Lys80p ofSaccharomyces cerevisiae, previously proposed as a specific repressor ofLYS genes, is a pleiotropic regulatory factor identical to Mks1p

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. 13: 1337–1346 (1997)

Lys80p of Saccharomyces cerevisiae, Previously Proposed as a Specific Repressor of LYS Genes, is a Pleiotropic Regulatory Factor Identical to Mks1p ANDRE u FELLER, FERNANDO RAMOS, ANDRE u PIE u RARD AND EVELYNE DUBOIS* Laboratoire de Microbiologie, Faculte´ des Sciences, Universite´ Libre de Bruxelles and Institut de Recherches du CERIA, avenue E. Gryson 1, B-1070 Bruxelles, Belgium Received 4 February 1997; accepted 5 May 1997

In Saccharomyces cerevisiae, an intermediate of the lysine pathway, á-aminoadipate semialdehyde (áAASA), acts as a coinducer for the transcriptional activation of LYS genes by Lys14p. The limitation of the production of this intermediate through feedback inhibition of the first step of the pathway results in apparent repression by lysine. Previously, the lys80 mutations, reducing the lysine repression and increasing the production of lysine, were interpreted as impairing a repressor of LYS genes expression. In order to understand the role of Lys80p in the control of the lysine pathway, we have analysed the effects of mutations epistatic to lys80 mutations. The effects of lys80 mutations on LYS genes expression were dependent on the integrity of the activation system (Lys14p and áAASA). The increased production of lysine in lys80 mutants appeared to result from an improvement of the metabolic flux through the pathway and was correlated to an increase of the á-ketoglutarate pool and of the level of several enzymes of the tricarboxylic acid cycle. The LYS80 genes has been cloned and sequenced; it turned out to be identical to gene MKS1 cloned as a gene encoding a negative regulator of the RAS-cAMP pathway. We conclude that Lys80p is a pleiotropic regulatory factor rather than a specific repressor of LYS genes. ? 1997 John Wiley & Sons, Ltd. Yeast 13: 1337–1346, 1997. No. of Figures: 1. No. of Tables: 2.

No. of References: 35.

  — Saccharomyces cerevisiae; LYS80 gene; á-ketoglutarate; apparent repression; pleiotropic factor

INTRODUCTION Among the various genes involved in the homocitrate/á-aminoadipate pathway of lysine biosynthesis in Saccharomyces cerevisiae (for a review see Bhattacharjee, 1992), at least six (LYS20, LYS21, LYS4, LYS2, LYS9 and LYS1) are regulated by lysine (Ramos and Wiame, 1985; Urrestarazu et al., 1985; Ramos et al., 1996). This control is mediated by the Lys14p transcriptional activator according to the levels of the metabolic intermediate á-aminoadipate semialdehyde (áAASA) serving as a sensor of lysine availability (Ramos et al., 1988; Feller et al., 1994). We have *Correspondence to: E. Dubois, Institut de Recherches du CERIA, 1 Av. E. Gryson, B-1070 Bruxelles, Belgium. Tel: 32-2-5267277; fax: 32-2-5267273; email: [email protected] Contract grant sponsor: Research Council of the Universite´ Libre de Bruxelles. CCC 0749–503X/97/141337–10 $17.50 ? 1997 John Wiley & Sons, Ltd.

presented evidence that the activation function of Lys14p requires the presence of áAASA and is strongly reduced when lysine is added to the growth medium, thus resulting in apparent repression by lysine (Feller et al., 1994). A LexA-Lys14 fusion protein is indeed able to activate the expression of lacZ under the control of the lexA operator whereas high concentrations of lysine antagonize the activation function. Since homocitrate synthase catalysing the first step of lysine biosynthesis is feedback inhibited by lysine (Tucci and Ceci, 1972), high levels of lysine lead to low levels of intracellular áAASA which reduce the transcriptional activation of LYS genes by Lys14p. Homocitrate synthase is thus a key element in the control of áAASA supply and of Lys14p activity. Recently, the genes LYS20 and LYS21 encoding the two isoforms of homocitrate

