Eur. J. Biochem. 188, 597-603 (3990) 0FEBS 1990
Molecular analysis of the structural gene for yeast transaldolase Ine SCHAAFF, Stefan HOHMANN and Friedrich K. ZIMMERMANN Institut fur Mikrobiologie, Technische Hochschule Darmstadt, Federal Republic of Germany (Received August 16/November 27, 1989) - EJB 89 1020
We have cloned the structural gene for yeast transaldolase. Transformants carrying the TALl gene on a multicopy plasmid over-produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild-type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TALl is the only transaldolase structural gene in yeast. TALI is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase-deficient strains accumulated sedoheptulose 7-phosphate, an intermediate of the pentose-phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7-phosphate on medium containing fructose than on glucose medium. This shows that fructose 6-phosphate and glyceraldehyde 3-phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose-phosphate pathway. The pentose-phosphate pathway (Fig.1) supplies precursor
Evidence for a direct interaction between glycolysis and
molecules for several important biosynthetic pathways : ribose
the pentose-phosphate pathway comes from the observation
5-phosphate for the synthesis of nucleic acids and histidine, erythrose 4-phosphate for the synthesis of aromatic amino acids and NADPH as reducing agent [l].Several bacteria and yeast species use the nonoxidative part of the pentosephosphate pathway for the utilization of xylose as sole carbon and energy source. Xylose is converted to fructose 6-phosphate and glyceraldehyde 3-phosphate which can enter glycolysis or polysaccharide synthesis [2, 31. Baker’s yeast, Saccharomyces cerevisiae, is unable to metabolize xylose but its isomerization product xylulose can be fermented to ethanol [41. However, the pentose-phosphate pathway can not substitute for glycolysis in the breakdown of glucose in yeast since deletion mutants in the structural gene for phosphoglucose isomerase do not grow with glucose as carbon source [5]. In contrast, Escherichia coli mutants deficient in phosphoglucose isomerase can use the pentose-phosphate pathway for glucose degradation [6]. The oxidative part of the pentose-phosphate pathway seems to be dispensible in yeast. Mutants lacking both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are not impaired for growth on glucose [7]. Similar results were obtained for E. coli and Drosophilu [S, 91.
that yeast phosphofructokinase mutants accumulate sedoheptulose 7-phosphate [lo, 111. Since mutants lacking one of the two phosphofructokinase genes ferment glucose but do not show phosphofructokinase activity in vitro, a pathway bypassing this reaction was proposed. This bypass should include reactions of the nonoxidative part of the pentosephosphate pathway [12]. We want to investigate this pathway in yeast using genetic methods in order to determine the importance of the nonoxidative part of the pentose-phosphate pathway in yeast metabolism and how glycolysis and the pentose-phosphate pathway interact. In this paper, we present the molecular analysis of TALI, the structural gene for transaldolase and describe the properties of a yeast mutant deleted for TALI.
Correspondence to I. Schaaff, Institut fur Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, D-6100 Darmstadt, Federal Republic of Germany Enzymes. Acetohydroxyacid reductoisomerase (EC 1.1 .I 26); fructose-bisphosphate aldolase (EC 4.1.2.13); glucose 6-phosphate 1); dehydrogcnase (EC 1.I .1.49); 6-phosphofructokinase (EC 2.7.1.I 6-phosphogluconate dehydrogenase (EC 1.1 .I .44) ; phosphoglucose isomerase (EC 5.3.1.9); D-ribose 5-phosphate ketol-isomerase (EC 5.3.3.6); ~-ribulose-5-phosphate3-epimerase (EC 5.1.3.1); transketolase (EC 2.2.1.1). Note. The sequence data described in Fig. 6 appear in the EMBL/ GenBank/DDBJ Nucleotide Sequence Databases under the acession number XI5953 S. cerevisiae TALI.
NADP+NADPH+H+ Glucose 6-P
-
H-0
G - ~ P D Hgluconolodone Loctonase
6-Phosphogluconate
N P @OH$ - ~ ~
NADPH*H,COz
Ribiose 5-P Isomerase/
\pimerose Tronsketolase
fhrxtose]Erythrose 4-P Fig. 1. Diagram o j t h e pentose-phosphate pathway. ~-Glucose-6-phosphate dehydrogenase (G-6-P DH), 6-phosphogluconolactonase (lactonase) and 6-phosphogluconate dehydrogenase (6-PG DH) are the enzymes of the oxidative part. ~-Ribulose-5-phosphate 3epimerase, n-ribose-5-phosphate ketol-isomerase (isomerase), transketolase and transaldolase are the enzymes of the nonoxidative part
598 MATERIALS AND METHODS Strains and media
P
B
S
E
1,
1 1 .
