Vol. 174, No. 14
JOURNAL OF BACTERIOLOGY, July 1992, p. 4701-4706 0021-9193/92/144701-06$02.00/0 Copyright X 1992, American Society for Microbiology
A Positive Regulatory Gene, THI3, Is Required for Thiamine Metabolism in Saccharomyces cerevisiae YUKO KAWASAKI,1 YOSHINOBU KANEKO,2 KAZUTO NOSAKA,1 AND AKIO IWASHIMA' Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602,1 and
HIROSHI
NISHIMURA,"*
Institute for Fermentation,
Osaka, Yodogawa-ku, Osaka 532, Japan
Received 1 October 1991/Accepted 5 May 1992
We have isolated a thiamine auxotrophic mutant carrying a recessive mutation which lacks the positive gene, THI3, which differs in the regulation of thiamine transport from the THI2 (PHO6) gene described previously (Y. Kawasaki, K. Nosaka, Y. Kaneko, H. Nishimura, and A. Iwashima, J. Bacteriol. 172:6145-6147, 1990) for expression of thiamine metabolism in Saccharomyces cerevisiae. The mutant (thi3) had a markedly reduced thiamine transport system as well as reduced activity of thiamine-repressible acid phosphatase and of several enzymes for thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-1-hydroxyethylthiazole. These results suggest that thiamine metabolism in S. cerevisiae is subject to two positive regulatory genes, THI2 (PH06) and THI3. We have also isolated a hybrid plasmid, pTTRl, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements thiamine auxotrophy in the thi3 mutant. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. Complementation of the activities for thiamine metabolism in the thi3 mutant transformed by some plasmids with the THI3 gene was also examined.
regulatory
The pathway for biosynthesis of thiamine from 2-methyl4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and 4-methyl-5-13-hydroxyethylthiazole (hydroxyethylthiazole) in Saccharomyces cerevisiae is catalyzed by several enzymes, including hydroxymethylpyrimidine kinase (EC 2.7.1.49), phosphomethylpyrimidine kinase (EC2.7.4.7), hydroxyethylthiazole kinase (EC 2.7.1.50), and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (3, 4, 18, 19). Our previous study showed that the activity of these enzymes is coordinately repressed by exogenous thiamine and that these activities are decreased by increased intracellular thiamine pyrophosphate levels in yeast cells (9). Recently, we isolated a thi80 mutant which has a partial thiamine pyrophosphokinase (EC 2.7.6.2) deficiency. The mutant has the constitutive phenotype for thiamine transport, thiamine-repressible acid phosphatase (T-rAPase), and enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole (15). In the previous report, we clarified that thiamine pyrophosphate is a negative effector of the regulatory mechanism of thiamine metabolism in S. cerevisiae. On the other hand, apho6 mutant defective in the regulatory gene for the synthesis of periplasmic T-rAPase encoded by the PHO3 gene (26, 27) is auxotrophic for thiamine, and the enzyme activities involved in thiamine synthesis described above are markedly low in crude extracts of this mutant (9). This indicated that the expression of structural genes for T-rAPase and the enzymes involved in thiamine biosynthesis was regulated positively by PHO6. Recently, it was elucidated that T-rAPase predominantly catalyzes the hydrolysis of thiamine phosphates under physiological conditions (17). The PHO6 gene is not required for the transcriptional control of the repressible acid phosphatase encoded by the PHOS gene (10) or the Pi transport system encoded by the PHO84 gene (2, 25) in the phosphate metabolism of S. *
cerevisiae. Since PHO6 is closely related to thiamine metabolism, we described this gene as THI2 (PHO6). However, the thi2 (pho6) mutant defective in the regulatory gene for enzymes involved in thiamine synthesis retained sufficient thiamine transport activity (16). Another thiamine auxotrophic strain (thil mutant) was reported (5), and it was found that the allelic thil locus is located in chromosome XIII (14). In this article, we describe the isolation of a thi3 mutant of S. cerevisiae which has a recessive thiamine auxotrophic phenotype similar to that of the thi2 (pho6) mutant. However, unlike the thi2 (pho6) mutant, which retained thiamine transport activity, the thi3 mutant had decreased activities not only of T-rAPase and the enzymes involved in thiamine
synthesis from hydroxylmethylpyrimidine and hydroxyethylthiazole, but also of thiamine transport. These results suggest that thiamine transport as well as thiamine biosynthesis and T-rAPase in S. cerevisiae are subject to the positive regulatory gene THI3. We have also isolated a hybrid plasmid, pTTR1, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements the thi3 mutation of S. cerevisiae. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. We propose a working hypothesis that thiamine metabolism in S. cerevisiae is controlled by the positive factors THI2 (PHO6) and THI3, whose actions are regulated negatively by the intracellular thiamine pyrophosphate level. MATERIALS AND METHODS Strains and culture conditions. The S. cerevisiae and Escherichia coli strains used in the present study are listed in Table 1. E. coli cells were cultured in LB medium (0.5% yeast extract, 1% Bacto-Peptone, 1% NaCl, 0.2% glucose) at 37°C. When necessary, the medium was supplemented with ampicillin (20 p,g/ml). Yeast strains were cultured at 30°C in YPD medium (1% yeast extract, 2% Bacto-Peptone, 2% glucose) or in a defined medium containing 0.67% yeast
Corresponding author. 4701
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NISHIMURA ET AL.
J. BACTERIOL. TABLE 1. Strains used
Strain
Relevant genotype
S. cerevisiae
IFO 10481 IFO 10482 IFO 10483 TRS3 T49-2D KYC319 K269-5c KYC307 NK1
MATa his4-519 ga12 MATa his4-519 gal2 MATa leu2-3, 112 gal2 MATa thi3 leu2-3,112gal2 MATa thi3 leu2-3,112 ura3-52 trpl-A63 lys2-801 his3-A200 MATa thi3 leu2-3,112 ura3-52 trpl-A63 lys2-801 MATa thi2(pho6-7)leu2-3,112 ura3-1,2 trpl-A63 MATa thi2(pho6-7)trpl-A&63 + MAa + his4-51 gal2 + MATa thi3 leu2-3,112 gal2
NK2
ALITa + MA Ta thi3 MATa + MATTa thi3
T114 E. coli
DH5aL MV1184
thi2(poL-7 leu2-3.112 ura3-1.2 trpl-A63ga leu2-3,112
+
thi2po6-7
+
+
+
gal2
tr2l-A63 gal2
+ leu2-3,112 gal2 supE44 AlacU169 (,0801acZAM15)hsdRl7 recAl endAl gyrA96 thi-1 reLAI +
A(lac-proAB) rpsL thi (+80JacZAM15) A(sH-recA)306::TnlO (TetD F' (traD36 proAB+ laclq lacZAM15)
ara
nitrogen base (Difco Laboratories) supplemented with essential amino acids or in Wickerham's synthetic medium supplemented with essential amino acids with or without thiamine (28). Yeast strains auxotrophic for thiamine were cultured in minimal medium with thiamine at a concentration of 10-8 M, which does not cause thiamine repression. Yeast strains harboring derivatives of plasmids YCp5O and pRS316 were cultured in medium lacking uracil. Isolation of mutants. Chemical mutagenesis with ethyl methanesulfonate was performed essentially as described by Lindegren et al. with some modifications (11). The parent strain, IFO 10483, was cultured in YPD (5 ml) overnight at 30°C. The cells were washed twice with sterile water and suspended in 4.6 ml of 0.2 M potassium phosphate buffer (pH 8.0). Ethyl methanesulfonate (0.15 ml) and 4% glucose (0.25 ml) were added, and the reaction mixtures were incubated at 30°C for 60 min with shaking. The cells were pelleted and washed five times with sterile water. This was followed by nystatin (35 ,ug/ml) treatment for mutant enrichment (24). The cells were then washed twice with sterile water and spread on agar containing Wickerham's synthetic medium with thiamine (10-8 M). The colonies in the master plate were replica plated on minimal medium without thiamine. After 2 to 3 days of incubation at 30°C, the colonies that did not thrive were selected from the master plate and transferred to a fresh plate for storage and further analysis. The genetic techniques used were those described by Sherman et al. (23). Enzyme assays with hydroxymethylpyrimidine and hydroxyethylthiazole in a crude extract. Yeast cells were harvested, washed once with cold water, and then suspended in 0.05 M potassium phosphate buffer (pH 7.5) containing 1 mM 2-mercaptoethanol and 1 mM EDTA. The cell suspensions were sonicated, and after centrifugation at 28,000 x g for 30 min, the supernatant was used as a crude extract. Overall thiamine-synthesizing activity from hydroxymethylpyrimidine and hydroxyethylthiazole and the activities of each of the four enzymes involved in the formation of thiamine monophosphate (hydroxymethylpyrimidine kinase, phosphomethylpyrimidine kinase, hydroxyethylthiazole kinase, and thiamine-phosphate pyrophosphorylase) were assayed as described previously (9). The protein concentrations were determined by the method of Lowry et al. (12).