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1338 synthase have been characterized (Ramos et al., 1996; G. Volckaert, pers. comm.; F. Ramos, unpublished results). A double lys20, lys21 mutant strain is unable to grow in the absence of lysine. Lys14p is a 89 kDa DNA binding protein that belongs to the Zn2Cys6 binuclear cluster family of fungal transcriptional activators. The promoters of the co-regulated LYS genes contain a sequence with CC and GG doublets separated by 3 bp which is required for Lys14p activation and lysine apparent repression (Becker, in preparation). It was reported previously that the repression by lysine was dependent on a negative regulator encoded by LYS80 (Ramos and Wiame, 1985). The lys80 mutations were obtained during the search for mutants defective in threonine catabolism. A first lys80-1 mutation was present in addition to the cha1 mutation (catabolism of hydroxy amino acids) in a mutant selected for the absence of growth on threonine as the nitrogen source. The cha1 gene encodes the catabolic -serine (-threonine) deaminase and is regulated by transcriptional induction by serine or threonine (Ramos and Wiame, 1985; Petersen et al., 1988) mediated by the Cha4p, a transcriptional activator belonging to the Cys6 zinc cluster class (Holmberg and Schierling, 1996). Another mutation, lys80-2, was isolated independently of a cha1 mutation and caused a reduction of the growth rate on threonine. The generation time was 6 h instead of 4 h for a wild-type strain. These lys80 mutations led to a considerable increase in cellular lysine pools, to an increase of LYS gene expression on minimal medium and to an impairment of LYS gene repression by lysine. These mutations were interpreted as affecting a specific repressor of LYS gene expression and were therefore called lys80. Recently, we have shown that the antagonistic effect of lysine on the activation ability of the LexA-Lys14 fusion protein is strongly reduced in a lys80 mutant strain (Feller et al., 1994), a behaviour which seems incompatible with that of a strain mutated in a repressor gene. The aim of the present work was to reinvestigate the nature of the LYS80 gene product. Evidence was obtained that the ability of lys80 mutations to increase the lysine pool is correlated to an increase of the cellular concentration of á-ketoglutarate, a precursor of the lysine pathway. Moreover cloning of the LYS80 gene revealed that LYS80 is identical to the MKS1 gene encoding a negative regulator of the RAS-cAMP pathway. 

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MATERIALS AND METHODS Strains and media The Escherichia coli strain used for plasmid maintenance and for propagation was XL1-Blue from Stratagene (recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac [F* proAB lacIqZÄM15 Tn10 (Tetr)]). All strains of S. cerevisiae were derivatives of the MATá wild-type strain Ó1278b (Be´chet et al., 1970): 8761c (lys80-1), 8953b (lys80-2), 12T7c (ura3), 13T6d (ura3,lys80-1), 17T9c (ura3,lys801,lys2), 13T7d (ura3,ilv1), 13T9a (ura3,lms,ilv1), 14TOd (ura3,lms,ilv1,lys80-1), 17T7a (ura3, lys2,lys9,lys80-1) and 14T3c (lys4 issued from MG360, lys1 issued from MG762). All yeast strains were grown on minimal medium containing 10 m-(NH4)2SO4, 3% glucose, vitamins and mineral traces as described previously (Messenguy, 1976). Where specified, 66 ìg of -lysine per ml, 25 ìg of uracil per ml and 25 ìg of -isoleucine per ml were added to the minimal medium. Enzyme assays and measurement of metabolite pools All the protein extracts were dialysed before determination of the different enzymatic activities. Each value of enzyme specific activity and metabolite concentration is the mean of two of three independent measurements. The level of variability is about 10% for enzyme specific activity and 15% for metabolite concentration. Saccharopine dehydrogenase (NADP + , glutamate forming; EC 1.5.1.10) was assayed as described by Jones and Broquist (1965). Saccharopine dehydrogenase (NAD + lysine forming; EC 1.5.1.7) was assayed as described by Fujioka and Nakatami (1970). Argininosuccinate lyase (EC 4.3.2.1) was assayed as described by Delbecq et al. (1994) but the argininosuccinate concentration was 2 m instead of 10 m. Citrate synthase (EC 4.1.3.7), isocitrate dehydrogenase (EC 1.1.1.41, EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37) and aconitase (aconitate hydratase; EC 4.2.1.3) were assayed as described in Parvin (1969), Kornberg (1955) and Fansler and Lowenstein (1969) respectively. For lysine pool measurements, exponentially growing cells were extracted with ice-cold 0·3 HClO4. The resulting extract was neutralized with K3PO4, centrifuged to eliminate the cells and ? 1997 John Wiley & Sons, Ltd.

