separated on 1.5% agarose/MOPS/ formaldehyde gels . For Southern and Northern blot analyses we used standard procedures . Single-stranded DNA templates were sequenced with the dideoxy chain termination method of Sanger . Western blot analysis
An antiserum raised against purified isocitrate lyase was kindly provided by Y. Lopez-Boado (University of Oviedo). For Western blot analyses, a modification of the method of Burnette  was followed. Crude extracts were prepared from cells grown overnight on SCE. The proteins were separated on 12% SDSjPAGE and electroblotted onto nitrocellulose filters. The filters were incubated with the antiserum, washed
Sequencing of the N-terminal amino acids of ICL
Crude extracts were prepared from a yeast strain transformed with a multicopy plasmid carrying the ZCLl gene as well as from the wild-type strain 10.7-11A. The proteins were separated on 12% SDS/PAGE, transferred to a polyvinylidene difluoride membrane from Millipore and stained with Coomassie brilliant blue. A protein band with a significantly increased intensity in the transformed strain as compared to the wild-type was cut out and used directly for sequencing with a sequenator from Applied Biosystems model 477A, equipped with an on-line phenylthiohydantoin analyzer, model 120A [44, 451.
RESULTS AND DISCUSSION Isolation and characterization of icZl mutants
Mutants defective in the gene encoding isocitrate lyase should be unable to grow on ethanol as the sole carbon source, but should do so on pyruvate. Based on this assumption, ten independent mutant alleles were isolated (see Materials and Methods). Three of them, designated icll-I, icll-2 and icll-3, showed isocitrate lyase activities below the level of detection (Table 1). They still had wild-type levels of malate synthase and acetyl-CoA synthetase (data not shown). Tetrad analysis showed that all three isolates carried single recessive mutations. Thus, in a cross between the icll-1 mutant strain with a wild-type strain, a 2 : 2 seggregation for ethanol utilization was observed in a total of 20 tetrads tested. Two complete tetrads were also assayed for isocitrate lyase activity and confirmed the 2: 2 seggregation of the growth pattern. All other markers in the cross seggregdted as expected. Further evidence that the obtained mutants were indeed defective in the structural gene of isocitrate lyase was obtained by determining the specific activities in heterozygous diploids (Table 1). They showed about half the activities of homozygous diploids derived from two wild strains, as expected for a mutation affecting the structural gene. In addition specific antibodies raised against isocitrate lyase detected the same band with similar intensity in the mutants than in the wild-type control. This
Table 1. Specific activities of isocitrate lyase in mutant and diploid strains. bd, below detection.
10.7-llA eth22 ethlh ethl5 YTGT-1 A x 10.7-11A YTGT-1A x eth22 YTGT-1A xethl5 eth22 x e t h l 5 eth22 x eth22 ethl5 x e t h l 5
ICLl icll- I icll-2 icll-3 ICLl x ICLl ICLI x icll-l ICLl x icll-3 irll-1 x icll-3 icll-1 x icll-1 icll-3 x icll-3
mU/w 300 bd bd bd 274 150 135 32 bd bd
985 H H H H J '
H P Sp
H P Sp
2 ' 4
XbBgXE I " '
' " ' I
' XbXbX 1
Xb X b X I
P S I
P S ,
Table 2. Specific activities of isocitrate lyase in wild-type strains and transformants. bd, below detection.
10.7-11A eth22-5B eth22-5B eth22-5B eth22-5B eth22-5B cth22-5B eth22-5B
ICLl icll leu2 icll leu2 icll leu2 id1 leu2 icll leu2 icll leu2 icll leu2
pICL1.l pICL1.2 pICL1.l-HpICLl.l-SpICLl.2-H-
H P Sp
Xb X b X I
P S E
' ' L
Fig. 1. Restriction maps of inserted sequences. Restriction enzymes used were: BamHI (B), BgnI (Bg), SalI (S), XhoI (X), XbaI (Xb), PstI (P), EccoRT (E), Hind111 (H) and SphI (Sp). H and Sp external to the inserts refer to the orientation in YEpl3.