Thiamine transport and T-rAPase activities in yeast cells. Thiamine transport was determined by the method described previously (7, 8). T-rAPase activity withp-nitrophenyl phosphate as a substrate was determined from the amount of p-nitrophenol produced, as described earlier (22). Transformation and DNA preparations. Yeast strains were transformed after lithium acetate treatment as described by Ito et al. (6). Bacterial transformations were performed as described by Maniatis et al. (13). Plasmid DNA from yeast cells was isolated as described by Sherman et al. (23) with some modifications. Bacterial plasmids were prepared by the alkaline lysis method of Maniatis et al. (13). Isolation of a plasmid able to complement THI3 mutations. The TRS3 strain (thi3 mutant) was crossed to strain YPH499, and a recipient strain, T49-2D, carrying several selectable auxotrophic amino acid markers and allowing transformation at high frequency was generated. This strain was transformed with a gene library of S. cerevisiae YCp50 "CEN BANK" A, constructed by partial digestion of the genomic DNA of S. cerevisiae GRF88 with Sau3AI and its ligation with YCpSO at the unique BamHI site. YCp5O was obtained from the American Type Culture Collection (Rockville, Md.). Transformants were screened for the ability to grow without uracil and thiamine on Wickerham's synthetic medium. After 3 days of incubation at 30°C, colonies were selected for further analysis. Plasmid pTTR1, which complemented THI3 mutations, was obtained from one colony among six transformants. Construction of plasmids. All procedures were performed under the conditions recommended by the suppliers or by standard methods as described by Maniatis et al. (13). For plasmid pTTR1-a, the BamHI fragment (6.2 kb) from pTTR1 was cloned into pRS316. For plasmid pTTR2, the HindIII fragment (5.3 kb) from pTTR1 was cloned into pRS316. For plasmid pTTR3, the ClaI-HindIII fragment (4.5 kb) from pflTR1 was cloned into pRS316. For plasmid pTTR4, the Sall-HindIII fragment (3.5 kb) from pTTRl1 was cloned into pRS316. For plasmid pTTRS, the ClaI-BglII fragment (3.0 kb) from plTR3 was cloned into pRS316. Plasmids, enzymes, and chemicals. Plasmid YCpS0 (21) was obtained from the American Type Culture Collection (Rockville, Md.), and plasmid pRS316, which was used as the vector for subcloning, was provided by Philip Hieter (Johns
VOL. 174, 1992
THI3 REQUIRED FOR THIAMINE METABOLISM IN S. CEREVISL4E
Hopkins University School of Medicine, Baltimore, Md.). Plasmid YIpS (1) was used in yeast integrative transformation. ApaI was purchased from Toyobo, Co., Ltd. (Osaka, Japan); BamHI, BglII, HindIII, and Sall were obtained from Nippon Gene (Toyama, Japan); ClaI, Kpnl, and PvuII were purchased from Takara Shuzo (Kyoto, Japan). [14C]thiamine ([thiazole-2-[14C]thiamine hydrochloride, 24.3 Ci/mol) was purchased from Amersham International (Buckinghamshire, United Kingdom). Nystatin was purchased from Sigma Chemical Co. All other chemicals were purchased from commercial suppliers. RESULTS Isolation and selection of the thi3 mutant. Yeast cells (IFO 10483) were treated with the chemical mutagen ethyl methanesulfonate and then enriched for mutants with nystatin in Wickerham's synthetic medium supplemented with hydroxymethylpyrimidine (2 x 10-7 M) plus hydroxyethylthiazole (2 x 10' M) or with thiamine (2 x 10-8 M) for selection and recovery, respectively. Two mutant colonies that grew on our minimal medium containing thiamine (2 x 10-8 M) but not on thiamine-free medium were isolated. One of these mutants, TRS3, was crossed with the wild-type strain, IFO 10482, and the resulting diploid, NK1, was prototrophic for thiamine, as is the wild type, indicating that this mutation was recessive. Four-spored asci were dissected from the diploid. All tetrads (19 asci were tested for each cross) showed 2+ :2- segregation for thiamine auxotrophy, confirming that a single chromosomal mutation was responsible for this phenotype. The mutation in TRS3 was designated thi3. Phenotypes (defects in thiamine transport and T-rAPase activity) other than thiamine auxotrophy in the thi3 mutant. Although, as noted above, the thi3 mutant was auxotrophic for thiamine, this mutant also showed decreased thiamine transport and T-rAPase activities compared with wild-type strains. Thiamine transport rates were 153.3 and 23.8 pmol/ 106 cells/min for the wild-type (IFO 10483) and thi3 mutant (TRS3) strains, respectively; T-rAPase activities were 4.2 and 1.0 nmol/106 cells/5 min, respectively. Therefore, we analyzed the segregation of the phenotypes for thiamine transport and T-rAPase activities in order to determine whether the defects in these activities cosegregated with thiamine auxotrophy in the thi3 mutant strain. The thi3 mutant was crossed with the wild-type strain (IFO 10482), and the resultant diploid strain NK1 was subjected to tetrad analysis. The phenotypes for thiamine transport and T-rAPase activity and thiamine auxotrophy cosegregated 2+:2in all 76 asci tested. These results indicate that the thi3 mutant, which is impaired in the synthesis of the thiamine transport system and periplasmic T-rAPase in addition to the de novo synthesis of thiamine, resulted from the mutation of a single gene in the nucleus. Another thiamine auxotrophic mutant carrying thi2 (pho6) (9) also showed decreased T-rAPase activity, whereas thiamine transport activity was retained in this mutant (16). A complementation test between the thi2 (pho6) mutant (K269Sc) and the thi3 mutant (TRS3) isolated in this study was then carried out. To check the allelism of thi2 (pho6) and thi3, a cross produced a thi2 (pho6)Ithi3 diploid (NK2), which showed thiamine prototrophy together with thiamine transport and T-rAPase activities (data not shown). Furthermore, an allelism test between thi2 (pho6) and the thi3 mutation was carried out. A diploid, T114, obtained by crossing the TRS3 (thi3 mutant) and KYC307 (thi2 mutant)
4703
TABLE 2. Enzyme activities involved in thiamine monophosphate synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in a wild-type strain (IFO 10483) and TRS3 (thi3 mutant) Sp act' (nmol/mg of Enzyme
Hydroxymethylpyrimidine kinase Phosphomethylpyrimidine kinase Hydroxyethylthiazole kinase Thiamine-phosphate pyrophosphorylase
protein/30 min) in: IFO 10483
thi3 mutant"
10.5 18.6 36.0 25.6
0.4 1.2 7.7 2.2
aEach value is the mean of two experiments. b The thi3 mutant was cultured in Wickerham's synthetic medium supplemented with 10-8 M thiamine.
