(Received for publication, February 18, 1997, and in revised form, April 18, 1997)
From the Department of Biochemistry, We isolated a thiamin transporter gene,
THI10, from a genomic library of Saccharomyces
cerevisiae by the complementation of a yeast mutant defective in
thiamin transport activity. The THI10 gene contained an
open reading frame of 1,794 base pairs encoding a 598-amino acid
polypeptide with a calculated molecular weight of 66,903. The
nucleotide sequence of THI10 is completely identical to
that of an anonymous DNA (open reading frame L8083.2)
mapped to chromosome XII; two other genes (open reading frames
YOR071c and YOR192c) in chromosome XV are
extremely similar to THI10. Moreover, the THI10
gene product showed significant sequence homology with yeast allantoin
and uracil transporters. Hydropathy profile suggested that THI10
product is highly hydrophobic and contains many transmembrane regions.
Gene disruption of the THI10 locus completely abolished the
thiamin transport activity and thiamin binding activity in yeast plasma
membrane fraction. Both the transport and thiamin binding activities
were restored in the disrupted cells when the THI10 open
reading frame was expressed by yeast GAL1 promoter,
suggesting that the THI10 gene encodes for the thiamin
transport carrier protein. Northern blot analysis demonstrated that
THI10 gene expression is regulated at the mRNA level by
intracellular thiamin pyrophosphate and that it requires a positive
regulatory factor encoded by THI3 gene.
Yeast cells take up thiamin from the extracellular environment by
an active transport system with a pH optimum of 4.5 and a
Km value of 0.18 µM, and thiamin in
the cells is concentrated ~10,000-fold over extracellular levels (1).
The thiamin transport system is more specific for the chemical
structure of the pyrimidine moiety than the thiazole moiety of thiamin
(2). Saccharomyces cerevisiae also secretes a
thiamin-repressible acid phosphatase encoded by PHO3 gene
(3, 4) in a periplasmic space. Since yeast cells cannot take up thiamin
phosphate esters, the PHO3 protein with a high affinity for thiamin
phosphate esters appears to hydrolyze them before the uptake (5, 6),
and then thiamin is converted to thiamin pyrophosphate
(TPP)1 by thiamin pyrophosphokinase (EC
2.7.6.2) encoded by the THI80 gene (7). TPP is an important
cofactor in the energy metabolism (8) and is a negative effector of the
regulation mechanism of thiamin metabolism in yeast cells (9).
We report here the isolation and characterization of S. cerevisiae THI10 gene and provide evidence that the
THI10 gene encodes for a thiamin transport carrier protein.
The predicted THI10 protein is highly hydrophobic and shows significant
sequence similarities to yeast uracil and allantoin transport proteins.
The thiamin transport activity and the thiamin binding activity in the
yeast plasma membrane fraction in thi10 null strain cells
were restored when the THI10 open reading frame (ORF) was
expressed by yeast GAL1 promoter. The regulation of the
THI10 gene expression was also investigated.
Table I shows the
S. cerevisiae strains used in this study. The thiamin
transport mutant was isolated as a strain resistant to pyrithiamin
after the chemical mutagenesis with ethyl methanesulfonate as described
(10). The E. coli strain DH5 was used to amplify plasmids.
The media and the growth conditions for the yeast and bacterial cells
were as described previously (7). Glucose in the yeast minimal medium
was replaced by 1% galactose for inducing the THI10,
YOR071c, and YOR192c transcription from
GAL1 promoter. Yeast strains auxotrophic for thiamin as
thi2 and thi3 mutants were cultured in the
minimal medium with thiamin at a concentration of 10 Table I.
Yeast strains used
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENT
REFERENCES
Organisms and Cultures
8
M, which does not cause thiamin repression.
Strain
Genotype
Source
X2180-1A
MATa SUC2 mal gal2
CUP1
YGSCa
IFO10482
MATa his4-519
gal2
IFOb
YPH499
MATa ura3-52 his3-
200 leu2-
1
trp1-
63 ade2-101 lys2-801
Y. Ohyac
YPH500
MAT
ura3-52 his3-
200 leu2-
1 trp1-
63 ade2-101 lys2-801
Y.