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acidified to pH 6·2 with HCl. The solution obtained was used in a bioassay for growing the lysine auxotroph 14T3c containing two mutations (lys4 and lys1) in order to avoid the selection of prototrophic revertants. Glutamate and á-ketoglutarate pools were determined as described in Delforge et al. (1975) and Dubois et al. (1974) respectively. DNA techniques All procedures for DNA manipulations were carried out by standard procedures (Sambrook et al., 1989). All DNA sequences of double-stranded templates were determined by the method of dideoxy chain termination (Sanger et al., 1977) using Sequenase DNA polymerase (USB), [35S]dATP and synthetic oligonucleotides as walking primers. Cloning of the LYS80 gene A 4·9 kb SalI-SalI fragment bearing the LYS2 gene isolated from plasmid pDP6 (Fleig et al., 1986; a gift from P. Phillipsen) was inserted in the unique SalI restriction site of pFL38 plasmid (pUC19-ARS CEN-URA3; Bonneaud et al., 1991) yielding plasmid pBB17. To destroy the two ClaI restriction sites flanking the ARS CEN region, the pBB17 was digested by ClaI and after ligation and E. coli transformation, a plasmid named pLAF58 containing the ARS CEN region and having lost the two ClaI restriction sites was selected. The plasmid pLAF59 (pUC19-ARS CEN-URA3LYS2) was obtained by inserting, in the unique PstI site of pLAF58, an oligonucleotide bearing the following restriction sites: PstI-MluI-ClaIMluI-PstI. The genomic DNA of the wild-type Ó1278b was isolated (Cryer et al., 1975) and partially digested by TaqI. The genomic TaqI fragments were inserted in the unique ClaI site of pLAF59 to give a low copy number library. Yeast strain 17T7a (ura3,lys2,lys9,lys80-1) was transformed by this DNA library on minimal medium plus lysine by the method of Ito et al. (1983). The pLAF67 plasmid containing a 8 kb PstI-PstI fragment was isolated from this library. The subcloning of this insert was performed by insertion of the following fragments in pFL38 vector: the 4·7 kb BamHIBamHI in the BamHI restriction site, the 2·6 kb EcoRI-EcoRI in the EcoRI restriction site, the 3·1 kb NcoI-BamHI blunt-ended by the Klenow ? 1997 John Wiley & Sons, Ltd.

enzyme in the SmaI restriction site, the 2·3 kb EcoRV-XbaI in the SmaI-XbaI restriction sites and the 2·9 kb BamHI-HindIII in the BamHI-HindIII restriction sites. Cloning and sequencing of lys80 mutants To determine the sequence of the LYS80 gene and of the mutant loci lys80-1 and lys80-2, the LYS80 region was amplified by PCR using as template the chromosomal DNA of strains Ó1278b (wild-type), 13T2d (ura3,lys80-1) and 8953b (lys80-2) isolated by the method of Karier and Auer (1993). We used the oligonucleotides OA52 (gcggatccCTCAAACTTGTGCAGATTGC) and OA53 (gcggatccGTGTGTCATTAGAAGGAA CT) derived from the published sequence of the yeast genome and extended with a BamHI restriction site. These oligonucleotides are located respectively at 533 bp upstream from the ATG and 113 bp downstream from the stop codon. The 3·3 kb BamHI-BamHI fragments were inserted in the BamHI site of pFL44L vector (pUC192ì-URA3; Bonneaud et al., 1991) and entirely sequenced. Deletion of the LYS80 gene The PCR-strategy of Wach (1996) was followed to disrupt the LYS80 gene. A disruption cassette containing the KanMX4 cassette conferring to yeast a resistance to geneticin (G418) with long flanking regions homologous to LYS80 was synthesized by PCR. The yeast strains, PCR protocols and plasmids are identical to those described in the original articles (Wach et al., 1994; Wach, 1996). The first two PCRs were made with oligonucleotides P5*/P5*L and P3*/P3*L using genomic DNA as template. The P5* oligonucleotide (TCAACCG GGATCGTGTCATA) is located 450 bp upstream from the start codon. The P5*L oligonucleotide (ggggatccgtcgacctgcagcCATTTTCAGGTTCCAG TCTCTC) is located at the start codon of LYS80. The P3* oligonucleotide (GTTGCAGTTGAGA ATCTTGT) is located at 374 bp downstream from the stop codon. The P3*L oligonucleotide (cgagctc gaattcatcgatgaTGATTACTAAGTTAAATAAA TCAG) is located at the stop codon of LYS80. The lower-case letters are identical to the borders of the cassette. The second PCR was performed using as template NotI-digested pFA4-KanMX4 and each product from the first PCR together with the oligonucleotide P3* and P5*. 