strains was sporulated, and the resulting asci were subjected to tetrad analysis. Of the 24 tetrads tested so far, 3 segregated 2+:2-, 14 segregated 1+:3-, and 7 segregated 0+:4for the thiamine requirement. These results confirmed that the two mutations, thi2 and thi3, occurred in two nonallelic genes. Activities of several enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in the crude extract of the thi3 mutant. Overall enzyme activity for thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in the presence of ATP and Mg2e was not detected in the crude extract from the thi3 mutant cells (data not shown). The activities of four enzymes involved in the formation of thiamine monophosphate from hydroxymethylpyrimidine and hydroxyethylthiazole, namely, hydroxymethylpyrimidine kinase (EC 2.7.1.49), phosphomethylpyrimidine kinase (EC 2.7.4.7), hydroxyethylthiazole kinase (EC 2.7.2.50), and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (Table 2), were determined. All four activities were markedly lower in the thi3 mutant cell extract than in the extract from the parent cells (IFO 10483). We previously reported that the activities of T-rAPase and the four enzymes involved in the formation of thiamine monophosphate from hydroxymethylpyrimidine and hydroxyethylthiazole in a thi2 (pho6) mutant were markedly low or undetectable (9). This indicated that the THI2 (PH06) gene positively regulates not only the expression of the PH03 gene, encoding T-rAPase, but the expression of at least four genes coding for the enzymes synthesizing thiamine monophosphate indicated above. Similarly, these results demonstrated that thiamine transport and biosynthesis and TrAPase activity in S. cerevisiae are subject to the positive regulatory gene THI3. Molecular cloning of S. cerevisule DNA fragment carrying the THI3 gene. The THI3 gene was identified on a recombinant DNA plasmid by its functional complementation of a thi3 mutation in T49-2D. The S. cerevisiae genomic DNA library, YCp5O "CEN BANK" A, was used to transform T49-2D (thi3 ura3). After transformation by alkaline cation treatment (6), the treated cells were washed twice with cold sterile water, incubated overnight at 30°C in minimal medium lacking thiamine and uracil, and then plated on agar containing Wickerham's synthetic medium without thiamine and uracil. The plates were incubated at 30°C for 3 days, and a total of six Thi+ Ura+ colonies were isolated. DNA prepared from these transformants was used to transform E. coli DH5ao to Ampr. A plasmid obtained from the Ampr transformant conferred the thiamine prototrophic phenotype on yeast strain T49-2D (thi3 ura3) in minimal medium
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NISHIMURA ET AL.
J. BA=rRIOL.
Complementat
Plasmid H BKPv C
S
Pv
Bg
H
pTRRI
pTfR1-a,
J+
+
B
B
pTTRI-a
pTTR2
pTTR1, respectively, into the multicloning vector pRS316. Plasmids pTTR2, and pTTR3, constructed by subcloning a 4.4-kb Clal-HindIII fragment from the 6.2-kb fragment into pRS316, complemented the thi3 mutation (Fig. 1). Plasmid pTTR4, bearing the 3.6-kb Sail-HindIII fragment from the 6.2-kb fragment in pRS316, did not complement the thi3 mutation. Furthermore, plasmid pTTR5, which was constructed by subcloning the 3.0-kb ClaI-BgiII fragment from pTI'R3, also complemented this mutation. These results suggest that the SalI site is located within a region necessary for restoring THI3 function. The 6.2-kb BamHIBamHI DNA fragment derived from plasmid pTJR1 used as a probe against yeast chromosomes that had been resolved by pulsed-field electrophoresis hybridized only to one chromosome, which was identified, by comparison with a standard, as chromosome IV (data not shown).
BOf J
+
H
H
+
L H
c
+
plTR3 H
S
pTTR4
Bg
c
pITR5
+ 1
kb
Expression of thiamine transport, T-rAPase, and enzyme activities involved in thiamine synthesis in the thi3 mutant transformed by the THI3 gene. The effect of some plasmids bearing the THI3 gene on thiamine metabolism was examined by measuring the activities of thiamine transport and T-rAPase. Transformants of the thi3 mutant T49-2D (thi3 ura3), harboring the low-copy-number vector plasmid YCp5O, pRS316, and two plasmids bearing the THI3 gene (pTTR1 and pTIR3) were cultured in the absence or presence of thiamine and assayed for thiamine transport and T-rAPase activity (Table 3). The transformants with pTTR1 and pTTR3 had far more potent thiamine transport activity than a wild-type strain (IFO 10483), whereas T-rAPase activity in the transformants harboring the plasmid with the THI3 gene was lower than in the wild-type strain. Both thiamine transport and T-rAPase activities in the recipient cells transformed with the control plasmids YCp5O and pRS316 were as markedly low as in the thi3 mutant (TRS3). Furthermore, the activities in transformants with pTTR1 and pTTR3 were repressed by thiamine (2 x 10' M) in minimal medium in the same manner as those of the wild-type strain (IFO 10483). These results indicated that the expression of thiamine transport and T-rAPase by pTTR1 and pTTR3 in the thi3 mutant was controlled by the regulatory system owing to the signal of the intracellular thiamine pyrophosphate. The overall enzyme activity for thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole and the activity of an enzyme involved in the formation of thiamine monophosphate (thiamine-phosphate pyrophosphorylase) in crude extracts of these transformants cultured in thiaminedeficient Wickerham's synthetic medium were assayed (Table 4). Thiamine-phosphate pyrophosphorylase activity of
FIG. 1. Restriction map of an S. cerevisiae DNA inisert in pTR1 and subcloning of DNA fragments with and without the ability to complement the thiM mutation. Restriction enzyme c-leavage sites: B, BamHI; C, CMaI; Bg, BglII; H, HindIII; K, KpnI; Pv, PvuII; S, Sall.
without thiamine and uracil. This plasmid, pTlrRl (Fig. 1) had an insertion of the 6.2-kb BamHI-BamHI fragment in YCp5O. That pTTR1 contains THI3 was confirmed b2 y integration of the DNA fragment complementing the thi3 nnutation into the yeast chromosome. We fractionated the 6.2 -kb fragment between two BamHI sites from pTTR1 (Fig. 1) and inserted it into integrative vector YIp5 to generate p INHK1. The recombinant plasmid was then transformed i nto the thi3 mutant KYC319, and Ura+ transformants were selected. All the Ura+ colonies were also Thi+. Both pheniotypes were stably inherited during mitotic growth in nonscelective conditions for many generations, suggesting integiration of the recombinant molecule into a nuclear chromosome. The strain transformed with pNHK1 was crosseid with IFO 10481, and the resultant diploids were subjectted to tetrad analysis. Of the 18 tetrads tested, 17 segregatecd 4+:0- and 1 segregated 3+:1- for thiamine requirement and 1 segregated 4+:0-, 12 segregated 3+:1-, and 5 segrelgated 2+:2for uracil requirement. These results indicate th at pNHK1 is integrated at or close to the thi3 locus. Thus, Mie concluded that pT'l1 bears the THI3 gene. Several deletion fragments were constructced from the cloned 6.2-kb fragment (Fig. 1). Plasmids p'ITR1-a and pTTR2 were constructed by subcloning a 6.2'-kb inserted DNA and a 5.3-kb HindIII-HindIII fragment f'rom plasmid
TABLE 3. Effect of thiamine added to the growth medium on thiamine transport and T-rAPase activities in the thi3 mutant strain transformed by various plasmid-borne DNA fragmnents Strain
IFO 10483 T49-2D (thi3 mutant)
Thiamine transport activity"
T-rAPase activity'
(pmol/106 cells/min)
(nmol/106 cells/5 min)
Plasmid
None
YCp5Ob (vector) pTTRl pRS316b (vector) pTIR3
No addition
0.2 FM thiamine
No addition
0.2 FM thiamine
142.4 8.3 206.7 3.9 176.8
3.7 NDc 10.1 ND 17.4
19.4 2.4 17.6 2.4 11.2
0.5 ND 1.0 ND 1.4
aEach value is the mean of two experiments. b Strains harboring a control vector were cultured in Wickerham's synthetic medium supplemented with 10-8 M thiamine. ND, not detectable.