result rules out a defect in a regulatory gene, that would lead to a down regulation of isocitrate lyase synthesis. The icll-I and icll-2 alleles, belong to the same complementation group since a icll-I/icll-2 diploid strain did not grow on medium containing ethanol as the carbon source. Diploid strains carrying the alleles icll-1 and icll-3 or icll-2 and icll-3 did grow on ethanol. However, icll-3 is allelic to icll-1 and icll-2 since the heterozygous diploid carrying the mutated alleles icll-1 and icll-3 has very low activities when compared with that found in a heterozygous diploid, constructed from a wild-type and a mutant strain (Table 1). We assume that an interallelic complementation accounts for these findings. Cloning of the ZCLl gene
For the isolation of the ICLl gene from the genomic library of Nasmyth and Tatchell, we used the strain eth225B (MATu leu2-3,112 icll-I) constructed by crossing the originally isolated icll-1 mutant strain with H44-3D (MATa leu23,112). Since yeast cells grow very poorly on synthetic media with ethanol as the sole carbon source, transformants were first selected for leucine prototrophy (conferred by the vector YEpl3 used for the construction of the genomic library) on glucose medium. They were then replica plated onto ethanol medium. From 14400 original transformants, 8 were able to grow on ethanol as the sole carbon source. They were shown to lose both the LEU' phenotype and the ability to grow on ethanol, when grown under non-selective conditions (YEPD). Two different plasmids, designated pICLl.l and pICLl.2 could be isolated from these transformants and were amplified in E. coli. Retransformation into the yeast strain eth22-5B confirmed their ability to complement the icll-1 mutation. The restriction maps of the yeast DNA insertions in the two pkdsmids showed an overlapping region with a length of about 8 kb (Fig. 1). From the molecular mass of the isocitrate lyase subunits, the ICL1 gene has an estimated length of about 2 kb. To delimit the coding region further, we deleted parts of the yeast DNA insertions. For this purpose, plasmids pICLl.l and plCL1.2 were completely digested with SphI and the larger fragments were religated to yield plCLl .l-S- and pICLl.2-S-. Similarily, through digestion with HindIII, plasmids pICLl .l-H- and pICL1.2-H-, were constructed. Only pICLl.l -S- was still able to complement the icll-1 defect
Activity in wild-type
250 bd 3140 2970 bd 3200 bd pICLl.2-S- bd
1 12.6 11.9 12.8
locating the ICLl gene to the DNA fragment retained in this plasmid (Fig. 1). The specific activities of isocitrate lyase were determined in transformants with the various plasmids after growth under inducing conditions (Table 2). As expected, about a 12-fold increase was observed in transformants carrying a functional copy of fCL1 as compared to the non-transformed wild-type. Under repressing conditions, the enzyme was not detectable even in the multicopy transformants. Gene disruption Further evidence for the nature of the cloned gene was obtained by constructing a deletion-substitution mutant in a haploid strain. As the method is based on homologous recombination, we first confirmed by Southern blot analysis that the organization of restriction sites in the isolated DNA fragments reflects the chromosomal organization of the ZCLl locus (data not shown). For the deletion of the chromosomal gene copy, plasmid YDp-icll was constructed