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1340 Haploid strain 12T7c (ura3) was transformed with 1 ìg of PCR product according to the method of Gietz et al. (1992) adapted for geneticin selection (Wach, 1996). Integration of the KanMX4 cassette at the target locus was confirmed by PCR using three oligonucleotides located 304 bp upstream from the LYS80 stop codon, 428 bp downstream from the LYS80 stop codon and 125 bp upstream from the 3* end of the KanMX cassette. The 5* region was checked by PCR using three other oligonucleotides located 142 bp downstream from the LYS80 start codon, 550 bp upstream from the LYS80 start codon and 126 bp downstream from the 5* end of the KanMX cassette. RESULTS Suppression of lys80 effects on LYS genes expression Since we have evidence that repression of LYS genes by lysine is not independent of activation by Lys14p and áAASA, we have analysed the expression of LYS genes in the Lys80,lys2 double mutant strain in which there is no production of áAASA. As shown in Table 1, the lys2 mutation is epistatic to lys80 mutations, the impairment of LYS gene repression by lysine in a lys80 strain being abolished in the lys80,lys2 strain. The effects of lys80 mutation on LYS gene expression are consequently dependent on a functional activation system (Lys14p plus á-AASA). We have tried to select other mutations leading to the suppression of the lys80 phenotype. The selection was based on the following observations. An ilv1 mutant strain, lacking the anabolic threonine deaminase is unable to grow on minimal medium without isoleucine whereas it is able to grow on serine as sole nitrogen source. Under these conditions, the catabolic threonine/serine deaminase is induced and converts intracellular threonine into á-ketobutyrate, the precursor of isoleucine. We did however observe that the presence of a lys80 mutation strongly reduces the growth rate of a ilv1 mutant strain on serine (7 h instead of 3 h). The difference in growth rate allowed us to select a spontaneous mutation conferring the ability to grow on serine to the ilv,lys80 mutant strain. This new mutation was called lms for lys80 mutation suppressor. As shown in Table 1, the lms mutation prevents not only the high production of lysine due to the lys80 mutation but also strongly reduces that production as compared 

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to a wild-type strain. The lms mutation is recessive, monogenic and not linked to lys80. In this strain, an extremely low lysine pool and a reduced glutamate pool are consequences of a six-fold reduction of the á-ketoglutarate pool. In contrast, in a lys80 mutant, the á-ketoglutarate intracellular concentration is increased three- to six-fold as compared to a wild-type strain. A correlation can consequently be established between the pools of á-ketoglutarate and of lysine. The increased production of á-ketoglutarate augments the entry of metabolites in the lysine pathway and consequently the synthesis of lysine. The normal repression of the LYS gene by lysine is restored in the lys80,lms double mutant strain since the synthesis of saccharopine dehydrogenase (lysine forming) is six-fold repressed by lysine like in a wild-type strain (see Table 1). The higher level on minimal medium is the result of derepression of the general control as indicated by a higher level of argininosuccinate lyase, the product of the gene ARG4 which was chosen as a reference enzyme since it only obeys the general control of amino acid biosynthesis. This derepression is probably due to lysine starvation but also to starvation for another amino acid because the presence of lysine in the growth medium does not allow complete restoration of the repression of LYS1 expression. All our data indicate that the effect of the lys80 mutation on expression of LYS genes and on lysine production is a consequence of the improvement of the metabolic flux through the lysine pathway. This leads to an increase of áAASA concentration and to a more efficient activation of LYS genes by Lys14p. Role of LYS80 and LMS gene products in the expression of genes encoding the tricarboxylic acid cycle enzymes Since the á-ketoglutarate intracellular pool is increased in the lys80 mutant and decreased in the lms mutant strain, we have measured the levels of the enzyme activities involved in the production of á-ketoglutarate such as citrate synthase, aconitase and NAD isocitrate dehydrogenase. The lys80 mutation leads to a two- to three-fold increase of these different enzymatic activities whereas in the lms mutant strain, the synthesis of citrate synthase is reduced four-fold and the level of aconitase is two-fold weaker than in the wild-type strain (see Table 2). The synthesis of the other enzymes of the tricarboxylic acid cycle such as malate ? 1997 John Wiley & Sons, Ltd.

