Yeast Yeast 2000; 16: 539±545. Research Article Identi®cation by functional analysis of the gene encoding a-isopropylmalate synthase II (LEU9) in Saccharomyces cerevisiae Enrico Casalone2*, Claudia Barberio1, Duccio Cavalieri1 and Mario Polsinelli1 1 2 Dipartimento di Biologia Animale e Genetica, UniversitaÁ di Firenze, via Romana 17±19, I-50125 Firenze, Italy Dipartimento di Scienze Biomediche, UniversitaÁ di Chieti, via dei Vestini, I-66100 Chieti, Italy * Correspondence to: E. Casalone, Dipartimento di Biologia Animale e Genetica, UniversitaÁ di Firenze via Romana 17, 50125 Florence, Italy. E-mail: firstname.lastname@example.org®.it Received: 2 November 1999 Accepted: 23 January 2000 Abstract The function of the open reading frame (ORF) YOR108w of Saccharomyces cerevisiae has been analysed. The deletion of this ORF from chromosome XV did not give an identi®able phenotype. A mutant in which both ORF YOR108w and LEU4 gene have been deleted proved to be leucine auxotrophic and a-isopropylmalate synthase (a-IPMS)-negative. This mutant recovered a-IPMS activity and a Leu+ phenotype when transformed with a plasmid copy of YOR108w. These data and the sequence homology indicated that YOR108w is the structural gene for a-IPMS II, responsible for the residual a-IPMS activity found in a leu4D strain. The leu4D strain appeared to be very sensitive to the leucine analogue tri¯uoroleucine. In the absence of leucine, its growth was not much impaired in glucose but more on non-fermentable carbon sources. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: disruption a-isopropylmalate synthase; leucine metabolism; LEU4; YOR108w; gene Introduction In Saccharomyces cerevisiae the activity of aisopropylmalate synthase (a-IPMS), the ®rst enzyme of the leucine biosynthetic pathway, is provided by at least three forms of a-IPMS (Voss et al., 1997; Beltzer et al., 1988; Chang et al., 1985). a-IPMS I, the major isozyme responsible for about 80% of the total activity, is encoded by LEU4, a gene that has been extensively characterized (Chang et al., 1984; Beltzer et al., 1986; Beltzer et al., 1988; Cavalieri et al., 1999). The gene LEU4 encodes mitochondrial and non-mitochondrial forms of a-IPMS (Beltzer et al., 1988). In mutant strains carrying the total deletion of the LEU4 gene, a residual a-IPMS activity of about 20%, designated a-IPMS II activity, is still present (Chang et al., 1985). Four genes, LEU5 (Baichwal et al., 1983; Chang et al., 1984, 1985), LEU6, LEU7 and LEU8 (Drain and Schimmel, 1988), whose mutations led to a Leux phenotype in a leu4 background, were considered candidates to encode a-IPMS II. LEU5 Copyright # 2000 John Wiley & Sons, Ltd. and LEU6 are required for both a-IPMS II activity and non-fermentable energy source metabolism, but genetic and biochemical analysis showed that LEU5 does not actually encode an a-IPMS (Drain and Schimmel, 1986, 1988). LEU7 and LEU8 are required only for a-IPMS II activity (Drain and Schimmel, 1988). The recent sequencing of the entire S. cerevisiae genome has shown the occurrence of many duplicate genes; among them, the putative ORF YOR108w, on chromosome XV, is highly homologous to LEU4 (Voss et al., 1997) and for this reason it could be a good candidate to encode aIPMS II. However, computational analysis cannot be the sole criterion to assign a function to a gene; deletions of all the homologous genes are needed. We report here the construction of three knock-out mutants, two carrying a deletion in YOR108w or in LEU4 ORF, and one carrying both the deletions. YOR108w has been cloned and used to establish its role in encoding a-IPMS II in S. cerevisiae. The deletion mutants have been characterized to establish their capability of growth on different media. 