as shown in Fig. 2 A. It was digested with Hind111 and PstI prior to transformation of the yeast strain H44-3D (leu2-3,112). One leucine prototroph transformant, called iclld, was used for further experiments. It did not grow on media containing ethanol as the sole carbon source and did not show any detectable isocitrate lyase activity. This confirms the essential role of isocitrate lyase for the utilization of ethanol. By Southern blot analysis no sequences hybridizing to ICLl in strain iclld were detected (Fig. 2B). Similarily, neither specific mRNA nor the isocitrate lyase protein could be detected in the iclld deletion strain (Fig. 2 C and D>. Nucleotide sequence of the ZCLl gene The 3.1-kb BglIIjSphI from plasmid pICLl .l-S- was completely sequenced for both strands. The nucleotide and the deduced amino acid sequences are listed in Fig. 3. A large open reading frame of 1671 nucleotides was detected, encoding 557 amino acids. The predicted molecular mass of the putative protein with 62515 Da is lower than the one determined by SDSjPAGE . This phenomenon has been observed for a large number of proteins. The initiation codon is contained within the 5'-AAAAATGCCT-3'sequence,which is in good agreement with 5'-A/CAA/CAATGTCT-3', reported for optimal initiation of translation in yeast . A putative TATA box is located at position - 142 bp and a possible polyadenyla-
H H H
I l l
I I I 1
H P S p I 1 I
1 - B1
P S I I
Fig. 2. Deletion of the ICLl gene. (A) Restriction map of the YDp-icll insertion. The two EcoRI fragments of pICL1.l containig the original LEU2 gene and 2 micro-sequences were deleted. For deletion of the chromosomal copy of the ICLl gene the 5.3-kb BgfII-XhoI fragment of plasmid pICLl.1 containing the complete ZCLl gene and its flanking regions was substituted for the 2.6-kb BgDI-XhoI fragment of YEpl3 carrying the LEU2 gene. The arrows indicate the enzymes used for digestion prior to transformation o f a haploid yeast strain (see Results and Discussion). (B) Southern blot analysis. AMW-13C (ZCL1)was crossed with iclld (icll: :LEU2),sporulated and subjected to tetrad analysis. Genomic DNA digested with XhoI was prepared from AMW-13C (ZCL1, lane l), iclld (icll: :LEU2, lane 2 ) and different segregants (lanes 3-6). The 4.9-kb XhoI fragment of pICL1.l (1) and the 2.6-kb EglII-XhoI fragment of YEpl3 containing the LEU2 gene (2) were used as radioactive probes. (+) and (-) refer to the growth on media containing ethanol as the sole carbon source. (C) Northern blot analysis. Total RNA from strains iclld (icll: :LEU2, lane l), 10.7-llA (ICL1, lane 2) and a multicopy transformant with pICL1.1-S- (lane 3) was extracted from cells grown overnight on SCE (see Materials and Methods). 10 pg was separated on an 1.5% agaroselMOPSlformaldehyde gel in the presence of ethidium bromide. The rRNA bands had similar intensities in all lanes. The gel was then transferred to a nitrocellulose filter and hybridized to the 1.7-kb EcoRI-Hind111 fragment of pICL1.1, labeled as described in Materials and Methods. (D) Western blot analysis. Crude extracts from iclld (icll: :LEU2, lane 1) and 10.7-llA (ZCL1, lane 2) containing 60 pg protein, each, were separated on 12% SDS/ PAGE. A control gel stained with Commassie brillant blue confirmed that similar amounts of protein were present in the two lanes. The gel was transferred to a nitrocellulose filter. Isocitrate lyase protein was detected with polyclonal antibodies as described in Materials and Methods