Ohyac
T50-1B
MATa thi10-1 ura3-52 his3-
200 leu2-
1
trp1-
63 lys2-801
This study
NKC6
MAT
thi10::HIS3 ura3-52 his3-
200 leu2-
1 trp1-
63 ade2-101
lys2-801
This study
O58-M5
MATa thi2(pho6) gal4
Y.
Oshimad
TRS3
MAT
thi3 leu2-3,112 gal2
Our stock
T36-2A
MATa thi80-2 pho2-8 pho4-1 trp1 gal2
Our stock
ND10
MATa/MAT
thi10-1/thi10::HIS3 ura3-52/ura3-52
his3-
200/his3-
200 leu2-
1/leu2-
1 trp1-
63/trp1-
63
lys2-801/lys2-801 +/ade2-101
This study
a
Yeast Genetic Stock Center, University of California,
Berkeley, CA.
b
Institute for Fermentation, Osaka, Yodogawa-ku, Osaka 532, Japan.
c
University of Tokyo.
d
University of Osaka.
Mating, sporulation, and dissection of yeast cells were carried out according to the standard procedures for yeast genetics (11). The thiamin transport activity on agar plates was detected using the staining method based on the reduction of triphenyltetrazolium chloride that is known to be taken up by yeast cells via the thiamin transport system (12).
Plasmids and Nucleic Acid ManipulationA gene library of
S. cerevisiae constructed by partial digestion of the
genomic DNA with Sau3AI and its ligation with YEp13 (13) at
the unique BamHI site was purchased from the American Type
Culture Collection (Rockville, MD). Shuttle vector pRS316 (14) was used
for expression in yeast as a single-copy vector. To construct the
thi10 disrupted mutant, the 4.0-kilobase pair (kb)
EcoRI-SphI fragment of pTTS1 (see Fig. 1)
containing the THI10 gene was ligated into the 3.1-kb pUC118
vector (15) to yield pUC118-THI10, and a 1.4-kb
XbaI-XhoI fragment containing yeast
HIS3 gene of pJJ217 (16) was inserted into pUC118-THI10 at
the corresponding sites within the THI10 coding sequence.
The linear 3.0-kb SpeI-SpeI fragment of the
resultant plasmid pUC-thi10::HIS3 was used to transform the
haploid yeast YPH500. His+ colonies were selected, and gene
disruption was confirmed by Southern blotting. To express
THI10, YOR071c, and YOR192c
genes from the GAL1 (17) promoter, a yeast expression vector
pYES2 (Invitrogen) carrying the GAL1 portion of the
divergent GAL1/GAL10 promoter region from
S. cerevisiae and the yeast CYC1 (18)
transcription termination signal sequence, a 2-µm origin of
replication was used. For subcloning of THI10, the ORF was
amplified by polymerase chain reaction (PCR, Perkin-Elmer Cetus
Instruments) from a plasmid pTTS6 (see Fig. 1): the 5-end primer,
CTCGAATTCATGAGTTTCGGTAGTAAAGT was immediately upstream of the
THI10 ATG with the Ec0RI linker, and the 3
-end
primer, CTCGCATGCCTAAGCAGCTTTTTCACTGG, was downstream of the
THI10 TAG with the SphI linker. For isolation of
YOR071c and YOR192c genes by PCR, specific
primers targeted to these untranslated regions were designed according
to the sequences of the GenBankTM data base.