540 E. Casalone et al. Materials and methods Strains, media and methods Yeast strains used are listed in Table 1; they were routinely grown on the complete medium YPD (Rose et al., 1990). Synthetic minimal medium (SD), glycerol complete medium (YPG) and sporulation medium (SM) were prepared as reported by Rose et al. (1990). SD medium supplemented with appropriate requirements was used to make COM minus media (Rose et al., 1990). COM minus leucine and threonine was used to test strain sensitivity to tri¯uoroleucine (TFL). G418-resistant strains were grown on YPD plates containing 200 mg/ml G418 (Roche). Escherichia coli XL1-blue supercompetent cells from Stratagene were used for the propagation of plasmids. All procedures for manipulating DNA employed standard methods. Mating, sporulation and tetrad analysis were performed as described by Spencer and Spencer (1988). Transformation of yeast was performed by the lithium±acetate procedure, according to Gietz and Woods (1994). After transformation with the yor108w::HIS3MX6 cassette, washed cells were plated directly on COM minus histidine. For the selection of the leu4::kanMX4 transformants, cells were resuspended in 2.5 ml YPD and grown for 5 h at 30uC before spreading on YPD containing 200 mg/ml G418. Plasmids Plasmid pDCT2 (Cavalieri et al., 1999) was the source of LEU4 sequence. Plasmids pFA6a± KanMX4 (Wach et al., 1994) and pFA6a± HIS3MX6 (Wach et al., 1997) were the source of the heterologous markers KanMX4, conferring geneticin-resistance (G418R), and HIS3MX6 complementing his3 mutation, respectively. Plasmid pYCG_YOR108w is a derivative of pRS416 (Sikorski and Hieter, 1989), in which a 2643 bp long EcoRI±HindIII DNA fragment containing ORF YOR108w was inserted into the unique EcoRI and HindIII restriction sites. The EcoRI±HindIII DNA fragment was synthesized by PCR using two oligonucleotides, WT1 GGGAATTCGCTCTTCCTTCTGCATGGCG and WT2 GGAAGCTTGCTAGCCTCCTTTCAGGTTC, derived from the published ORF YOR108w sequence (Voss et al., 1997) and extended with EcoRI (WT1) and HindIII (WT2) recognition sequences (underlined). The 2643 bp long PCRsynthesized fragment contained the entire YOR108w ORF plus 345 bp upstream and 477 bp downstream. Procedure of disruption of LEU4 and YOR108w Deletion cassettes were constructed by PCR following the long ¯anking homology (LFH) regions procedure of Wach (1996). This PCR procedure generated the 3.2 Kb long leu4::KanMX4 and 2.2 Kb long yor108w::HIS3MX6 deletion cassettes, in which 1650 bp of the coding sequences of LEU4 (from the 21st codon after ATG to the 4th codon before the stop codon) and 1754 bp of that of YOR108w (from the 5th codon after ATG to the 14th before stop codon) were replaced by KanMX4 (1510 bp) and HIS3MX6 (1350 bp) sequences, respectively. Correct integration of the deletion cassettes leu4::KanMX4 and yor108w::HIS3MX6 into the genome was veri®ed by PCR, using whole yeast cells as a source of target DNA (Wach et al., 1994). Two sets of three primers each were used to analyse G418-resistant leu4::KanMX4 transformants and His+ yor108w::HIS3MX6 transformants. Table 1. Saccharomyces cerevisiae strains Strain Genotype Source FY1679 YNN281 YNN282 YMR1 YMR2 YMRX12 a/a ura3-52/ura3-52 leu2D1/+ trp1D63/+ his3D200/+ MATa trp1-D his3-D200 ura 3-52 lys2-801 ade2-1 MATa trp1-D his3-D200 ura3-52 lys2-801 ade2-1 MATa trp1-D his3-D200 ura 3-52 lys2-801 ade2-1 leu4::KanMX4 MATa trp1-D his3-D200 ura3-52 lys2-801 ade2-1 yor108w::HIS3MX6 MATa/MATa trp1-D/trp1-D his3-D200/his3-D200 ura3-52/ ura3-52 lys2-801/lys2-801 ade2-1/ade2-1 leu4::KanMX4/ LEU4 yor108w::HIS3MX6/ YOR108w MATa trp1-D his3-D200 ura3-52 lys2-801 