tion site is 227 b p downstream f r o m the translation s t o p codon. The codon bias index according to Bennetzen a n d Hall  is 0.33, reflecting a codon usage for moderately wellexpressed yeast genes. The start of the isocitrate lyase was confirmed by sequencing the N-terminal amino acids. T h e first seven amino acids of the enzyme were found t o correspond with codons
2-8 of the DNA sequence. The initial methionine residue predicted t o be present b y the DNA sequence was n o t found
in the purified protein, suggesting that it is removed by processing . Comparison of the amino acid sequence with isocitrate lyases f r o m other sources (Fig. 4) shows 32%, 65% and 48% similarity with the enzymes from E. cofi , C. tropicalis  and castor bean , respectively. There are highly conserved regions possibly confined t o the catalytic domain. Among them is the hexapeptide KKCGHM that has been used as a recognition pattern for isocitrate lyases .
987 AGAT -890
790 800 810 820 830 840 ACTTTGATTAGCTCAACCATCGATACCAGAGATCATTATTTCATTGTCGGTGCCAC~T ThrLeUIleSerSerThrIleAspThrArgAspHisTyrPheIleValGlyAlaThrAsn 850
-770 -760 -750 -740 -730 CAAACATAATCATACGTGTTTCACTCCAAATTTGATCACAAATGGCCACCCATTATAGCT
910 920 930 940 950 960 CAAGAACTAGCTGACATTGAACAAA?ATGGTGTAGAGACGCTGGACTCAAGTTATTCCAT GlnGluLeuAlaAspIleGluGlnLysTrpCysArgAspAlaGlyLeuLysLeuPheHis
-710 -700 -690 -680 -670 TGTTATTATTTACTTCTTCTTGAGTTCATTTTATTGAAAGATGCCGCTGGGM~CA -650
AGTTAAAATGACAATGTTAGAAGGAAAAACTAAATCAAGCTCCCATAGGmTTTCmT -530 -520 -510 -500 -490 GTCAGTAATATGTCTCGAGAAAATAATAATAATTGAATCTATTTATTGWGTmTAT
1040 1050 1060 1070 1080 AAATTCAC TCTAAAGTGGGTCCATTGACTGAAACATCCCACAGAGAAGCCAAGAAGCTC L sPheTh erLysVa1GlyProLeuThrGluThrSerHisArgGluAlaLysLysLeu
-470 -460 -450 -440 -430 CTCGTAACCCGGATGCTTTGGGCGGTCGGTCGGGTTTTGCTACTCG~~~~~~~~TGAGWC 1150 1160 1170 1180 1190 1200 GGGTTGTACCGTTACAGAGGTGGGACGCAATGTTCTATCATGAGGGCCCG~CAT~CT -410 -400 -390 -380 -370 GlyLeuTyrArgTyrArgGlyGlyThrGlnCysSerIleMetArgAlaArgAlaPh~la TGTTCCCTTTTGCCCCAGGTTTCCATTFRTFFGAGCGATCACTTATCTGA~CGTCA~ 1210 1220 1230 1240 1250 1260 -350 -340 -330 -320 -310 CCATATGCTGATTTGGTATGGATGGAATCTAACTACCCAGACTTC~CAGGCCAAGGAG TTTTCATTTFTCCC+CAATCAAAACTGAAGCCAATCACCACA?+AATTMCACTmC ProTyrAlaAspLeuValTrpMetGluSerAsnTyrProAspPheGlnGlnAlaLysGlu -290 -280 -270 -260 -250 GTCATCTTTCACTACCCTTTACAGAAGAAAATATCCATAGTCCGGACTAGCATCCCAGTA -230 -230 -210 -200 -190 TGTGACTCAATATTGGTGCAAAAGAGAAAAGCATAAGTCAGTCCAAAGTCCGCCCTTMC -170
TTTGCAGAAGGTGTTAAAGAGAAATTCCCCTGACCAATGGCTAGCTTACAACTTGTCTCCA PheAlaGluGlyValLysGluLysPheProAspGlnTrpLo 1330
-80 -70 AAATTCTTATTATTTGTCTTGGCTTGCTAATTTCATCTTATCCTTTTTTTCTTTTCACAC -110
1390 1400 1410 1420 1430 1440 GGTGATCTAGGTTACATCTGGCAATTTATCACATTGGCCGGTTTACACACTMCGCTTTA GlyAspLeuGlyTyrIleTrpGlnPheIleThrLeuAlaGlyLeuHisThrAs~laLeu 1450 1460 1470 1480 1490 1500 GCTGTCCATAACTTCTCTCGTGACTTTGCCAAGGATGGGATGAAAGCTTATGCCCAGAAT
AlaVa1HisAsnPheSerArgAspPheAlaLysAspGlyMetLysAla~rAlaGlnAsn 10 20 30 40 50 60 ATGCCTATCCCCGTTGGAAATACGAAGAACGATTTTGCAGCTTTACAAG~CTAGAT MetProIleProValGlyAsnThrLysAsnAspPheAlaAlaLeuGl~laLysLeuAsp
70 80 90 100 110 120 GCAGATGCTGCCGAAATTGAGAAATGGTGGTCTGACTCACGTTGGAGTAAGACTAAGAGA AlaAspAlaAlaGluIleGluLysTrpTrpSerAspSerAr~rpSerLysThrLysArg 130
GTTCAGCAGAGGGAAATGGACGATGGTGTTGATGTGTTGAAACATCAAAAATGGTCTGGT ValGlnGlnArgGluMetAspAspGlyValAspValLeuLysHisGlnLysTrpSe~ly 1570 1580 1590 1600 1610 1620 GCGGAGTACATCGATGGGTTATTGAAGTTAGCT~GGTGGTGTTAGCGCMCAG~GCT