I
Yeast strain UTL-7A MATa leu2-3/112 ura3-52 trpl (H. D. Schmitt) was used as recipient for transformation experiments and the diploid strain M4 MATa leu2-3/112 ura3-52 trpl MATol leu2-31112 U R A 3 T R P l
was used to introduce the deletion of T A L I . The phosphoglucose-isomerase-deficient strain G I 1 MATu pgil A 34: LEU2 his4 ura3 GAL2 [5] was used for the construction of the double mutants. Yeast strains were grown either in rich media (2% peptone, 1% yeast extract) or in synthetic media (0.5% ammonium sulfate, 0.17% Difco yeast nitrogen base without amino acids, supplemented with amino acids, adenine and uracil). Carbon sources were added as indicated in the results. The pgil deletion mutant requires trace amounts of glucose for growth and was grown in medium containing 2% fructose as carbon source supplemented with 0.1 YOglucose
PI.
E. coli strains Y1088, Y1090 and BTA282 [I31 were used for handling the &$I1 library. Nucleic acid manipulations
Standard procedures were used unless otherwise indicated 1141. For hybridization experiments DNA or RNA were transferred to nylon filters (Amersham) by vacuum blotting (Pharmacia-LKB). For DNA sequencing fragments were subcloned into M13mp18 and M13mp19 [I51 and sequenced on both strands (Fig. 2) by the chain-termination method [I61 using the T7 polymerase sequencing kit from PharmaciaLKB. Yeast sphaeroblasts were transformed according to Beggs ~71. Preparation of antiserum and screening ofI.gtl1 library
Purified yeast trdnsaldolase was obtained from Boehringer Mannheim and used for the immunization of rabbits. The specificity of the antiserum was assayed by ELISA, Western blot analysis and inactivation of transaldolase activity in yeast crude extracts. Anti-E. coli antibodies were removed from the antiserum by incubation with E. coli lysates for 2 h and sedimentation of the precipitate. The Agtl 1 expression library (YL1001) contained genomic DNA fragments from yeast strain X2380 and was obtained from Clontech Laboratories Inc. (Palo Alto CA). 7 x lo5 recombinant phages were screened as described by Young and Davis [ 131 except that alkaline-phosphatase-linked secondary antibody (Sigma) was used for detection of positive phage plaques. Two identical clones were identified and shown to produce an immunopositive fl-galactosidase fusion protein of 145 kDa. The 2.0-kb insert of yeast DNA was subcloned into pUC19 [IS] and used as a radioactive probe to isolate larger DNA fragments by colony hybridization [I81 from a yeast genomic library constructed by Nasmyth and Tatchell [19] in the vector YEpl3 [20]. Deletion of TALl
The transaldolase gene was deleted following the method of Rothstein [21]. A 6-kb SphI-Sac1 fragment from clone
E
S
,I/
BIE I' XX$"ElBH B
\
SpBaH
1
LEU2
\
Xh
P BI XIE
'\\,
1
Sa
BaXP
\
1 kb
TA L1 c
_ c
ILVS
_
--
-
a _ c
.