THI3 REQUIRED FOR THIAMINE METABOLISM IN S. CEREVISIAE
VOL. 174, 1992
TABLE 4. Activity of thiamine-synthesizing enzymes in the thi3 mutant strain transformed by various plasmid-borne DNA fragmentsa Strain
IF010483 T49-2D (thi3 mutant)
Plasmid
None
YCpS0 (vector) pTTR1 pRS316 (vector) pTTR3
Enzyme activity (nmol/mg of proteinl30 min)
Overall reaction
Thiamine-phosphate pyrophosphorylase
1.19 0 0.06 0 0.64
12.4 0.3 7.4 0.4 11.3
4705
(effector)
(CTI3
THJ2(PHO6)
FIG. 2. Working hypothesis for the interaction between the regulatory factors for transmission of the thiamine pyrophosphate (TPP) signals. Square boxes indicate genes, and ovals represent regulatory factors. Arrows, stimulatory effect; horizontal bar, repressive effect; ?, negative regulatory factor(s).
a See Table 3, footnotes a and b.
transformants harboring plasmids with the THI3 gene was expressed at the same level as that of the wild-type strain. In the crude extract of cells transformed with pTTR3, the overall enzyme and thiamine-phosphate pyrophosphorylase activities were also the same as those of the wild-type strain, but neither activity in the crude extract of the transformant with pTTRl was as potent as that of the wild-type strain, although the reason for this remains unknown. These activities in the extracts of recipient cells transformed with the control plasmids YCp50 and pRS316 were also as markedly low as those of the thi3 mutant (TRS3). DISCUSSION This work indicates that thiamine metabolism, including the thiamine transport system, in S. cerevisiae is also controlled by the positive regulatory gene THI3. This is an alternative to the THI2 (PH06) gene, which is indispensable to yeast thiamine metabolism except for the thiamine transport system. We have already elucidated that expression of the structural genes for T-rAPase and the enzymes involved in thiamine biosynthesis in S. cerevisiae is regulated positively by THI2 (PH06) (9), whereas expression of the genes for thiamine metabolism is controlled negatively by the intracellular thiamine pyrophosphate level (15). However, as described above, the THI2 (PH06) gene seems not to be essential for expression of the thiamine transport system (16), and therefore we have attempted to obtain a thiamine auxotrophic mutant with a defect in the thiamine transport system in order to identify a positive regulatory gene for thiamine metabolism, including thiamine transport. As expected, the mutant (TRS3) isolated in this study was auxotrophic for thiamine, with defective T-rAPase and thiamine transport activities. These phenotypes occurred as the result of the same mutation of a single gene in the nucleus, which was designated thi3. The distinguishing difference between the thi2 (pho6) and thi3 mutations was the phenotype for thiamine transport. It was possible that these mutations were produced in two different genes of the nucleus. Furthermore, a complementation test and a tetrad analysis between thi2 and thi3 mutants implied that the mutations occurred in two nonallelic genes. The THI3 gene was located on chromosome IV, whereas the THI2 (PH06) gene was situated on the right arm of chromosome II (16). These data indicated that the thi3 and thi2 (pho6) loci were indeed distinct, since mutations in each generated distinguishable phenotypes for thiamine transport. Another allelic locus (thil), which shows thiamine auxotrophy as a phenotype in S. cerevisiae, has been reported (5), but the THII gene was situated on the right arm of chromosome XIII (14). These results confirmed
that THI3 was a novel gene that is indispensable for thiamine prototrophy and that controlled the expression of the thiamine transport system simultaneously. Accordingly, it was considered that the THI2 (PH06) and THI3 genes are both required for expression of T-rAPase and several enzymes involved in thiamine synthesis, whereas the thiamine transport system was regulated only by the THI3 gene as a positive factor. Two positive regulatory genes, PH02 and PHO4, whose products are indispensable for the transcriptional control of the structural genes for repressible acid phosphatase encoded by PHOS and the Pi transport system encoded by PH084 in S. cerevisiae, have been reported by Oshima and coworkers (20, 29, 30). The PHO regulatory system in S. cerevisiae appears to be of interest for further study of this thiamine regulatory system. To examine whether several plasmids containing the THI3 gene complemented the biochemical phenotypes of the thi3 mutation (loss of thiamine transport and T-rAPase activity), transformants of strain T49-2D were grown in the absence or presence of thiamine and assayed for thiamine transport and T-rAPase activities. When the cells were cultured in the absence of thiamine, complementation of the thi3 mutation by these plasmid-borne genes was always nearly equal to complementation of the wild-type strain, as shown in Table 3. However, after growth in minimal medium supplemented with thiamine (2 x 10' M), thiamine transport and TrAPase activities in transformant cells carrying a plasmidborne THI3 gene were significantly repressed, as in the wild-type strain. These results are of interest in connection with the regulatory mechanisms of thiamine metabolism in S. cerevisiae. In our previous studies (8, 9), the thiamine transport system, T-rAPase activity, and the enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in S. cerevisiae were found to be repressible by exogenous thiamine. We then demonstrated that thiamine metabolism in S. cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level (15). At present, as shown in Fig. 2, we presume that thiamine metabolism in yeast cells is controlled by the positive factors encoded by THI2 and THI3, which might be negatively regulated by the intracellular thiamine pyrophosphate level via some gene(s). In order to make sure this assumption, the mode of expression of two positive regulatory genes (THI2 and THI3) and the interaction between the positive factors and a negative factor(s) should be investigated. Although we have been trying to determine the nucleotide sequence of the THI3 gene, further study at the molecular level is required to clarify the detailed mechanism of the regulation of yeast thiamine metabolism.
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REFERENCES 1. Botstein, D., S. C. Falco, S. E. Stewart, M. Brennan, S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24. 2. Bun-ya, M., M. Nishimura, S. Harashima, and Y. Oshima. 1991. The PH084 gene of Saccharomyces cerevisiae encodes an inorganic phosphate transporter. Mol. Cell. Biol. 11:3229-3238. 3. Camiener, G. W., and G. M. Brown. 1959. The biosynthesis of thiamine and thiamine phosphates-by extracts of baker's yeast. J. Am. Chem. Soc. 81:3800. 4. Camiener, G. W., and G. M. Brown. 1960. The biosynthesis of thiamine. II. Fractionation of enzyme system and identification of thiazole monophosphate and thiamine monophosphate as intermediates. J. Biol. Chem. 235:2411-2417. 5. Hawthorne, D. C., and R. K. Mortimer. 1960. Chromosome mapping in Saccharomyces: centromere-linked genes. Genetics 45:1085-1110. 6. Ito, H., Y. Fukuda, K. Murata, and A. Kimura. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153:163-168. 7. Iwashima, A., H. Nishino, and Y. Nose. 1973. Carrier-mediated transport of thiamine in baker's yeast. Biochim. Biophys. Acta 330:222-234. 8. Iwashima, A., Y. Wakabayashi, and Y. Nose. 1975. Thiamine transport mutants of Saccharomyces cerevisiae. Biochim. Biophys. Acta 413:243-247. 9. Kawasaki, Y., K. Nosaka, Y. Kaneko, H. Nishimura, and A. Iwashima. 1990. Regulation of thiamine biosynthesis in Saccharomyces cerevisiae. J. Bacteriol. 172:6145-6147. 10. Lemire, J. M., T. Wilicocks, H. 0. Halvorson, and K. A. Bostian. 1985. Regulation of repressible acid phosphatase gene transcription in Saccharomyces cerevisiae. Mol. Cell. Biol. 5:2131-2141. 11. Lindegren, G., Y. L. Hwang, Y. Oshima, and C. C. Lindegren. 1965. Genetical mutants induced by ethyl methanesulfonate in Saccharomyces. Can. J. Genet. Cytol. 7:491-499. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 14. Mortimer, R. K., D. Schild, C. R. Contopoulou, and J. A. Kans. 1989. Genetic map of Saccharomyces cerevisiae, edition 10. Yeast 5:321-404. 15. Nishimura, H., Y. Kawasaki, K. Nosaka, Y. Kaneko, and A. Iwashima. 1991. A constitutive thiamine metabolism mutation, thi8O, causing reduced thiamine pyrophosphokinase activity in Saccharomyces cerevisiae. J. Bacteriol. 173:2716-2719. 16. Nishimura, H., Y. Kawasaki, Y. Kaneko, K. Nosaka, and A. Iwashima. 1992. Cloning and characteristics of a positive regulatory gene, THI2(PH06), of thiamin biosynthesis in Saccharo-