AlaGluTyrIleAspGlyLeuLeuLysLeuAlaGlnGlyGlyValSerAlaThrAlaAla 1630 1640 1650 1660 1670 1680 ATGGGAACCGGTGTCACAGAAGATCAATTCAAAGAAAATGGCGTAAAGAAATAGGACAAG MetGlyThrGlyValThrGluAspGlnPheLysGluAsnGlyValLysLysEND 1690 1700 1710 1720 1730 1740 GATTMCCATTAGMCTGCCGGCATTTCCTGACAAGTATATATATTCGTAATCT~TGT
SerSerValMetAlaArgLysLeuPheLysValLeuGluLysHisHisAsnGluGlyT~ 1750 250 260 270 280 290 300 GTCTCTWCTTTCGGTGCCCTAGATCCTGTCCAGATTTCTCAAATGG~TACTTA ValSerLysThrPheGlyAlaLeuAspProValGlnIleSerGlnMetAlaLysTyrLeu
CAATGTTCTCTAATGGCATCTCTCTAATGGTACTCCAACTAAAGTCAACATTCTCTCCGT 320 330 340 350 360 GACACAATCTATATTTCTGGTTGGCAGTGTTCATCAACTGCTTCCACCTCAAATG~CCT 310
1870 1880 1890 1900 1910 1920 GCAAGAATGCCTTTCTCTAGTTGCAAAAACGAGTC~CATCTTTGATCGAGTCGCC
370 380 390 400 410 420 GGTCCAGACTTAGCTGATTATCCAATGGACACCGTTCCAAACAAAGTGGAACA~TGTTC
1930 1940 1950 1960 1970 1980 TCATTTTTGTTGACTCGGTGACTAAAATCAGTGCCATTTCTGTCCATGCCTCGTGCGCGA
GlyProAspLeuAlaAspTyrProMetAspThrValProAsnLysValGl~isLeuPhe 430 440 450 460 470 480 AAGGCCCAATTGTTTCACGACAGAAAACAACTAGAGGCACGGTCAAAGGCTAAATCTCAG LysAlaGlnLeuPheHisAspArgLysGlnLeuGluAlaAlgSerLysAlaLysSerGln
ATGATTGAATCGAACTAATCCCGAACACAATCATAGTATCGTATCGCCCATTAGTGCCGT 2050 2060 2070 2080 2090 2100 GAGTGTACGCGAGGCAAAAGGCACCATGGAARATATTCCCCTTAGTCATCTTGGAAATCAT
490 500 510 520 530 540 GAAGAACTCGATGAGATGGGTGCCCCAATTGACTACCTAACACCAATTGTCGCTGATGCA GluGluLeuAspGluMetGlyAlaProIleAspTyrLeuThrProI1eValAlaAspAla
2110 2120 2130 2140 2150 2160 TGAGACTGTAAAGGAACTCGCAAACGGGTAACCGATATTTACCCCACCTTTGGTCGAGAA
550 560 570 580 590 600 GACGCAGGCCACGGCGGTTTAACCGCAGTCTTCAAATTGACC~GATGTTCATTGAGCGT
2170 2180 2190 2200 2210 2220 ATTCCACCTGTTCGGCAATTTTTGCCGCTCTTCT~GAG~GGCGTTTTTTTTTCT
AspAlaGlyHisGlyGlyLeuThrAlaValPheLysLeuThrLysMetPheIleGluArg 610 620 630 640 650 660 GGTGCTGCTGGGATCCACATGGAAGACCAGA~TCTA~TAAGAAATGTGGGCATATG GlyAlaAlaGlyIleHisMetGluAspGlnThrSerThrAsnLysLysCysGlyHisMet
2230 2240 GACACGCCTCTTCTCTCGCATGC
Fig.3. Nucleotide and deduced amino acid sequences of lCL1. Sequences underlined in the 5’ and the 3’ non-coding regions indicate the TATA element and the polyadenylation sites. Three directed reAlaGlyArgCysValIleProValGlnGluHisValAsnArgLeuVa1ThrIleArgMet peats are indicated by the dashed lines. The GAGGAGA sequence 730 740 750 760 770 780 TGTG~GATATCATGCATTCGACTTAATTGTCGTTGCTAGGACTGATTCAG~GCAG~~ mentioned in the test is overlined. The two putative CAMP phosC y s A l a A s p I l e M e t H i s S e r A s p L e u I l e V a l V a l A l a ~ g T h r A s p S e r G l u A l a A l a phorylation sites are boxed. 670 680 690 700 710 720 GCAGGAAGATGTGTTATACCCGTTCAGGAACATGTTAACAGATTGGTGACTATTAGAATG
988 castor bean E.COli c.tropicalie s.cerevisiae castor bean E. COli c.tropicalis s.cereviaiae
MAASFSGPSM-IMEEEGRFEAEVAEVQAWWNSERFKLT~YTARDWACRGNLKQ-SYA mTR--------------- TQQIEELQKEWTQPRWEGITRPYSAEDVVIURGSVNPECTL AYTKIDINQEEADFQKEVAEI~SEPRWRKTKRIYSAEDIAKKRGTLK-IAYP
castor bean E.coli c.tropicalis S.cerevisiae
caator bean E.coli c.tropicalis s.cerevisiae
castor bean E. coli C.tropicalis S-cerevisiae
castor bean E-coli C.tropicalis S.cerevieiae
KTGAELQATEDNWLAMAQLKTFPECVMDAIKNMNAGEDEK-RRRMNEWMNHTSYDKCLSY IYGDELARIETEWTKKAGLKLFHEAVIDEIKAGNYSNKBALIKKFTDKVNPLSHT---SH ASGQELADIEQKWCRDAGLKLPHEAVIDEIERSALSNKQELIKKFTSKVGPLTET---SH
castor bean E. COli c.tropicalis S.cerevisiae
EQGREIADRMGLKNLFWDWDLPRTRGFYRFKGSVMAAWRGRAFAPHADIIWMETAKPD RTSEGFFRTHAGIEQAISRGLAYAPYADLVWCETSTPD KBAICKLRKELTGRDIYFNV~GYYRYPGGTQCAVPD RBARKWU(EILGHEIFFDWELPRVREGLYRYRGGTQCS1MRARAFAPYADLVWXESNYPD