ade2-1 leu4::KanMX4 yor108w::HIS3MX6 Winston et al. (1995) YGSC (Berkeley) YGSC (Berkeley) This work This work This work YMRX-3B Copyright # 2000 John Wiley & Sons, Ltd. This work Yeast 2000; 16: 539±545. Identi®cation of the a-isopropylmalate synthase II gene 541 Table 2. Growth characteristics and a-IPMS activity of S. cerevisiae strains Growth Strain YPD YPD+G418 COM-histidine COM-leucine YPG a-IPMS activityb (%) YNN281 YNN282 YMR1 YMR2 YMRX12 YMRX-3B YMRX-3B/pYCG_YOR108w + + + + + + + x x + x + + + x x x + + + + + + + + + x + + + + + + + + 100 98 20 79 ndc 0 29 a a a a a a See Strains and media section. Activity is reported as a percentage of YNN281 values; speci®c activities are 0.325, 0.318, 0.256, 0.065, 0.000, 0.003 and 0.094 mmol CoA released per hour per mg protein for strains YNN281, YNN282, YMR1, YMR2, YMRX-3B and YMRX-3B/pYCG_YOR108w, respectively. Values are the mean of two independent determinations. c nd=not determined. b Enzyme activity Crude extracts were obtained by grinding pre-weighted humid cells, from a 70 ml COM minus leucine and threonine culture containing 2r108 cells/ml, in a cold mortar with alumina (type 305, Sigma); 50 mM Tris±HCl±1.5 mM phenylmethylsulphonyl ¯uoride, pH 7.5, as added 1 : 2 (w/v) and the suspension was centrifuged at 4uC for 30 min at 39 000rg. The supernatants were collected and stored at x20uC until needed. aIPMS activity was measured according to the end point assay of Kohlhaw (1988) by determining the amount of CoA liberated in 10 min with Ellman's reagent (Sigma). Results and discussion Identi®cation of YOR108w as the gene encoding a-IPMS II To determine the function of the YOR108w gene product, the leu4::KanMX4 and yor108w::HIS3MX6 cassettes were constructed (see Materials and methods) and used to transform YNN281 to G418 resistance and YNN282 to histidine prototrophy, generating the YMR1 (leu4::KanMX4) and YMR2 (yor108w::HIS3MX6) deletion mutants, respectively. The occurrence of homologous recombination events was veri®ed by PCR (data not shown). Strains YMR1 and YMR2 were crossed, diploids were selected for G418-resistance and histidine prototrophy, and the G418R His+ YMRX12 diploid strain was isolated. Ten asci from Copyright # 2000 John Wiley & Sons, Ltd. YMRX12 were dissected and their spores analysed; all the tetrads segregated 2 : 2 for both G418R: G418S and His+:Hisx (data not shown). One G418R His+ segregant, the YMRX-3B (leu4:: KanMX4 yor108w::HIS3MX6) deletion mutant, was further analysed. The deletion mutants YMR1, YMR2, YMRX12, YMRX-3B, the parental strains YNN281 and YNN282 and the plasmid transformed strain YMRX±3B/pYCG_YOR108w were characterized for their ability to grow on different media and for a-IPMS activity. The results, reported in Table 2 and Figure 1, showed that YMR1 (leu4::KanMX4) and YMR2 (yor108w::HIS3MX6) still retained part of a-IPMS activity (79% and 20% of that of the YNN282 wild-type strain, respectively) and were Leu+; YMRX-3B (leu4::KanMX4 yor108w:: HIS3MX6) showed no enzyme activity and was Leux. When a plasmid copy of YOR108w was introduced in the deletion mutant YMRX-3B, aIPMS activity, as well as the Leu+ phenotype, were recovered. These results indicated that YOR108w is the structural gene for a-IPMS II, responsible for the residual a-IPMS activity in the leu4::KanMX4 strain, and that only two a-IPMS encoding genes are present in S. cerevisiae. Furthermore, the growth of YMR2 on nonfermentable carbon sources (Figure 1) indicated that the YOR108w gene is different from LEU6, in fact leu6 mutants did not growth on nonfermentable carbon sources, showing a petite phenotype (Drain and Schimmel, 1988). Mutants bearing leu7 or leu8 alleles were able to grow on non-fermentable carbon sources (Drain and Yeast 2000; 16: 539±545. 