Fig. 2. Restriction mup of the cloned TALI locus. In the upper part the strategy for the deletion of the transaldolase gene by replacement with LEU2 is indicated. The SphI site a t the left side derives from the vector YEpl3 [20]. Below the restriction map the sequencing strategy is shown. The close linkage to the ZLV5 gene was found by sequence analysis. Restriction enzyme abbreviations: B, Bgnl ; Ba, BumHl; BI, Bun; E, EcoRI; H, HindIII; P, PstI; S, Sufi; Sa, Sucl; Sp, SphI; X, XbuI; Xh, XhoI
TALI-2 (Fig. 3 ) was subcloned into pUC19 [I61 (Fig.2). The 1.45-kb PstI - XhoI fragment containing the entire coding region of TALI was replaced by the 3.43-kb PstI- XhoI fragment carrying the LEU2 gene from YEpl3 [20]. The resulting plasmid was digested with HindIII prior to yeast transformation. Western blot analysis
Yeast crude extracts were denatured and electrophoresed in 8% polyacrylamide gels [22]. Proteins were transferred to nitrocellulose by electroblotting. Immunoblot analysis was performed according to Burnette [23] using non-fat dried milk as blocking agent and alkaline-phosphatase-linked secondary antibody for detection. Trunsaldolase enzyme assay
Yeast crude extracts were prepared according to Ciriacy and Breitenbach [24] using 50 mM imidazole pH 7.6. Specific transaldolase activity was determined as described by Bergmeyer [25]. Protein was measured by the microbiuret method [26]. Determination of metabolite levels
Extracts for metabolite determinations were prepared as described by Ciriacy and Breitenbach [24]. Levels of sugar phosphates were determined as described by Bergmeyer [25]. RESULTS Cloning qfTAL1
Three overlapping clones were isolated from the yeast genomic library in vector YEpl3 (Fig.3). YEpl3 is a multicopy yeastlE. coli shuttle vector [20]. These three plasmids were transformed into strain UTL-7A and transaldolase specific activity was determined. Transformants with plasmids TALI-I and TALI-2 over-produced transaldolase activity, indicating that they carry the structural gene for transaldolase. Plasmid TALI-3 does not contain the entire T A L l gene. Fig. 4A shows a Western blot analysis of transformants carrying plasmids TALI-1 and TALI-2. Both immunopositive proteins present in the crude extract of the untransformed strain are overproduced in the transformants.
599 trnnsaldalase octlvlty
c_I
E
Ba
E
EE
E
M E
EE
E
EE
Ba
E
Ba
Table 1. Tetrad analysis of diploid transformants Spore
LEU-phenotypeltransaldolase activity in tetrad
pTAL1-1
1400
13 4
pTAL1-2
635
61
3
pTAL1-3
110
11
- / m u . mg protein-
E
'
"+
2
3
4
5
6
-191 +/
Molecular analysis of the structural gene for yeast transaldolase Ine SCHAAFF, Stefan HOHMANN and Friedrich K. ZIMMERMANN Institut fur Mikrobiologie, Technische Hochschule Darmstadt, Federal Republic of Germany (Received August 16/November 27, 1989) - EJB 89 1020
We have cloned the structural gene for yeast transaldolase. Transformants carrying the TALl gene on a multicopy plasmid over-produced transaldolase. A deletion mutant which was constructed using the cloned gene did not show any detectable transaldolase activity in vitro. Furthermore, both transaldolase isoenzymes which were detected in wild-type crude extracts by immunoblotting were missing in the deletion mutants. Thus, TALl is the only transaldolase structural gene in yeast. TALI is not an essential gene. Deletion of the transaldolase gene did not affect growth on complete media with different carbon sources or on synthetic media. However, the transaldolase-deficient strains accumulated sedoheptulose 7-phosphate, an intermediate of the pentose-phosphate pathway. Mutants lacking both transaldolase and phosphoglucose isomerase grew more slowly than the single mutants. They accumulated more sedoheptulose 7-phosphate on medium containing fructose than on glucose medium. This shows that fructose 6-phosphate and glyceraldehyde 3-phosphate, metabolites of glycolysis, can enter the nonoxidative part of the pentose-phosphate pathway. The pentose-phosphate pathway (Fig.1) supplies precursor
Evidence for a direct interaction between glycolysis and
molecules for several important biosynthetic pathways : ribose
the pentose-phosphate pathway comes from the observation
5-phosphate for the synthesis of nucleic acids and histidine, erythrose 4-phosphate for the synthesis of aromatic amino acids and NADPH as reducing agent [l].Several bacteria and yeast species use the nonoxidative part of the pentosephosphate pathway for the utilization of xylose as sole carbon and energy source. Xylose is converted to fructose 6-phosphate and glyceraldehyde 3-phosphate which can enter glycolysis or polysaccharide synthesis [2, 31. Baker’s yeast, Saccharomyces cerevisiae, is unable to metabolize xylose but its isomerization product xylulose can be fermented to ethanol [41. However, the pentose-phosphate pathway can not substitute for glycolysis in the breakdown of glucose in yeast since deletion mutants in the structural gene for phosphoglucose isomerase do not grow with glucose as carbon source [5]. In contrast, Escherichia coli mutants deficient in phosphoglucose isomerase can use the pentose-phosphate pathway for glucose degradation [6]. The oxidative part of the pentose-phosphate pathway seems to be dispensible in yeast. Mutants lacking both glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are not impaired for growth on glucose [7]. Similar results were obtained for E. coli and Drosophilu [S, 91.