castor bean E. coli C.tropicalis S.cerevisiae
FAECTAFAEGVKSMHPEIMLAYNLSPSFNWDASGMTDEQMFIPRIARLGFCWQFITLG LELRRRFAPAIHAKYPGKLLAYNCSPSFNWQ-KNLDDKTIASFQEQLSDMGYKFQFITLA YTQAKEFADGVKAAVPDQWLAYNLSPSF"N-Ud4PADEQETYIKRLGQLGYVWQFITLA
castor bean E.coli C.tropicalis S.cerevisiae
GFHADALVIDTFAK!JYAR-RGMLAYVERIQREER---KNGVDTLAHQKWSGANYYDRYLK GIHSMWFNMFDLANAYAQGEGMKHYVEKVWPEFAAAKDGYTFVSHQQEVGTGYFDKVTT -LHTTALAVDDFANQYSQ-IGMPAYGQTVQQPEI---EKGVEVVKHQIWSGANYIDGLLR GLHTNALAVHNFSRDFAK-DGMKAYAQNVQQREM---DDGVDVLKHQKWSGAEYIDGLLK
caator bean E.coli C.tropicalis S.cerevisiae
Fig. 4. Alignments for maximal amino acid similarities of isocitrate lyase from different organisms. Identical residues among all isocitrate lyase enzymes are indicated with an asterisk (*). Conservative changes are marked with a black point ( 0 ) .
The synthesis of isocitrate lyase in yeast is induced by ethanol and repressed by glucose . Presumably, sequences located in the 5' non-translated region are responsible for this regulation. The derepression of isocitrate lyase, fructose-1,6bisphosphatase, alcohol dehydrogenase I1 and malate dehydrogenase is strongly affected in various mutants such as some of the cat  and ccr  mutants. The promoter sequence of the gene encoding the malate dehydrogenase that is controlled by this mechanism, has only been determined up to - 158 bp  relative to the ATG translation start codon. ?lus, as most regulatory elements are expected further upstream, a detailed comparison was not possible. For the other genes, we found a region of similarity among the ICL1, ADH2  and the FBP  promoters (Fig. 5A). A second region that shows significant similarity between the ZCLl and A D H 2 promoters does not seem to be present in the FBP promoter (Fig. 5 B). Although no detailed promoter analysis of FBP has been published, yet, deletion of a DNA fragment including these two regions from the ADH2 promoter resulted in a twofold decrease of specific activity under derepressing con-
ditions . Thus, we assume that it could function as a regulatory element in the ZCLl promoter, too. The sequence 5'-GAGGAGA-3', located at -893 bp relative to the ATG, is also found in the promoter of ADH2. There, it has been implicated in ADRl -dependent transcriptional activation as part of a palindromic sequence [%I. However, in the ICLl promoter it is not part of such a palindrom and might not be a functional A D R l binding site. Finally, three direct repeats of eight nucleotides each were found between position -442 and -343 in the I C L l promoter (Fig. 3). These sequences are neither found in the FBP promoter, nor in the ADH2 promoter. The physiological meaning of the above-mentioned sequences is currently under investigation. Isocitrate lyase is one of the enzymes subject to catabolitc inactivation by glucose . Recently its reversible inactivation was shown to be mediated by a phosphorylation of the enzyme . Two possible CAMP-dependent protein-kinase phosphorylation sites of threonine are present at amino acid positions 50 (RRGT) and 340 (KKFT) in isocitrate lyase. These sites are also conserved in the C. tropicalis enzyme,
989 Gene promoter
Fig.5. Comparison of the promoter regions of the ICLl, FBP and ADH2 genes. (A) and (B) refer to the homologous regions mentioned in Results and Discussion. The numbers indicate the positions relative to the initiation codons.