GTGTTGCTACCGCTATTTGTTCTTCGG (forward, 225 bp upstream from the presumed
initiating codon) and TCTCTTGCTATATGGATGGCATAATTAGC (reverse, 259 bp
downstream from the stop codon) were used for YOR071c, and
ACCATTCACATAAGATAGACTGTTCCGC (forward, 561 bp upstream from the
presumed initiating codon) and AATCAATGAAAATATCATCCTCAACAGAAATA (reverse, 252 bp downstream from the stop codon) were used for YOR192c gene. These amplified products from the genome DNA
of wild-type yeast strain YPH500 were used as the templates for a second PCR to obtain the following ORFs. Forward primers were immediately upstream of the ATG with the HindIII linker,
CTCAAGCTTATGAGCTTCAGTAGTATAGT (YOR071c) and
CTCAAGCTTATGAGTTTCGGTACGAGAAT (YOR-192c), and the reverse
primers were immediately downstream of the stop codons with the
EcoRI linker, CTCGAATTCTCAGACAACTTGTTGAGTGG
(YOR071c) and CTCGAATTCTTAGGCAATTTGTTTTTCAC
(YOR192c). All PCR products were purified using
WizardTM PCR Preps (Promega), and the obtained ORFs
treated with the appropriate restriction enzymes were inserted at the
corresponding sites of pYES2. General methods for DNA and RNA
manipulation, blot analysis, and yeast or bacterial transformation were
as described elsewhere (7, 15).
DNA Sequencing
The DNA sequence was determined by primer walking using synthesized nucleotides on an automated laser fluorescent DNA sequencer (Pharmacia LKB Biotechnology Inc.) as described (7). The sequencing was performed twice for each primer on both orientations using the purified plasmid pTTS6 (see Fig. 1) as a template. Nucleotide and deduced amino acid sequences were analyzed with programs from a GENETYX software package (Software Development, Tokyo).
Determination of Thiamin Transport and Thiamin-binding ActivitiesThe uptake of the radioactive thiamin into the yeast
cells was measured as described previously (1). The thiamin binding activity was determined by an equilibrium dialysis against
107 M [14C]thiamin overnight in
the cold as described (19) after subtracting nonspecific thiamin
binding performed in the same manner except that 10
4
M nonlabeled thiamin was added in the outer solution of
dialysis containing the labeled thiamin. Yeast plasma membrane was
prepared by a sucrose gradient centrifugation method from the crude
membrane fraction as previously reported (20). Protein concentration in
membrane preparations was measured by the procedure of Markwell et al. (21) using bovine serum albumin as standard.
DNA probes were labeled with
[-32P]dCTP (3,000 Ci/mmol) purchased from ICN
Biomedicals, and thiazole-2-[14C]thiamin hydrochloride
(24.3 Ci/mol) was from Amersham International. All other chemicals were
purchased from commercial suppliers.
A yeast strain T50-1B resistant to pyrithiamin, a strong thiamin antagonist bearing high affinity for thiamin transport system in yeast, is a recessive mutant defective in thiamin transport activity. T50-1B was transformed with a yeast genomic library and plated on the agar medium without thiamin and leucine. The plates were incubated at 30 °C for 3 days, and a total of 15,000 Leu+ colonies were examined for the thiamin transport activity by staining with triphenyltetrazolium chloride. Only one colony was found to have the thiamin transport activity (by turning the colony red), and by the plasmid-curing experiment, the colony was confirmed to have a plasmid complementing the mutation of T50-1B. A plasmid prepared from the transformant was used to transform Escherichia coli DH5 to Ampr. The plasmid obtained from the Ampr, pTTS1 (Fig. 1), had an insertion of a 5.2-kb fragment in YEp13. The insert DNA contained an ORF (see below), and thus the gene obtained here was named THI10. Transformant carrying pTTS1 exhibited restoration of the thiamin transport activity (Table II).
|
To define the limits of the
THI10 gene, various subclones were constructed using the
pRS316 vector from the cloned plasmid pTTS1. The yeast transformants
carrying the subcloned plasmids were tested for their thiamin transport
activities (Fig. 1). Plasmid pTTS6 containing the 2.6-kb
SpeI-NdeI fragment complemented the mutation of
T50-1B, whereas plasmid pTTS5, constructed by subcloning the 2.3-kb
SpeI-XhoI fragment, did not significantly
suppress the phenotype of the mutation. Therefore, we concluded that
the 2.6-kb SpeI-NdeI region contains the
THI10 gene and that the nucleotide sequence of this region
was determined using pTTS6 as a DNA template. As shown in Fig.