  Lys80p

? 1997 John Wiley & Sons, Ltd.

Table 1.

Effect of mutations in the LYS80 genes and of a suppressor of the lys80 mutation on LYS gene expression and lysine production. Specific activity (ìmol/h per mg protein)

Strain (genotype) Ó1278b (w.t.) 8761c (lys80-1) 8953b (lys80-2) 12T7cL80* (ura3,lys80::KanMX4) 17T9c* (ura3,lys2,lys80-1) 13T7d* (ura3,ilv1) 13T9a* (ura3,ilv1,lms) 14TOd* (ura3,ilv1,lys80-1,lms)

Media



M.am M.am+lys M.am M.am+lys M.am M.am+lys M.am M.am+lys M.am+lys M.am M.am+lys M.am M.am+lys M.am M.am+lys

Saccharopine dehydrogenase (glutamate forming)

Saccharopine dehydrogenase (lysine forming)

1·8 0·2 3·1 1·0 4·4 2·5 4·2 2·1 0·1

15·0 2·5 22·0 9·0 51·0 28·0 3·0 15·0 3·0 26·0 9·0 34·0 6·0

Pool (nmol/mg dry cells)

Argininosuccinate lyase

Lysine

á-Ketoglutarate

Glutamate

18

1·5

124

110

5·0

200

150

10·0

210

10·0

1·2

18

6·0 1·3

3·0 2·5 3·5

1

0·25

1

0·25

20

*If required, uracil (25 ìg/ml) or isoleucine (50 ìg/ml) were added to the growth medium. M.am, minimal medium.

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Table 2. Effect of mutations in the LYS80 genes and of a suppressor of lys80 mutation on the levels of enzymes involved in the production of á-ketoglutarate. All specific activities are expressed in ìmol of formed product/h per mg protein. Isocitrate dehydrogenases Strain (genotype) Ó1278b (w.t.) 8761c (lys80-1) 8953b (lys80-2) 12T7cL80* (ura3,lys80::KanMX4) 13T7d* (ura3,ilv1) 13T9a* (ura3,ilv1,lms)

Citrate synthase

Aconitase

NAD

NADP

Malate dehydrogenase

2·1 4·3 7·0 10·7 2·3 0·4

1·1 2·0 2·4

1·0 2·6 3·7

2·3 3·3 3·7

9·8 9·2 9·2

1·3 0·6

1·8 1·7

4·3 4·8

All the strains are grown on minimal medium. *If required, uracil (25 ìg/ml) or isoleucine (50 ìg/ml) were added to the growth medium.