suggesting that its activity is regulated by phosphorylation, too. The consensus sequences do not appear in the isocitrate lyase of E. coli, where the enzyme is activated by phosphorylation of histidine residues . They are also not found in the isocitrate lyase from castor bean. The intracellular location of isocitrate lyase in S. cerevisiue is still controversial. Early reports have located it in the peroxisomes . More recently, McCammon et al.  did not find isocitrate lyase within peroxisomes obtained from oleic-acid induced cells [SS], using a biochemical approach. In eukaryotic organisms the signal for import of a protein into peroxisomes consists of a tripeptide located at the C-terminus with serine, alanine or cysteine at the first, lysine, histidine or arginine at the second and leucine at the third position . The tripeptide VKK present at the C-terminus of the isocitrate lyase from S. cerevisiae does not coincide, suggesting a nonperoxisomal location of the enzyme. In this context it is important to mention that although isocitrate lyase from C. tropicalis and S. cerevisiue share extensive similarities, they differ in the C-terminal amino acids. For isocitrate lyase from C. tropicalis both a peroxisomal location and a C-terminal sequence, AKV, equivalent to SKL, have been reported . In S. cerevisiue only the amino acid sequences of a few peroxisomal proteins are known. Whereas the peroxisomal citrate synthase contains SKL at its C-terminus , catalase A does not . However, the SKF present in the latter sequence also differs from that of the isocitrate lyase of S. cerevisiue. It has been suggested that more than one type of signal may exist 1601. Therefore, conclusive evidence for the intracellular location of isocitrate lyase from S. cerevisiae can only be obtained from further investigations. We wish to thank Professor S . Gascon for many interesting suggestions and Jiirgen Heinisch for the critical reading of the manuscript. We are especially grateful to Carlos Lopez for his help in determining the N-terminus of the protein and for providing oligonucleotides for DNA sequencing. We also like to thank Yolanda Lopez-Boado for providing the antiserum against isocitrate lyase. Finally, we appreciate the help of Michael von Pein, Ullrich Kleinhans and Ricardo Sanchez in the computer analysis. E. F. is a recipient of a fellowship of the Ministerio de Educacidn y Cienciu de EFpufia.This work was funded by a grant from Comisidn Assesoru de Investigacibn Cientifica y Tkcnica (PB87-0191).
REFERENCES 1. Vanni, P., Giachetti, E., Pinzauti, G. & McFadden, B. A. (1990) Comp. Biochem. Physiol. 958, 431 -458. 2. Robertson, E. F. & Reeves, H. C. (1987) Curr. Microhiol. 14, 347 - 350. 3. Conder, M. J., KO,Y. & McFadden, B. A. (1988) Prep. Biochern. 18,431 -442. 4. Johanson, R. A., Hill, J. M. & McFadden, B. A. (1974) Biochem. Biophys. Actu 364,341 -352. 5. Uchida, M., Ueda, M., Matsuki, T., Okada, A. & Fukui, S. (1986) Agric. Biol. Chem. 50, 127- 134. 6. Rua, J., de Arriaga, D., Busto, F. & Soler, J. (1990) Biochem. J . 272,359-367. 7. Lopez-Boado, Y., Herrero, P., Fernandez, M. T., Fernandez, R. & Moreno, F. (1988) Yeast 4,41-46. 8. Herrero, P., Fernbndez, R. & Moreno, F. (1985) Arch. Microhiol. 143,216-219. 9. Kornberg, H. L. (1966) Biochem. J. 99, 1 - 11. 10. Matsuoka, M., Ueda, Y. & Aiba, S. (1980) J. Bacteriol. 144,692697. 11. Armitt, S., McCullough, W. & Roberts, C. F. (1976) J . Gen. Microbiol. 92, 263 -282. 12. Gonzalez, E. (1977) J . Bacteriol. 129, 1343-1348. 13. Lopez-Boado, Y. S., Herrero, P., Gascon, S . & Moreno, F. (1987) Arch. Microbiol. 147, 231 -234. 14. Lopez-Boado, Y. S., Herrero, P., Fernindcz, T., Fernandez, R. & Moreno, F. (1988) J . Gen. Microbiol. 