2, an ORF of 1,794 bp starting at position 548 and
ending at 2,341 was found. A putative TATA box was found at positions
404-408, and two CCAAT transcription elements (22) were located at
positions 101-105 and 529-533 in the 5-upstream region. The
polyadenylation signal AATAAA was found at positions 2,350-2,355,
2,375-2,380, 2,507-2,512, and 2,572-2,577 in the 3
-noncoding
region. The yeast intron splice signal, TACTAAC (23), was not found.
GenBankTM data base search revealed that the nucleotide
sequence of THI10 is completely identical to that of a DNA
coding an unknown protein (L8083.2 gene, accession number
U19027), which has been mapped to chromosome XII. We also found
YOR071c (1,794 bp of ORF, accession number Z74979) and
YOR192c (1,797 bp, accession number Z75100) genes, which are
both located at S. cerevisiae chromosome XV and code for
proteins of unknown function. The nucleotide sequences of both genes
match over the entire length of THI10 with no gaps and about
84% sequence identity (data not shown), suggesting that THI10, YOR071c, and YOR192c genes are
from a common evolutionary origin.
Structure of the Predicted Protein
The predicted protein
encoded by the THI10 ORF contains 598 amino acid residues
with a calculated molecular weight of 66,903. By comparison with
sequences in the SWISS-PROT data base, the predicted THI10 protein was
found to have significant sequence similarity to allantoin
(DAL4) (24) and uracil transporter (FUR4) (25) of
S. cerevisiae over almost the entire region with 30.2 and
27.9% identity, respectively (Fig. 3). However, neither
allantoin nor uracil inhibited [14C]thiamin uptake into
yeast cells when each base of 1,000 times the concentration of labeled
thiamin was added to the assay mixture for thiamin transport activity
(data not shown). Hydropathy analysis of the deduced THI10 protein
indicated that it consists of many potential transmembrane stretches,
with the NH2- and COOH-terminal regions being mostly
hydrophilic (Fig. 4). According to the algorithm developed by Kyte and Doolittle (26), 10 segments of 19 sequential residues with an average hydropathy above +1.2 would span the membrane.
The average hydropathy values of the transmembrane candidates are as
follows: II (amino acids 78-96), 1.61; III (amino acids 107-125),
1.37; IV (amino acids 127-145), 1.25; V (amino acids 175-193), 1.45;
VI (amino acids 198-216), 2.02; VII (amino acids 278-296), 2.04; VIII
(amino acids 330-348), 1.26; X (amino acids 402-420), 1.88; XI (amino
acids 448-466), 1.21; XII (amino acids 488-506), 1.89. Although less
extended, there are two hydrophobic segments: I (amino acids 53-71)
with the value of 1.04 and IX (amino acids 361-379) with the value of
0.82. Like many other transporter proteins, the NH2
terminus of the THI10 gene product apparently does not
contain any structure that resembles a signal peptide. Potential
N-linked glycosylation sites, Asn-X-(Ser/Thr) (27), were found at amino acid positions 39, 323, and 390. They were
all located in hydrophilic regions at the NH2 terminus or between two adjacent membrane-spanning regions.
Disruption of THI10
To further confirm the identity of the
cloned gene, we disrupted the corresponding gene locus. The
HIS3 fragment was inserted within the THI10
coding region, and the disrupted thi10 fragment was
introduced into the his3 strain (Fig.
5A). DNA was extracted from a
His+ transformant strain (NKC6) and simultaneously digested
with NdeI and SpeI. The digest was separated by
agarose gel electrophoresis and Southern blotted. Fig. 5B
shows that the THI10 locus of NKC6 was correctly disrupted
(lane 2). NKC6 could not uptake [14C]thiamin
in the transport assay (Table II), indicating that the THI10 protein is
indispensable for the thiamin transport system in S. cerevisiae. Hence, we examined whether or not THI10 was the wild-type allele of the original mutation. NKC6 was crossed with
T50-1B. The resultant diploid (ND10) was resistant to pyrithiamin, indicating that complementation did not occur. ND10 was then
sporulated, and its tetrads were analyzed. The transport activity of
all 10 tetrads was <1.0% that of the wild-type level (data not
shown). Thus, the original isolate was concluded to be thi10
mutant.