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dehydrogenase and isocitrate dehydrogenase (NADP + ) is not affected by the lys80 and lms mutations (see Table 2). It is noteworthy that these mutations are not linked to CIT1 (mitochondrial citrate synthase), CIT2 (peroxisomal citrate synthase) or ACO1 (aconitase) loci. The products of LYS80 and LMS thus play directly or indirectly a role in the synthesis of enzymes leading to á-ketoglutarate production. In order to obtain more information about the function of Lys80p, we have undertaken the cloning and sequencing of the LYS80 gene. Cloning and sequencing of LYS80 gene We chose to clone the LYS80 gene by complementation of a peculiar phenotype resulting from the lys80 mutation. We observed that the combination of mutations lys80 and lys9 causes a loss of cell viability, a behaviour which is not observed when the strains also carry a lys2 mutation. This probably results from the fact that these strains possess an increased metabolic flux that causes a toxic accumulation of áAASA (Zaret and Sherman, 1985), this accumulation being avoided in the presence of the lys2 mutation which prevents the production of áAASA. The cloning was achieved by transforming the triple lys80,lys9,lys2 mutant strain with a genomic library of S. cerevisiae inserted in a plasmid bearing the wild-type LYS2 gene. To this end, we have inserted the product of a partial Taq1 digestion of chromosomal DNA from the wild-type strain Ó1278b in the ClaI restriction site of plasmid pLAF59 (pUC19-ARS CENLYS2-URA3; see Materials and Methods for construction of this vector). The strain 17T7a (lys80,lys9,lys2,ura3) was transformed with the genomic library and the transformants were selected on minimal medium+lysine. Since all the URA + transformants also contain the LYS2 gene, they are unable to grow on this medium. All the transformants except one contained a plasmid bearing the LYS9 gene. From the colony growing on minimal medium+lysine which is still lys9, we recovered a plasmid (pLAF67) with an insert of about 8 kb that was able to restore the cellular growth of the lys80,lys9,lys2,ura3 strain. The PstI-PstI fragment containing the complete insert was introduced in the pFL38 vector (pUC19-ARS CEN-URA3), leading to the plasmid pLAF70. In the lys80,ura3 strain (13T6d) transformed with the plasmid PLAF70, the pro? 1997 John Wiley & Sons, Ltd.

duction of lysine and á-ketoglutarate and the expression of LYS genes in the presence or not of lysine were similar to those of a wild-type strain (see Table 1). The nucleotide sequence of one extremity of the insert was determined and compared to the sequence of the yeast genome in the MIPS data base. This revealed that the cloned DNA fragment is located on the left arm of chromosome XIV and contains four open reading frames YNL074c, YNL075w, YNL076w and YNL077w. This DNA fragment has been sequenced during the sequencing of the complete yeast genome but only the YNL076w ORF has been previously described as the product of the MKS1 gene (Matsuura and Anraku, 1993). MKS1 encodes a protein of 584 aa whereas the YNL076w is described as a protein of 599 aa. The difference in length of the two ORFs results from the presence of an intron not mentioned by Matsuura and Anraku (1993). Through a combination of subcloning experiments (see Materials and Methods) and back transformation of the lys80,ura3 strain (13T6d), the complementing activity was located on a 3·1 kb NcoI-BamHI fragment containing the MKS1 gene (see Figure 1). Our data consequently demonstrate that the LYS80 and MKS1 loci are identical. The complete coding sequence of LYS80 isolated from the Ó1278b wild-type strain was determined using synthetic oligonucleotides as primers. This sequence is 99% identical to the YNL076w sequence. There are 17 base substitutions, six of them being located in the intron and five of them leading to amino acid replacements (His90 by Tyr, Pro159 by Ala, Asp169 by Glu, Pro126 by Thr and Glu465 by Gly). Complete inactivation of the LYS80/MKS1 gene was achieved by replacing its coding region with the KanMX4 cassette (see Materials and Methods). The previously characterized lys80 mutations and the lys80 deletion affected similarly the production of á-ketoglutarate and the expression of the genes of lysine biosynthesis and of the tricarboxylic acid cycle. This result confirms that Lys80p is not essential for cellular growth. The mutated lys80-1 and lys80-2 genes were cloned and sequenced. Both mutations consisted of G]A transversions, yielding a stop codon at amino acid position 347 in the case of lys80-1 and the replacement of Ser73 by Asn for lys80-2. 

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Figure 1. Subcloning analysis of the genomic area around the MKS1/LYS80 locus. Subclones represented by the black lines were constructed as described in Materials and Methods. All plasmids were tested for lys80 complementing activity by transformation of strain 13T6d (lys80,ura3). Complementation was scored by growth on minimal medium with 10 ìg/ml AEC. The open boxes represent the open reading frames present on the 8 kb PstI-PstI insert from plasmid pLAF70.