134,2499-2505. 15. Maloy, S . R. & Nunn, W. D. (1981) J . Bacteriol. 148, 83-90. 16. Barth, G. (1987) Curr. Genet. 10, 119-124. 17. Matsuoka, M., Ueda, Y. & Aiba, S . (1984)J. Bacteriol. 157.899908. 18. Leckic, B. J. & Fincham, J. R. S. (1971) J . Bucteriol. 65, 35-43. 19. King, H. B. & Casselton, L. A. (1977) Mol. Gen. Genet. 157,319325. 20. Matsuoka, M. & McFadden, B. A. (1988) J . Bacteriol. 170,4528 4536. 21. Atomi, H., Ueda, M., Hikida, M., Teranishi, Y. & Tamaka, A. (1990) J . Biochem. 107,262-266. 22. Ballace, D. J. & Turner, G. (1986) Mol. Gen. Genet. 202, 271 275. 23. Mcllon, M. F., Little, F. R. & Cassclton, L. A. (1987) Mol. Gen. Genet. 210, 352 - 357. 24. Ciriacy, M. (1977) Mot. Gen. Genet. 154, 213-220. 25. Zimmermann, F. K., Kaufmann, I., Rasenberger, H. & Haussmann, P. (1977) Mol. Gen. Genet. 151,95-103. 26. Rodicio, R. & Zimmermann, F. K. (1985) Curr. Genetics 9, 539545. 27. Yanisch-Perron, C., Vieira, J. &Messing, J. (1985) Gene 33,103119. 28. Bullock, W. O., Fernandez, J. M. & Short, J. M. (1987) BioTechniques 5,376- 379. 29. Bolivar, F. & Backman, K. (1979) Methods Enzymol. 68, 245250. 30. Nasmyth, K. & Tatchell, K. (1980) Cell 19, 753-764. 31. Dixon, G. H. & Kornberg, H. L. (1959) Biochem. J . 72, 3 P. 32. Lowry, 0. H., Rosebrough, N. J. J., Farr, A. L. & Randal, R. J. (1951) J . B i d . Chem. 193, 265-275. 33. Hanahan, D. (1985) in DNA cloning: apructicalupprouch (Glover, G. M., ed.) vol. 1, pp. 309 - 135, IRL Press, Oxford. 34. Birnboim, H. C. & Doli, J. (1979) Nucleic Acids Res. 7, 15131523. 35. Clewell, D. B. (1972) J . Bacteriol. 110, 667-676. 36. Smith, H. 0. (1980) Methods Enzymol. 65, 371 - 380. 37. Ito, H., Fukada, Y., Murata, K. & Kimura, A. (1983) J . Bacrpriol. 153,163-168. 38. Hoffman, C. S. & Winston, F. (1987) Gene 57, 267-272. 39. Sherman, F., Fink, G. R. & Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics, Cold Spring Harbor Laboratory, New York. 40. Thomas, P. S. (1980) Proc. Natl Acad. Sci. USA 77, 5201 5205.
41. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory,
New York. 42. Sanger, F., Hicklen, S. & Coulson, A. R. (1977) Proc. Natl Acad. Sci. USA 74, 5463 - 5467. 43. Burnette, W. N.(l981) Anal. Biochem. 112,195-203. 44. Matsudaira, P. (1987) J . Biol. Chem. 262, 10035-10038. 45. Hewick, R. M., Hunkapiller, M. W., Hood, L. E. & Dreyer, W. J. (1981) J. Biol. Chem. 256, 7990-7997. 46. Hamilton, R., Watanabe, C. K. & de Boer, H. A. (1987) Annu. Rev. Biochem. 55,913-953. 47. Bennetzen, J. L. & Hall, B. D. (1982) J . Biol. Chem. 257, 30263031. 48. Ben-Bassat, A. & Bauer, K. (1987) Nature 326, 315. 49. Beeching, J. R. & Northcote, D. H. (1987) Plant Mol. Biol. 8, 471 -475. 50. Beeching, J. R. (1989) Protein Seq. Data Anal. 2,463-466. 51. Minard, K. I. & McAlister-Henn, L. (1991) Mol. Cell. Biol. 11, 370- 380.
52. Russell, D. W., Smith, M., Williamson, V. M. & Young, E. T. (1983) J. Biol. Chem. 258, 2674-2682. 53. Rogers, D. T., Hiller, E., Mitsock, L. & Orr, E. (1988) J . Biol. Chem. 263,6051 -6057. 54. Beier, D. R., Sledziewski, A. & Young, E. T. (1985) Mol. Cell. Biol. 5, 1743 - 1749. 55. Shuster, J., Yu, Y., Cox, D., Chan, R. V. L., Smith, M. & Young, E. (1986) Mol. Cell. Biol. 6, 1894- 1902. 56. Szabo, A. S. & Avers, C. J. (1969) Ann. N . Y . Acad. Sci. 168,302 312. 57. McCamrnon, M. T., Veenhuis, M., Trapp, S. B. & Goodman, J. M. (1990) J . Bacteriol. 172, 5816-5827. 58. Veenhuis, M., Mateblowski, M., Kunau, W.-H. & Harder, W. (1987) Yeast 3,77-84. 59. Could, S. J., Keller, G.-A,, Hosken, N., Wilkinson, J. & Subramani, s. (1989) J. Cell. Biol. 108, 1657-1664. 60. Lewin, A. S., Hines, V. & Small, G. M. (1990) Mol Cell. Biol. 10, 1399-1405. 61. Cohen, G., Rapatz, W. & Ruis, H. (1988) Eur. J . Biochem. 72, 248 - 254.