Thiamin Binding Activity of THI10 Protein
We have previously
reported the occurrence of a thiamin binding activity in the plasma
membrane of S. cerevisiae (20, 28). The thiamin binding
activity is repressed by exogenous thiamin, and the dissociation
constant (Kd) value for thiaminis of 0.11 µM is close to the apparent Km (0.18 µM) of thiamin transport in S. cerevisiae, but
the entity of the protein in the cell membranes remains unclear. We
investigated, therefore, the effect of the disruption of
THI10 gene on the thiamin binding activity in the yeast
plasma membrane fraction. As shown in Table II, thi10 null
strain completely lost the thiamin binding activity, indicating that
the THI10 protein is indispensable for the binding activity as well as
the thiamin incorporation. Then we tried to produce the entire THI10
protein in thi10 null strain from the GAL1
promoter. A vector pYES2-THI10 was constructed as described under
"Experimental Procedures" and transformed into NKC6, and the
transformed cells were cultivated in galactose medium with 106 M thiamin. We found that the plasma
membrane fractions prepared from the transformed cells contained high
thiamin binding activity, and the cells producing the THI10 protein
restored the thiamin transport activity (Table II). These findings
suggested that the THI10 protein is located in the plasma membrane of
yeast cells and has the recognition site for thiamin. From these
results and the similarity of THI10 protein with DAL4 and FUR4
proteins, we concluded that the THI10 gene of S. cerevisiae is a structural gene for a thiamin transport carrier
protein.
Although YOR071c and YOR192c genes are extremely similar to the THI10 gene, it is not clear whether both genes are actually expressed and concerned in thiamin transport in yeast cells. Then, we isolated YOR071c and YOR192c genes by PCR from yeast genome DNA and expressed both ORFs from the GAL1 promoter in thi10 null strain in the same way as with TH110 expression. As shown in Table II, NKC6 cells producing YOR071c or YOR192c protein showed the thiamin transport activities of 13.7% and 41.0%, respectively, compared with the cells expressing THI10 protein. These results suggested that both gene products function together in a thiamin transporter complex with THI10 product or that these genes code a thiamin analog transporter bearing a low affinty for thiamin.
Regulation of THI10 ExpressionThe thiamin transport activity
of S. cerevisiae is coordinately repressed with the enzymes
involved in thiamin metabolism by exogenous thiamin (29). We determined
that the uptake and synthesis of thiamin are controlled positively by a
regulatory gene, THI3 (10), and negatively by the
intracellular TPP level (9). However, a mutant defective in another
positive regulatory gene, THI2, associated with the
expression of activities of the enzymes involved in thiamin
biosynthesis and thiamin-repressible acid phosphatase retains
sufficient thiamin transport activity (31). To determine how
THI10 expression is regulated, we investigated the effect of
thiamin in the medium on the abundance of THI10 mRNA
using the wild-type strain as well as the thi2,
thi3, and thi80 mutants. Cells were cultured
under either low or high concentrations of thiamin, and total RNA was
extracted and Northern-blotted using THI10 and
URA3 DNAs as probes. As shown in Fig. 6,
THI10 mRNA was detected as a single 1.9-kb band, which
was almost consistent with the coding region. THI10 mRNA
of the wild-type strain grown in low thiamin medium was expressed
abundantly, but a high concentration of thiamin almost completely
repressed THI10 mRNA (Fig. 6, lanes 1 and
2). The thi3 mutant did not exhibit the
THI10 transcript (Fig. 6, lanes 7 and
8). On the other hand, the decrease in the THI10
mRNA level by thiamin was slight in the thi2 mutant
defective in thiamin biosynthesis and the thi80 mutant
carrying a level of thiamin pyrophosphokinase activity that was about
one-tenth of that in the wild-type strain (Fig. 6, lanes
3-6). This effect was thought to be caused by an insufficient
increase in the intracellular concentration of the co-repressor, TPP.
These results indicated that THI10 expression is controlled
at the mRNA level in S. cerevisiae by the TPP signal
transduction pathway including the THI3 protein.