542 Figure 1. Growth of deletion mutants and parental strains on COM solid media with 2% different carbon sources in the absence and in the presence of 0.1 mg/l leucine. Cells were grown overnight at 30uC on YPD, washed and resuspended in water at different cell density. Five ml each suspension were spotted on to the plates. The plates were incubated for 2 days at 30uC Schimmel, 1988), but unfortunately it was not possible to test the genetic identity of YOR108w to LEU7 or LEU8, since the suitable mutants were not available. We designated YOR108w as LEU9. Comparison of the sequences of LEU9 and LEU4 genes and of their protein products YOR108w (LEU9) is a member of a cluster of nine genes on chromosome XV that is related to a similar cluster of eight genes on chromosome XIV carrying the LEU4 gene. Wolfe and Shields (1997) formulated the hypothesis that clusters like this arise from whole-genome duplication events. LEU4 and LEU9 are highly homologous genes (82% Copyright # 2000 John Wiley & Sons, Ltd. E. Casalone et al. identity in the ORF regions), whereas no identity was found in the 1500 bp long region upstream from the start codon of LEU4 and LEU9. However, as in LEU4 (Beltzer et al., 1986) putative consensus sequences for Gcn4p (Hinnebusch, 1988) (TGACTCT x1173; AGTCA x762 and TGACTCA x257) and for Leu3p (Friden and Schimmel, 1987) (CCGGTAACGG x277 and GGCCTTGCC x232) were present in LEU9, which could put LEU9 under the general control of amino acid biosynthesis (Hinnebusch, 1992) and leucine-speci®c control (Baichwal et al., 1983; Brisco and Kohlhaw, 1990), respectively. Moreover, comparative analysis showed the presence in both LEU9 (x525) and LEU4 (x594) of the palindromic sequence (TTTACTTCATTT), whose role is unknown. However, as shown by the capability of plasmid pYCG_YOR108w to transform the Leux YMRX-3B strain to leucine prototrophy (see above), the ®rst 345 bp upstream from the start codon of YOR108w seem to be enough for the functionality of the gene. a-IPMS I and a-IPMS II showed a high degree of amino acid identity (82%) and conservation (97%). The putative catalytic site of a-IPMS I (Patek et al., 1994) is completely conserved in the N-terminal moiety of a-IPMS II. Major differences are present at the C-terminal end of the two isozymes, where the so-called regulatory region (R-region) of aIPMS I, of 39 amino acids, is also located. This region is responsible for the enzyme resistance to leucine inhibition and Zn++-mediated CoA inactivation in seven different T¯R mutants (Cavalieri et al., 1999). This region shares 87% identity with the corresponding region of a-IPMS II. In this region, a serine, whose absence is responsible for resistance to leucine feedback inhibition in a mutated a-IPMS I (Cavalieri et al., 1999), is replaced by an alanine in a-IPMS II. This amino acid difference could be responsible for the lower sensitivity to leucine observed in a-IPMS II respect to a-IPMS I (Chang et al., 1985). Physiological characterization of the deletion mutants a-IPMS activity was in¯uenced by the carbon source: (a) growth on acetate induced a form of aIPMS more sensitive to leucine inhibition (Brown et al., 1975), which could very likely be a-IPMS I; (b) induction of LEU4 but not of YOR108w Yeast 2000; 16: 539±545. Identi®cation of the a-isopropylmalate synthase II gene 543 Figure 2. Growth curves of deletion mutants and parental strains in COM medium in the presence or absence of 0.1 mg/l leucine at 30uC (LEU9) occurred in the correspondence of the metabolic shift from fermentation of glucose to respiration of ethanol (De Risi et al., 1997); (c) leucine is