J. BAcTERIOL. myces cerevisiae. FEBS Lett. 297:155-158. 17. Nosaka, K. 1990. High affinity of acid phosphatase encoded by PH03 gene in Saccharomyces cerevisiae for thiamin phosphates. Biochim. Biophys. Acta 1037:147-154. 18. Nose, Y., K. Ueda, and T. Kawasaid. 1959. Enzymic synthesis of thiamine. Biochim. Biophys. Acta 34:277-279. 19. Nose, Y., K. Ueda, T. Kawasaki, A. Iwashima, and T. Fujita. 1961. Enzymatic synthesis of thiamine. II. The thiamine synthesis from pyrimidine and thiazole phosphates and the enzymatic synthesis of pyrimidine mono- and diphosphate and thiazole monophosphate. J. Vitaminol. 7:98-114. 20. Oshima, Y. 1982. Regulatory circuits for gene expression: the metabolism of galactose and of phosphate, p. 159-180. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), The molecular biology of the yeast Saccharomyces: metabolism and gene expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 21. Rose, M. D., P. Novik, J. H. Thomas, D. Botstein, and G. R. Fink. 1987. A Saccharomyces cerevisiae genomic plasmid bank based on a centromere-containing shuttle vector. Gene 60:237243. 22. Schweingruber, M. E., R. Fluri, K. Maudrell, A. M. Schweingruber, and E. Dumermuth. 1986. Identification and characterization of thiamin repressible acid phosphatase in yeast. J. Biol. Chem. 193:265-275. 23. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Methods in yeast genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 24. Snow, R. 1966. An enrichment method for auxotrophic yeast mutant using antibiotic "Nystatin." Nature (London) 211:206207. 25. Tamai, Y., A. Toh-e, and Y. Oshima. 1985. Regulation of inorganic phosphate transport system in Saccharomyces cerevisiae. J. Bacteriol. 164:964-968. 26. Toh-e, A., S. Kakimoto, and Y. Oshima. 1975. Two new genes controlling the constitutive acid phosphatase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 141:81-83. 27. Toh-e, A., S. Kakimoto, and Y. Oshima. 1975. Genes coding for the structure of the acid phosphatase in Saccharomyces cerevisiae. Mol. Gen. Genet. 143:65-70. 28. Wickerham, L. J. 1951. Taxonomy of yeast. 1. Techniques of classification. 2. A classification of the genus Hansenula. U.S. Dept. Agric. Tech. Bull. 1029:1-56. 29. Yoshida, K., Z. Kuromitsu, N. Ogawa, K. Ogawa, and Y. Oshima. 1987. Regulatory circuit for phosphatase synthesis in Saccharomyces cerevisiae, p. 49-55. In A. Torriani-Gorini, F. G. Rothman, S. Silver, A. Wright, and E. Yagil (ed.), Phosphate metabolism and cellular regulation in microorganisms. American Society for Microbiology, Washington, D.C. 30. Yoshida, K., N. Ogawa, and Y. Oshima. 1989. Function of PHO regulatory genes for repressible acid phosphatase synthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 217:40-46.
JOURNAL OF BACTERIOLOGY, July 1992, p. 4701-4706 0021-9193/92/144701-06$02.00/0 Copyright X 1992, American Society for Microbiology
A Positive Regulatory Gene, THI3, Is Required for Thiamine Metabolism in Saccharomyces cerevisiae YUKO KAWASAKI,1 YOSHINOBU KANEKO,2 KAZUTO NOSAKA,1 AND AKIO IWASHIMA' Department of Biochemistry, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602,1 and
HIROSHI
NISHIMURA,"*
Institute for Fermentation,
Osaka, Yodogawa-ku, Osaka 532, Japan
Received 1 October 1991/Accepted 5 May 1992
We have isolated a thiamine auxotrophic mutant carrying a recessive mutation which lacks the positive gene, THI3, which differs in the regulation of thiamine transport from the THI2 (PHO6) gene described previously (Y. Kawasaki, K. Nosaka, Y. Kaneko, H. Nishimura, and A. Iwashima, J. Bacteriol. 172:6145-6147, 1990) for expression of thiamine metabolism in Saccharomyces cerevisiae. The mutant (thi3) had a markedly reduced thiamine transport system as well as reduced activity of thiamine-repressible acid phosphatase and of several enzymes for thiamine synthesis from 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-1-hydroxyethylthiazole. These results suggest that thiamine metabolism in S. cerevisiae is subject to two positive regulatory genes, THI2 (PH06) and THI3. We have also isolated a hybrid plasmid, pTTRl, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements thiamine auxotrophy in the thi3 mutant. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. Complementation of the activities for thiamine metabolism in the thi3 mutant transformed by some plasmids with the THI3 gene was also examined.
regulatory
The pathway for biosynthesis of thiamine from 2-methyl4-amino-5-hydroxymethylpyrimidine (hydroxymethylpyrimidine) and 4-methyl-5-13-hydroxyethylthiazole (hydroxyethylthiazole) in Saccharomyces cerevisiae is catalyzed by several enzymes, including hydroxymethylpyrimidine kinase (EC 2.7.1.49), phosphomethylpyrimidine kinase (EC2.7.4.7), hydroxyethylthiazole kinase (EC 2.7.1.50), and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (3, 4, 18, 19). Our previous study showed that the activity of these enzymes is coordinately repressed by exogenous thiamine and that these activities are decreased by increased intracellular thiamine pyrophosphate levels in yeast cells (9). Recently, we isolated a thi80 mutant which has a partial thiamine pyrophosphokinase (EC 2.7.6.2) deficiency. The mutant has the constitutive phenotype for thiamine transport, thiamine-repressible acid phosphatase (T-rAPase), and enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole (15). In the previous report, we clarified that thiamine pyrophosphate is a negative effector of the regulatory mechanism of thiamine metabolism in S. cerevisiae. On the other hand, apho6 mutant defective in the regulatory gene for the synthesis of periplasmic T-rAPase encoded by the PHO3 gene (26, 27) is auxotrophic for thiamine, and the enzyme activities involved in thiamine synthesis described above are markedly low in crude extracts of this mutant (9). This indicated that the expression of structural genes for T-rAPase and the enzymes involved in thiamine biosynthesis was regulated positively by PHO6. Recently, it was elucidated that T-rAPase predominantly catalyzes the hydrolysis of thiamine phosphates under physiological conditions (17). The PHO6 gene is not required for the transcriptional control of the repressible acid phosphatase encoded by the PHOS gene (10) or the Pi transport system encoded by the PHO84 gene (2, 25) in the phosphate metabolism of S. *
cerevisiae. Since PHO6 is closely related to thiamine metabolism, we described this gene as THI2 (PHO6). However, the thi2 (pho6) mutant defective in the regulatory gene for enzymes involved in thiamine synthesis retained sufficient thiamine transport activity (16). Another thiamine auxotrophic strain (thil mutant) was reported (5), and it was found that the allelic thil locus is located in chromosome XIII (14). In this article, we describe the isolation of a thi3 mutant of S. cerevisiae which has a recessive thiamine auxotrophic phenotype similar to that of the thi2 (pho6) mutant. However, unlike the thi2 (pho6) mutant, which retained thiamine transport activity, the thi3 mutant had decreased activities not only of T-rAPase and the enzymes involved in thiamine
synthesis from hydroxylmethylpyrimidine and hydroxyethylthiazole, but also of thiamine transport. These results suggest that thiamine transport as well as thiamine biosynthesis and T-rAPase in S. cerevisiae are subject to the positive regulatory gene THI3. We have also isolated a hybrid plasmid, pTTR1, containing a 6.2-kb DNA fragment from an S. cerevisiae genomic library which complements the thi3 mutation of S. cerevisiae. This gene was localized on a 3.0-kb ClaI-BglII fragment in the subclone pTTR5. We propose a working hypothesis that thiamine metabolism in S. cerevisiae is controlled by the positive factors THI2 (PHO6) and THI3, whose actions are regulated negatively by the intracellular thiamine pyrophosphate level. MATERIALS AND METHODS Strains and culture conditions. The S. cerevisiae and Escherichia coli strains used in the present study are listed in Table 1. E. coli cells were cultured in LB medium (0.5% yeast extract, 1% Bacto-Peptone, 1% NaCl, 0.2% glucose) at 37°C. When necessary, the medium was supplemented with ampicillin (20 p,g/ml). Yeast strains were cultured at 30°C in YPD medium (1% yeast extract, 2% Bacto-Peptone, 2% glucose) or in a defined medium containing 0.67% yeast
Corresponding author. 4701
4702
NISHIMURA ET AL.