DISCUSSION The early inference that gene LYS80 encodes a repressor of LYS genes expression was essentially based on the observation that lys80 mutations reduce the repression of LYS genes and increase the production of lysine without affecting the feedback inhibition of homocitrate synthase by lysine (Ramos and Wiame, 1985). This conclusion was, however, difficult to reconcile with more recent observations such as the influence of lys80 mutations on the activation ability of a LexALys14 fusion protein (Feller et al., 1994) or the suppression of lys80 effects in the absence of a functional activation system (Lys14p+áAASA). In this study, we show that the lys80 mutations, due probably to an increased production of several citrate cycle enzymes, lead to an increase of the á-ketoglutarate pool. Such mutations are consequently expected to augment the metabolic flux through the lysine pathway and to result in an increased concentration of áAASA. Therefore, our data demonstrate that Lys80p is not a repressor of LYS genes and agree with the conclusion that lysine does not work as a co-repressor but as a modulator of áAASA co-activator production. The products of LYS80 and LMS play directly or indirectly a role in the synthesis of enzymes 

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involved in the production of á-ketoglutarate. In S. cerevisiae, the synthesis of citrate synthase and aconitase is repressed by glucose alone and, synergistically, by glucose and a glutamate source (Kim et al., 1986; Gangloff et al., 1990). However, the decreased or increased levels of citrate cycle enzymes in lms and lys80 mutant cells are independent of the carbon source, indicating that these factors are not involved in catabolite repression (unpublished results). In contrast to the RTG2 gene which is a regulator of ACO1 expression only under catabolite repression conditions (Ve´lot et al., 1996), the LMS gene is required for maximal synthesis of aconitase and citrate synthase in the presence not only of glucose but also of galactose or lactate. That Lys80p is not a specific regulator of lysine biosynthesis is further supported by the demonstration of the identity of the LYS80 gene with the MKS1 gene. This gene was identified for its ability to inhibit growth of cells with diminished cyclicAMP-dependent kinase activity. These data are in favor of a negative regulatory function of Mks1p in transcription of several genes controlled by cAMP (Matsuura and Ankaru, 1993). It is difficult to correlate the data about MKS1 and the effect of lys80 mutations on the expression of citrate cycle genes; a positive or negative effect of cAMP on ? 1997 John Wiley & Sons, Ltd.

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expression of these genes has never been reported. Moreover, analysis of the effects of cAMP on protein synthesis showed that cAMP had only a minor effect on the protein pattern of cells growing exponentially on glucose (Boy-Marcotte et al., 1990). In contrast, deletion of the LYS80 gene affects the production of enzymes of the tricarboxylic acid cycle during growth on glucose medium. Although the primary target of the Lys80p remains unknown, it is clear that this 599 aa protein presenting no feature of DNA binding proteins is required for expression of a large set of genes. Moreover, it is noteworthy that the lys80 mutations were selected for a slower growth on threonine as sole nitrogen source. It would consequently be interesting to study the expression of the CHA1 gene encoding the catabolic -serine/-threonine deaminase in a lys80 deleted strain. ACKNOWLEDGEMENTS We thank Fabienne Vierendeels for excellent technical assistance in the sequencing of LYS80. We are grateful to M. Crabeel for helpful discussions and to P. Phillipsen for the gift of plasmid pDP6. This work was supported by the Research Council of the Universite´ Libre de Bruxelles. REFERENCES Be´chet, J., Grenson, M. and Wiame, J.-M. (1970). Mutations affecting the repressibility of arginine biosynthetic enzymes in Saccharomyces cerevisiae. Eur. J. Biochem. 12, 31–39. Bhattacharjee, J. K. (1992). Evolution of áaminoadipate pathway for the synthesis of lysine in fungi. In Mortlock, R. P. (Ed.), The Evolution of Metabolic Function. CRC Press, New York, pp. 47– 80. Bonneaud, N., Ozier-Kalogeropoulos, O., Li, G., Labouesse, M., Minvielle-Sebastia, L. and Lacroute, F. (1991). A family of low and high copy replicative, integrative and single stranded S. cerevisiae/E. coli shuttle vectors. Yeast 7, 609–615. Boy-Marcotte, E., Tadi, D., Perrot, M., Boucherie, H. and Jacquet, M. (1996). High cAMP levels antagonize the reprogramming of gene expression that occurs at the diauxic shift in Saccharomyces cerevisiae. Microbiology 142, 459–467. Cryer, D. R., Eccleshall, R. and Marmur, J. (1975). Isolation of yeast DNA. Meth. Cell. Biol. 12, 39–44. Delbecq, P., Werner, M., Feller, A., Filipkowski, R. K., Messenguy, F. and Pie´rard, A. (1994). A segment of ? 1997 John Wiley & Sons, Ltd.

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