released in the medium during the ®nal stage of fermentation (Henschke and Jiranek, 1993) when glucose is exhausted. In order to assess the effect of the deletion of LEU4 and LEU9 on the ability to grow on different carbon sources, YNN281, YNN282, YMR1, YMR2 and YMRX-3B were grown on plates of minimal medium containing glucose or one of the following non-fermentable carbon sources: glycerol, ethanol, lactate, pyruvate and acetate. The results are shown in Figure 1. The growth of all strains was always extremely low on pyruvate and practically undetectable on acetate (not shown). YMRX-3B always failed to grow in the absence of leucine. On glucose, in the presence or absence of leucine, no differences in the growth of YMR1 mutants was observed compared with its parental strain. On non-fermentable carbon sources in the absence of leucine, mutant YMR1 showed always reduced growth, particularly on glycerol; if leucine was added, all the strains grew worse but YMR1 and, as expected, YMRX-3B. YMR2 always grew like the parental strain. These results indicated that the presence of a Copyright # 2000 John Wiley & Sons, Ltd. functional LEU4 gene, but not of LEU9, was critical to maintain normal growth levels on nonfermentable carbon sources when leucine was absent. Conversely, the slight better growth of YMR1 on non-fermentable carbon sources in the presence of leucine could be due to the lack of a functional LEU4 gene, whose absence, reducing the unnecessary synthesis of leucine, makes acetyl CoA available for the Krebs cycle. Nevertheless, a role of the transcription factor Leu3p (Hu et al., 1995), whose activity is modulated by a-IPM, cannot be excluded. Unlike the solid glucose medium, in liquid COM medium with glucose, slight growth differences were observed among strains. In the absence of leucine, strain YMR1, unlike YMR2 (Figure 2B), which lacks a-IPMS I, had a longer initial lag-phase than its parental strain (Figure 2A). This phenotype is due to an impaired synthesis of leucine, as shown by its reversion following leucine addition. It should be also noted that, with the exception of YMR1, a slight decrease in the growth rate of all strains occurred when leucine was added to the medium, as already observed and discussed for the growth on plates with unfermentable carbon sources. Further characterization of the strains was performed by growing them on TFL-containing Yeast 2000; 16: 539±545. 544 E. Casalone et al. Figure 3. Sensitivity of deletion mutants and parental strains to TFL. Cells were grown overnight at 30uC on YPD, washed and resuspended in water at different cell density. Five ml of each suspension were spotted on COM minus leucine plates. The plates were incubated for 2 days at 30uC media. Results in Figure 3 showed that strain YMR1 was more sensitive to TFL than the parental and YMR2 strains. These results con®rmed the main role of a-IPMS I in counteracting TFL toxic effects by the production of the analogue competitor, leucine (Baichwal et al., 1983; Casalone et al., 1997; Cavalieri et al., 1999). The identi®cation of LEU9 as the gene encoding a-IPMS II, and the construction of deletion mutants lacking one or both the structural genes for a-IPMS activity in S. cerevisiae, can be very useful to study the physiological role of the two isozymes. Furthermore, a leu4D leu9D genetic background should be ideal to isolate and investigate new leucine transport mutants (Stella et al., 1999), or as a recipient strain of new vectors carrying LEU4 markers for leucine prototrophy and/or TFL resistance (Bendoni et al., 1999). References Baichwal VR, Cunningham TS, Gatzek PR, Kohlhaw GB. 1983. 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