J. BACTERIOL. TABLE 1. Strains used
Strain
Relevant genotype
S. cerevisiae
IFO 10481 IFO 10482 IFO 10483 TRS3 T49-2D KYC319 K269-5c KYC307 NK1
MATa his4-519 ga12 MATa his4-519 gal2 MATa leu2-3, 112 gal2 MATa thi3 leu2-3,112gal2 MATa thi3 leu2-3,112 ura3-52 trpl-A63 lys2-801 his3-A200 MATa thi3 leu2-3,112 ura3-52 trpl-A63 lys2-801 MATa thi2(pho6-7)leu2-3,112 ura3-1,2 trpl-A63 MATa thi2(pho6-7)trpl-A&63 + MAa + his4-51 gal2 + MATa thi3 leu2-3,112 gal2
NK2
ALITa + MA Ta thi3 MATa + MATTa thi3
T114 E. coli
DH5aL MV1184
thi2(poL-7 leu2-3.112 ura3-1.2 trpl-A63ga leu2-3,112
+
thi2po6-7
+
+
+
gal2
tr2l-A63 gal2
+ leu2-3,112 gal2 supE44 AlacU169 (,0801acZAM15)hsdRl7 recAl endAl gyrA96 thi-1 reLAI +
A(lac-proAB) rpsL thi (+80JacZAM15) A(sH-recA)306::TnlO (TetD F' (traD36 proAB+ laclq lacZAM15)
ara
nitrogen base (Difco Laboratories) supplemented with essential amino acids or in Wickerham's synthetic medium supplemented with essential amino acids with or without thiamine (28). Yeast strains auxotrophic for thiamine were cultured in minimal medium with thiamine at a concentration of 10-8 M, which does not cause thiamine repression. Yeast strains harboring derivatives of plasmids YCp5O and pRS316 were cultured in medium lacking uracil. Isolation of mutants. Chemical mutagenesis with ethyl methanesulfonate was performed essentially as described by Lindegren et al. with some modifications (11). The parent strain, IFO 10483, was cultured in YPD (5 ml) overnight at 30°C. The cells were washed twice with sterile water and suspended in 4.6 ml of 0.2 M potassium phosphate buffer (pH 8.0). Ethyl methanesulfonate (0.15 ml) and 4% glucose (0.25 ml) were added, and the reaction mixtures were incubated at 30°C for 60 min with shaking. The cells were pelleted and washed five times with sterile water. This was followed by nystatin (35 ,ug/ml) treatment for mutant enrichment (24). The cells were then washed twice with sterile water and spread on agar containing Wickerham's synthetic medium with thiamine (10-8 M). The colonies in the master plate were replica plated on minimal medium without thiamine. After 2 to 3 days of incubation at 30°C, the colonies that did not thrive were selected from the master plate and transferred to a fresh plate for storage and further analysis. The genetic techniques used were those described by Sherman et al. (23). Enzyme assays with hydroxymethylpyrimidine and hydroxyethylthiazole in a crude extract. Yeast cells were harvested, washed once with cold water, and then suspended in 0.05 M potassium phosphate buffer (pH 7.5) containing 1 mM 2-mercaptoethanol and 1 mM EDTA. The cell suspensions were sonicated, and after centrifugation at 28,000 x g for 30 min, the supernatant was used as a crude extract. Overall thiamine-synthesizing activity from hydroxymethylpyrimidine and hydroxyethylthiazole and the activities of each of the four enzymes involved in the formation of thiamine monophosphate (hydroxymethylpyrimidine kinase, phosphomethylpyrimidine kinase, hydroxyethylthiazole kinase, and thiamine-phosphate pyrophosphorylase) were assayed as described previously (9). The protein concentrations were determined by the method of Lowry et al. (12).
Thiamine transport and T-rAPase activities in yeast cells. Thiamine transport was determined by the method described previously (7, 8). T-rAPase activity withp-nitrophenyl phosphate as a substrate was determined from the amount of p-nitrophenol produced, as described earlier (22). Transformation and DNA preparations. Yeast strains were transformed after lithium acetate treatment as described by Ito et al. (6). Bacterial transformations were performed as described by Maniatis et al. (13). Plasmid DNA from yeast cells was isolated as described by Sherman et al. (23) with some modifications. Bacterial plasmids were prepared by the alkaline lysis method of Maniatis et al. (13). Isolation of a plasmid able to complement THI3 mutations. The TRS3 strain (thi3 mutant) was crossed to strain YPH499, and a recipient strain, T49-2D, carrying several selectable auxotrophic amino acid markers and allowing transformation at high frequency was generated. This strain was transformed with a gene library of S. cerevisiae YCp50 "CEN BANK" A, constructed by partial digestion of the genomic DNA of S. cerevisiae GRF88 with Sau3AI and its ligation with YCpSO at the unique BamHI site. YCp5O was obtained from the American Type Culture Collection (Rockville, Md.). Transformants were screened for the ability to grow without uracil and thiamine on Wickerham's synthetic medium. After 3 days of incubation at 30°C, colonies were selected for further analysis. Plasmid pTTR1, which complemented THI3 mutations, was obtained from one colony among six transformants. Construction of plasmids. All procedures were performed under the conditions recommended by the suppliers or by standard methods as described by Maniatis et al. (13). For plasmid pTTR1-a, the BamHI fragment (6.2 kb) from pTTR1 was cloned into pRS316. For plasmid pTTR2, the HindIII fragment (5.3 kb) from pTTR1 was cloned into pRS316. For plasmid pTTR3, the ClaI-HindIII fragment (4.5 kb) from pflTR1 was cloned into pRS316. For plasmid pTTR4, the Sall-HindIII fragment (3.5 kb) from pTTRl1 was cloned into pRS316. For plasmid pTTRS, the ClaI-BglII fragment (3.0 kb) from plTR3 was cloned into pRS316. Plasmids, enzymes, and chemicals. Plasmid YCpS0 (21) was obtained from the American Type Culture Collection (Rockville, Md.), and plasmid pRS316, which was used as the vector for subcloning, was provided by Philip Hieter (Johns
VOL. 174, 1992
THI3 REQUIRED FOR THIAMINE METABOLISM IN S. CEREVISL4E
Hopkins University School of Medicine, Baltimore, Md.). Plasmid YIpS (1) was used in yeast integrative transformation. ApaI was purchased from Toyobo, Co., Ltd. (Osaka, Japan); BamHI, BglII, HindIII, and Sall were obtained from Nippon Gene (Toyama, Japan); ClaI, Kpnl, and PvuII were purchased from Takara Shuzo (Kyoto, Japan). [14C]thiamine ([thiazole-2-[14C]thiamine hydrochloride, 24.3 Ci/mol) was purchased from Amersham International (Buckinghamshire, United Kingdom). Nystatin was purchased from Sigma Chemical Co. All other chemicals were purchased from commercial suppliers. RESULTS Isolation and selection of the thi3 mutant. Yeast cells (IFO 10483) were treated with the chemical mutagen ethyl methanesulfonate and then enriched for mutants with nystatin in Wickerham's synthetic medium supplemented with hydroxymethylpyrimidine (2 x 10-7 M) plus hydroxyethylthiazole (2 x 10' M) or with thiamine (2 x 10-8 M) for selection and recovery, respectively. Two mutant colonies that grew on our minimal medium containing thiamine (2 x 10-8 M) but not on thiamine-free medium were isolated. One of these mutants, TRS3, was crossed with the wild-type strain, IFO 10482, and the resulting diploid, NK1, was prototrophic for thiamine, as is the wild type, indicating that this mutation was recessive. Four-spored asci were dissected from the diploid. All tetrads (19 asci were tested for each cross) showed 2+ :2- segregation for thiamine auxotrophy, confirming that a single chromosomal mutation was responsible for this phenotype. The mutation in TRS3 was designated thi3. Phenotypes (defects in thiamine transport and T-rAPase activity) other than thiamine auxotrophy in the thi3 mutant. Although, as noted above, the thi3 mutant was auxotrophic for thiamine, this mutant also showed decreased thiamine transport and T-rAPase activities compared with wild-type strains. Thiamine transport rates were 153.3 and 23.8 pmol/ 106 cells/min for the wild-type (IFO 10483) and thi3 mutant (TRS3) strains, respectively; T-rAPase activities were 4.2 and 1.0 nmol/106 cells/5 min, respectively. Therefore, we analyzed the segregation of the phenotypes for thiamine transport and T-rAPase activities in order to determine whether the defects in these activities cosegregated with thiamine auxotrophy in the thi3 mutant strain. The thi3 mutant was crossed with the wild-type strain (IFO 10482), and the resultant diploid strain NK1 was subjected to tetrad analysis. The phenotypes for thiamine transport and T-rAPase activity and thiamine auxotrophy cosegregated 2+:2in all 76 asci tested. These results indicate that the thi3 mutant, which is impaired in the synthesis of the thiamine transport system and periplasmic T-rAPase in addition to the de novo synthesis of thiamine, resulted from the mutation of a single gene in the nucleus. Another thiamine auxotrophic mutant carrying thi2 (pho6) (9) also showed decreased T-rAPase activity, whereas thiamine transport activity was retained in this mutant (16). A complementation test between the thi2 (pho6) mutant (K269Sc) and the thi3 mutant (TRS3) isolated in this study was then carried out. To check the allelism of thi2 (pho6) and thi3, a cross produced a thi2 (pho6)Ithi3 diploid (NK2), which showed thiamine prototrophy together with thiamine transport and T-rAPase activities (data not shown). Furthermore, an allelism test between thi2 (pho6) and the thi3 mutation was carried out. A diploid, T114, obtained by crossing the TRS3 (thi3 mutant) and KYC307 (thi2 mutant)
4703
TABLE 2. Enzyme activities involved in thiamine monophosphate synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in a wild-type strain (IFO 10483) and TRS3 (thi3 mutant) Sp act' (nmol/mg of Enzyme
Hydroxymethylpyrimidine kinase Phosphomethylpyrimidine kinase Hydroxyethylthiazole kinase Thiamine-phosphate pyrophosphorylase
protein/30 min) in: IFO 10483
thi3 mutant"
10.5 18.6 36.0 25.6
0.4 1.2 7.7 2.2
aEach value is the mean of two experiments. b The thi3 mutant was cultured in Wickerham's synthetic medium supplemented with 10-8 M thiamine.