We cloned the THI10 gene by complementation of the phenotype of the thiamin transport defective mutant. The following findings strongly suggested that THI10 encodes a thiamin transport carrier protein. The amino acid sequence deduced from the nucleotide sequence of the gene possesses the structural characteristics of an integral membrane protein, and the thiamin transport activity as well as the thiamin binding activity in the plasma membrane fraction of thi10 null strain was restored when the THI10 ORF was expressed by the GAL1 promoter. This is the first report of the gene-encoding thiamin transport carrier protein for any species.
The predicted protein shows significant sequence similarity to allantoin (DAL4) and uracil transporter (FUR4) of S. cerevisiae. Andre (32) recently reported that the transporters of purines, pyrimidines, and derivatives can be classified into two superfamily (FUR and FCY) families. The FUR family contains DAL4, FUR4, YBL042c (uridine permease), and THI10, which is described as the L8083.2 gene in the review. The THI10 protein certainly seems to recognize the pyrimidine moiety of thiamin (2, 33). However, these four gene products appear not to share function. Cooper et al. (34) reported that allantoin accumulation was negligible in the dal4 mutant strain even though it possessed a wild-type FUR4 allele. We demonstrated here that both allantoin and uracil even at high concentrations did not inhibit thiamin uptake into yeast cells. On the other hand, adenosine and cordycepin, an analog of adenosine, share a common transport system with thiamin in growing S. cerevisiae cells (35). Among nucleosides other than adenosine, only cytidine was taken up by the thiamin transport system but to a level that was only 5.9% that of adenosine. It is probable that the THI10 product participates in the uptake of adenosine in S. cerevisiae.
Although the expression of YOR071c or YOR192c gene from GAL1 promoter partially compensated for the deficiency of THI10 protein in yeast cells, the functions of these genes remain unknown. For elucidation of the relationship between these genes and thiamin transport in yeast, it is essential to ascertain whether the disrupted strains of these genes abolish the thiamin transport activity and whether the expression of these genes is controlled by the thiamin regulatory pathway. Recently, Ruml and Silhankova (36) reported a recessive mutation, thp1, leading to complete loss of thiamin uptake in S. cerevisiae that was mapped to chromosome VII. It has not been clear whether the THP1 gene encodes a structural gene of the thiamin transporter or a positive regulatory factor specific for expression of THI10.
The results of Northern blot hybridization indicated that the
THI10 gene expression of S. cerevisiae is
regulated at the mRNA level by TPP, similar to other genes involved
in thiamin metabolism (7, 37). The expression of the PHO3
gene is known to be regulated at the transcriptional level, and we
analyzed the region responsible for the transcriptional activation of
the PHO3 gene in response to thiamin in the medium (38). The
presumed upstream activating sequence, AAA(A/C)TCAA, is found in the
5-upstream noncoding regions of PHO3 (3) and
THI6 (37) genes by two copies for each gene. The
THI6 gene product is a bifunctional enzyme with thiamin-phosphate pyrophosphorylase (EC 2.5.1.3) and
4-methyl-5-
-hydroxyethylthiazole kinase (EC 2.7.1.50) activities
(30). On the other hand, there is a TTGATTT sequence at positions
238-244 of the THI10 5
-upstream noncoding region (Fig. 2).
This sequence is an inverse complement of the AAATCAA. Although the
physiological significance of these sequences is not clear, a finding
that the THI10 does not require the positive factor THI2 for
its expression suggests that the regulatory mechanism of
THI10 expression is different from those of PHO3,
THI6, and THI80. The expressions of these
structural genes, except for THI10, are considered to be
controlled by both THI2 and THI3 factors. We expect that the detailed
analysis of THI10 gene expression will facilitate
elucidation of the TPP signal transduction pathway in yeast thiamin
metabolism.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D55634.
We thank Dr. Y. Kaneko, Osaka University, for constructing the yeast strain T50-1B and the gift of the plasmids pJJ217 and pJJ242 and Hiroshi Tatsumi for help in cloning THI10 gene.