that yeast phosphofructokinase mutants accumulate sedoheptulose 7-phosphate [lo, 111. Since mutants lacking one of the two phosphofructokinase genes ferment glucose but do not show phosphofructokinase activity in vitro, a pathway bypassing this reaction was proposed. This bypass should include reactions of the nonoxidative part of the pentosephosphate pathway [12]. We want to investigate this pathway in yeast using genetic methods in order to determine the importance of the nonoxidative part of the pentose-phosphate pathway in yeast metabolism and how glycolysis and the pentose-phosphate pathway interact. In this paper, we present the molecular analysis of TALI, the structural gene for transaldolase and describe the properties of a yeast mutant deleted for TALI.
Correspondence to I. Schaaff, Institut fur Mikrobiologie, Technische Hochschule Darmstadt, Schnittspahnstrasse 10, D-6100 Darmstadt, Federal Republic of Germany Enzymes. Acetohydroxyacid reductoisomerase (EC 1.1 .I 26); fructose-bisphosphate aldolase (EC 4.1.2.13); glucose 6-phosphate 1); dehydrogcnase (EC 1.I .1.49); 6-phosphofructokinase (EC 2.7.1.I 6-phosphogluconate dehydrogenase (EC 1.1 .I .44) ; phosphoglucose isomerase (EC 5.3.1.9); D-ribose 5-phosphate ketol-isomerase (EC 5.3.3.6); ~-ribulose-5-phosphate3-epimerase (EC 5.1.3.1); transketolase (EC 2.2.1.1). Note. The sequence data described in Fig. 6 appear in the EMBL/ GenBank/DDBJ Nucleotide Sequence Databases under the acession number XI5953 S. cerevisiae TALI.
NADP+NADPH+H+ Glucose 6-P
-
H-0
G - ~ P D Hgluconolodone Loctonase
6-Phosphogluconate
N P @OH$ - ~ ~
NADPH*H,COz
Ribiose 5-P Isomerase/
\pimerose Tronsketolase
fhrxtose]Erythrose 4-P Fig. 1. Diagram o j t h e pentose-phosphate pathway. ~-Glucose-6-phosphate dehydrogenase (G-6-P DH), 6-phosphogluconolactonase (lactonase) and 6-phosphogluconate dehydrogenase (6-PG DH) are the enzymes of the oxidative part. ~-Ribulose-5-phosphate 3epimerase, n-ribose-5-phosphate ketol-isomerase (isomerase), transketolase and transaldolase are the enzymes of the nonoxidative part
598 MATERIALS AND METHODS Strains and media
P
B
S
E
1,
1 1 .
I
Yeast strain UTL-7A MATa leu2-3/112 ura3-52 trpl (H. D. Schmitt) was used as recipient for transformation experiments and the diploid strain M4 MATa leu2-3/112 ura3-52 trpl MATol leu2-31112 U R A 3 T R P l
was used to introduce the deletion of T A L I . The phosphoglucose-isomerase-deficient strain G I 1 MATu pgil A 34: LEU2 his4 ura3 GAL2 [5] was used for the construction of the double mutants. Yeast strains were grown either in rich media (2% peptone, 1% yeast extract) or in synthetic media (0.5% ammonium sulfate, 0.17% Difco yeast nitrogen base without amino acids, supplemented with amino acids, adenine and uracil). Carbon sources were added as indicated in the results. The pgil deletion mutant requires trace amounts of glucose for growth and was grown in medium containing 2% fructose as carbon source supplemented with 0.1 YOglucose
PI.