strains was sporulated, and the resulting asci were subjected to tetrad analysis. Of the 24 tetrads tested so far, 3 segregated 2+:2-, 14 segregated 1+:3-, and 7 segregated 0+:4for the thiamine requirement. These results confirmed that the two mutations, thi2 and thi3, occurred in two nonallelic genes. Activities of several enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in the crude extract of the thi3 mutant. Overall enzyme activity for thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in the presence of ATP and Mg2e was not detected in the crude extract from the thi3 mutant cells (data not shown). The activities of four enzymes involved in the formation of thiamine monophosphate from hydroxymethylpyrimidine and hydroxyethylthiazole, namely, hydroxymethylpyrimidine kinase (EC 2.7.1.49), phosphomethylpyrimidine kinase (EC 2.7.4.7), hydroxyethylthiazole kinase (EC 2.7.2.50), and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (Table 2), were determined. All four activities were markedly lower in the thi3 mutant cell extract than in the extract from the parent cells (IFO 10483). We previously reported that the activities of T-rAPase and the four enzymes involved in the formation of thiamine monophosphate from hydroxymethylpyrimidine and hydroxyethylthiazole in a thi2 (pho6) mutant were markedly low or undetectable (9). This indicated that the THI2 (PH06) gene positively regulates not only the expression of the PH03 gene, encoding T-rAPase, but the expression of at least four genes coding for the enzymes synthesizing thiamine monophosphate indicated above. Similarly, these results demonstrated that thiamine transport and biosynthesis and TrAPase activity in S. cerevisiae are subject to the positive regulatory gene THI3. Molecular cloning of S. cerevisule DNA fragment carrying the THI3 gene. The THI3 gene was identified on a recombinant DNA plasmid by its functional complementation of a thi3 mutation in T49-2D. The S. cerevisiae genomic DNA library, YCp5O "CEN BANK" A, was used to transform T49-2D (thi3 ura3). After transformation by alkaline cation treatment (6), the treated cells were washed twice with cold sterile water, incubated overnight at 30°C in minimal medium lacking thiamine and uracil, and then plated on agar containing Wickerham's synthetic medium without thiamine and uracil. The plates were incubated at 30°C for 3 days, and a total of six Thi+ Ura+ colonies were isolated. DNA prepared from these transformants was used to transform E. coli DH5ao to Ampr. A plasmid obtained from the Ampr transformant conferred the thiamine prototrophic phenotype on yeast strain T49-2D (thi3 ura3) in minimal medium
4704
NISHIMURA ET AL.
J. BA=rRIOL.
Complementat
Plasmid H BKPv C
S
Pv
Bg
H
pTRRI
pTfR1-a,
J+
+
B
B
pTTRI-a
pTTR2
pTTR1, respectively, into the multicloning vector pRS316. Plasmids pTTR2, and pTTR3, constructed by subcloning a 4.4-kb Clal-HindIII fragment from the 6.2-kb fragment into pRS316, complemented the thi3 mutation (Fig. 1). Plasmid pTTR4, bearing the 3.6-kb Sail-HindIII fragment from the 6.2-kb fragment in pRS316, did not complement the thi3 mutation. Furthermore, plasmid pTTR5, which was constructed by subcloning the 3.0-kb ClaI-BgiII fragment from pTI'R3, also complemented this mutation. These results suggest that the SalI site is located within a region necessary for restoring THI3 function. The 6.2-kb BamHIBamHI DNA fragment derived from plasmid pTJR1 used as a probe against yeast chromosomes that had been resolved by pulsed-field electrophoresis hybridized only to one chromosome, which was identified, by comparison with a standard, as chromosome IV (data not shown).
BOf J
+
H
H
+
L H
c
+
plTR3 H
S
pTTR4
Bg
c
pITR5
+ 1
kb
Expression of thiamine transport, T-rAPase, and enzyme activities involved in thiamine synthesis in the thi3 mutant transformed by the THI3 gene. The effect of some plasmids bearing the THI3 gene on thiamine metabolism was examined by measuring the activities of thiamine transport and T-rAPase. Transformants of the thi3 mutant T49-2D (thi3 ura3), harboring the low-copy-number vector plasmid YCp5O, pRS316, and two plasmids bearing the THI3 gene (pTTR1 and pTIR3) were cultured in the absence or presence of thiamine and assayed for thiamine transport and T-rAPase activity (Table 3). The transformants with pTTR1 and pTTR3 had far more potent thiamine transport activity than a wild-type strain (IFO 10483), whereas T-rAPase activity in the transformants harboring the plasmid with the THI3 gene was lower than in the wild-type strain. Both thiamine transport and T-rAPase activities in the recipient cells transformed with the control plasmids YCp5O and pRS316 were as markedly low as in the thi3 mutant (TRS3). Furthermore, the activities in transformants with pTTR1 and pTTR3 were repressed by thiamine (2 x 10' M) in minimal medium in the same manner as those of the wild-type strain (IFO 10483). These results indicated that the expression of thiamine transport and T-rAPase by pTTR1 and pTTR3 in the thi3 mutant was controlled by the regulatory system owing to the signal of the intracellular thiamine pyrophosphate. The overall enzyme activity for thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole and the activity of an enzyme involved in the formation of thiamine monophosphate (thiamine-phosphate pyrophosphorylase) in crude extracts of these transformants cultured in thiaminedeficient Wickerham's synthetic medium were assayed (Table 4). Thiamine-phosphate pyrophosphorylase activity of
FIG. 1. Restriction map of an S. cerevisiae DNA inisert in pTR1 and subcloning of DNA fragments with and without the ability to complement the thiM mutation. Restriction enzyme c-leavage sites: B, BamHI; C, CMaI; Bg, BglII; H, HindIII; K, KpnI; Pv, PvuII; S, Sall.