E. coli strains Y1088, Y1090 and BTA282 [I31 were used for handling the &$I1 library. Nucleic acid manipulations
Standard procedures were used unless otherwise indicated 1141. For hybridization experiments DNA or RNA were transferred to nylon filters (Amersham) by vacuum blotting (Pharmacia-LKB). For DNA sequencing fragments were subcloned into M13mp18 and M13mp19 [I51 and sequenced on both strands (Fig. 2) by the chain-termination method [I61 using the T7 polymerase sequencing kit from PharmaciaLKB. Yeast sphaeroblasts were transformed according to Beggs ~71. Preparation of antiserum and screening ofI.gtl1 library
Purified yeast trdnsaldolase was obtained from Boehringer Mannheim and used for the immunization of rabbits. The specificity of the antiserum was assayed by ELISA, Western blot analysis and inactivation of transaldolase activity in yeast crude extracts. Anti-E. coli antibodies were removed from the antiserum by incubation with E. coli lysates for 2 h and sedimentation of the precipitate. The Agtl 1 expression library (YL1001) contained genomic DNA fragments from yeast strain X2380 and was obtained from Clontech Laboratories Inc. (Palo Alto CA). 7 x lo5 recombinant phages were screened as described by Young and Davis [ 131 except that alkaline-phosphatase-linked secondary antibody (Sigma) was used for detection of positive phage plaques. Two identical clones were identified and shown to produce an immunopositive fl-galactosidase fusion protein of 145 kDa. The 2.0-kb insert of yeast DNA was subcloned into pUC19 [IS] and used as a radioactive probe to isolate larger DNA fragments by colony hybridization [I81 from a yeast genomic library constructed by Nasmyth and Tatchell [19] in the vector YEpl3 [20]. Deletion of TALl
The transaldolase gene was deleted following the method of Rothstein [21]. A 6-kb SphI-Sac1 fragment from clone
E
S
,I/
BIE I' XX$"ElBH B
\
SpBaH
1
LEU2
\
Xh
P BI XIE
'\\,
1
Sa
BaXP
\
1 kb
TA L1 c
_ c
ILVS
_
--
-
a _ c
.
Fig. 2. Restriction mup of the cloned TALI locus. In the upper part the strategy for the deletion of the transaldolase gene by replacement with LEU2 is indicated. The SphI site a t the left side derives from the vector YEpl3 [20]. Below the restriction map the sequencing strategy is shown. The close linkage to the ZLV5 gene was found by sequence analysis. Restriction enzyme abbreviations: B, Bgnl ; Ba, BumHl; BI, Bun; E, EcoRI; H, HindIII; P, PstI; S, Sufi; Sa, Sucl; Sp, SphI; X, XbuI; Xh, XhoI
TALI-2 (Fig. 3 ) was subcloned into pUC19 [I61 (Fig.2). The 1.45-kb PstI - XhoI fragment containing the entire coding region of TALI was replaced by the 3.43-kb PstI- XhoI fragment carrying the LEU2 gene from YEpl3 [20]. The resulting plasmid was digested with HindIII prior to yeast transformation. Western blot analysis
Yeast crude extracts were denatured and electrophoresed in 8% polyacrylamide gels [22]. Proteins were transferred to nitrocellulose by electroblotting. Immunoblot analysis was performed according to Burnette [23] using non-fat dried milk as blocking agent and alkaline-phosphatase-linked secondary antibody for detection. Trunsaldolase enzyme assay
Yeast crude extracts were prepared according to Ciriacy and Breitenbach [24] using 50 mM imidazole pH 7.6. Specific transaldolase activity was determined as described by Bergmeyer [25]. Protein was measured by the microbiuret method [26]. Determination of metabolite levels
Extracts for metabolite determinations were prepared as described by Ciriacy and Breitenbach [24]. Levels of sugar phosphates were determined as described by Bergmeyer [25]. RESULTS Cloning qfTAL1
Three overlapping clones were isolated from the yeast genomic library in vector YEpl3 (Fig.3). YEpl3 is a multicopy yeastlE. coli shuttle vector [20]. These three plasmids were transformed into strain UTL-7A and transaldolase specific activity was determined. Transformants with plasmids TALI-I and TALI-2 over-produced transaldolase activity, indicating that they carry the structural gene for transaldolase. Plasmid TALI-3 does not contain the entire T A L l gene. Fig. 4A shows a Western blot analysis of transformants carrying plasmids TALI-1 and TALI-2. Both immunopositive proteins present in the crude extract of the untransformed strain are overproduced in the transformants.
599 trnnsaldalase octlvlty
c_I
E
Ba
E
EE
E
M E
EE
E
EE
Ba
E
Ba
Table 1. Tetrad analysis of diploid transformants Spore
LEU-phenotypeltransaldolase activity in tetrad
pTAL1-1
1400
13 4
pTAL1-2
635
61
3
pTAL1-3
110
11
- / m u . mg protein-
E
'
"+
2
3
4
5
6
-191 +/