without thiamine and uracil. This plasmid, pTlrRl (Fig. 1) had an insertion of the 6.2-kb BamHI-BamHI fragment in YCp5O. That pTTR1 contains THI3 was confirmed b2 y integration of the DNA fragment complementing the thi3 nnutation into the yeast chromosome. We fractionated the 6.2 -kb fragment between two BamHI sites from pTTR1 (Fig. 1) and inserted it into integrative vector YIp5 to generate p INHK1. The recombinant plasmid was then transformed i nto the thi3 mutant KYC319, and Ura+ transformants were selected. All the Ura+ colonies were also Thi+. Both pheniotypes were stably inherited during mitotic growth in nonscelective conditions for many generations, suggesting integiration of the recombinant molecule into a nuclear chromosome. The strain transformed with pNHK1 was crosseid with IFO 10481, and the resultant diploids were subjectted to tetrad analysis. Of the 18 tetrads tested, 17 segregatecd 4+:0- and 1 segregated 3+:1- for thiamine requirement and 1 segregated 4+:0-, 12 segregated 3+:1-, and 5 segrelgated 2+:2for uracil requirement. These results indicate th at pNHK1 is integrated at or close to the thi3 locus. Thus, Mie concluded that pT'l1 bears the THI3 gene. Several deletion fragments were constructced from the cloned 6.2-kb fragment (Fig. 1). Plasmids p'ITR1-a and pTTR2 were constructed by subcloning a 6.2'-kb inserted DNA and a 5.3-kb HindIII-HindIII fragment f'rom plasmid
TABLE 3. Effect of thiamine added to the growth medium on thiamine transport and T-rAPase activities in the thi3 mutant strain transformed by various plasmid-borne DNA fragmnents Strain
IFO 10483 T49-2D (thi3 mutant)
Thiamine transport activity"
T-rAPase activity'
(pmol/106 cells/min)
(nmol/106 cells/5 min)
Plasmid
None
YCp5Ob (vector) pTTRl pRS316b (vector) pTIR3
No addition
0.2 FM thiamine
No addition
0.2 FM thiamine
142.4 8.3 206.7 3.9 176.8
3.7 NDc 10.1 ND 17.4
19.4 2.4 17.6 2.4 11.2
0.5 ND 1.0 ND 1.4
aEach value is the mean of two experiments. b Strains harboring a control vector were cultured in Wickerham's synthetic medium supplemented with 10-8 M thiamine. ND, not detectable.
THI3 REQUIRED FOR THIAMINE METABOLISM IN S. CEREVISIAE
VOL. 174, 1992
TABLE 4. Activity of thiamine-synthesizing enzymes in the thi3 mutant strain transformed by various plasmid-borne DNA fragmentsa Strain
IF010483 T49-2D (thi3 mutant)
Plasmid
None
YCpS0 (vector) pTTR1 pRS316 (vector) pTTR3
Enzyme activity (nmol/mg of proteinl30 min)
Overall reaction
Thiamine-phosphate pyrophosphorylase
1.19 0 0.06 0 0.64
12.4 0.3 7.4 0.4 11.3
4705
(effector)
(CTI3
THJ2(PHO6)
FIG. 2. Working hypothesis for the interaction between the regulatory factors for transmission of the thiamine pyrophosphate (TPP) signals. Square boxes indicate genes, and ovals represent regulatory factors. Arrows, stimulatory effect; horizontal bar, repressive effect; ?, negative regulatory factor(s).
a See Table 3, footnotes a and b.
transformants harboring plasmids with the THI3 gene was expressed at the same level as that of the wild-type strain. In the crude extract of cells transformed with pTTR3, the overall enzyme and thiamine-phosphate pyrophosphorylase activities were also the same as those of the wild-type strain, but neither activity in the crude extract of the transformant with pTTRl was as potent as that of the wild-type strain, although the reason for this remains unknown. These activities in the extracts of recipient cells transformed with the control plasmids YCp50 and pRS316 were also as markedly low as those of the thi3 mutant (TRS3). DISCUSSION This work indicates that thiamine metabolism, including the thiamine transport system, in S. cerevisiae is also controlled by the positive regulatory gene THI3. This is an alternative to the THI2 (PH06) gene, which is indispensable to yeast thiamine metabolism except for the thiamine transport system. We have already elucidated that expression of the structural genes for T-rAPase and the enzymes involved in thiamine biosynthesis in S. cerevisiae is regulated positively by THI2 (PH06) (9), whereas expression of the genes for thiamine metabolism is controlled negatively by the intracellular thiamine pyrophosphate level (15). However, as described above, the THI2 (PH06) gene seems not to be essential for expression of the thiamine transport system (16), and therefore we have attempted to obtain a thiamine auxotrophic mutant with a defect in the thiamine transport system in order to identify a positive regulatory gene for thiamine metabolism, including thiamine transport. As expected, the mutant (TRS3) isolated in this study was auxotrophic for thiamine, with defective T-rAPase and thiamine transport activities. These phenotypes occurred as the result of the same mutation of a single gene in the nucleus, which was designated thi3. The distinguishing difference between the thi2 (pho6) and thi3 mutations was the phenotype for thiamine transport. It was possible that these mutations were produced in two different genes of the nucleus. Furthermore, a complementation test and a tetrad analysis between thi2 and thi3 mutants implied that the mutations occurred in two nonallelic genes. The THI3 gene was located on chromosome IV, whereas the THI2 (PH06) gene was situated on the right arm of chromosome II (16). These data indicated that the thi3 and thi2 (pho6) loci were indeed distinct, since mutations in each generated distinguishable phenotypes for thiamine transport. Another allelic locus (thil), which shows thiamine auxotrophy as a phenotype in S. cerevisiae, has been reported (5), but the THII gene was situated on the right arm of chromosome XIII (14). These results confirmed
that THI3 was a novel gene that is indispensable for thiamine prototrophy and that controlled the expression of the thiamine transport system simultaneously. Accordingly, it was considered that the THI2 (PH06) and THI3 genes are both required for expression of T-rAPase and several enzymes involved in thiamine synthesis, whereas the thiamine transport system was regulated only by the THI3 gene as a positive factor. Two positive regulatory genes, PH02 and PHO4, whose products are indispensable for the transcriptional control of the structural genes for repressible acid phosphatase encoded by PHOS and the Pi transport system encoded by PH084 in S. cerevisiae, have been reported by Oshima and coworkers (20, 29, 30). The PHO regulatory system in S. cerevisiae appears to be of interest for further study of this thiamine regulatory system. To examine whether several plasmids containing the THI3 gene complemented the biochemical phenotypes of the thi3 mutation (loss of thiamine transport and T-rAPase activity), transformants of strain T49-2D were grown in the absence or presence of thiamine and assayed for thiamine transport and T-rAPase activities. When the cells were cultured in the absence of thiamine, complementation of the thi3 mutation by these plasmid-borne genes was always nearly equal to complementation of the wild-type strain, as shown in Table 3. However, after growth in minimal medium supplemented with thiamine (2 x 10' M), thiamine transport and TrAPase activities in transformant cells carrying a plasmidborne THI3 gene were significantly repressed, as in the wild-type strain. These results are of interest in connection with the regulatory mechanisms of thiamine metabolism in S. cerevisiae. In our previous studies (8, 9), the thiamine transport system, T-rAPase activity, and the enzymes involved in thiamine synthesis from hydroxymethylpyrimidine and hydroxyethylthiazole in S. cerevisiae were found to be repressible by exogenous thiamine. We then demonstrated that thiamine metabolism in S. cerevisiae is controlled negatively by the intracellular thiamine pyrophosphate level (15). At present, as shown in Fig. 2, we presume that thiamine metabolism in yeast cells is controlled by the positive factors encoded by THI2 and THI3, which might be negatively regulated by the intracellular thiamine pyrophosphate level via some gene(s). In order to make sure this assumption, the mode of expression of two positive regulatory genes (THI2 and THI3) and the interaction between the positive factors and a negative factor(s) should be investigated. Although we have been trying to determine the nucleotide sequence of the THI3 gene, further study at the molecular level is required to clarify the detailed mechanism of the regulation of yeast thiamine metabolism.
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NISHIMURA ET AL.
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