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J. Biol. Chem., Vol. 275, Issue 40, 30987-30995, October 6, 2000
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,
From the School of Biochemistry and Molecular Genetics and the ¶ NMR Facility, The University of New South Wales, Sydney, New South Wales 2052, Australia
Received for publication, May 18, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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One-carbon metabolism in yeast is an essential
process that relies on at least one of three one-carbon donor
molecules: serine, glycine, or formate. By a combination of genetics
and biochemistry we have shown how cells regulate the balance of
one-carbon flow between the donors by regulating cytoplasmic serine
hydroxymethyltransferase activity in a side reaction occurring in the
presence of excess glycine. This control governs the level of
5,10-methylene tetrahydrofolate (5,10-CH2-H4folate) in the cytoplasm,
which has a direct role in signaling transcriptional control of the
expression of key genes, particularly those encoding the unique
components of the glycine decarboxylase complex (GCV1,
GCV2, and GCV3). Based on these and other
observations, we propose a model for how cells balance the need to
supplement their one-carbon pools when charged folates are limiting or
when glycine is in excess. We also propose that under normal
conditions, cytoplasmic 5,10-CH2-H4folate is mainly directed to generating methyl groups via methionine, whereas one-carbon units generated from glycine in mitochondria are more directed to purine biosynthesis. When glycine is in excess,
5,10-CH2-H4folate is decreased, and the
regulation loop shifts the balance of generation of one-carbon units
into the mitochondrion.
Tetrahydrofolate
(H4folate)1-mediated
one-carbon metabolism lies at the center of a large number of essential
cellular processes including methyl group biogenesis and the synthesis
of nucleotides, vitamins, and some amino acids. One-carbon units are
derived from catabolism of three donor molecules: serine, glycine, and
formate. These are then activated and compartmentalized by attachment
to H4folate for biosynthetic processes. In most organisms,
serine is the principle one-carbon donor (1) contributing to the pool of 5,10-CH2-H4folate by the action of serine
hydroxymethyltransferase (SHMT) enzymes (see Fig. 1 and Table I,
reactions 1 and 2). Glycine catabolism via the
mitochondrial glycine decarboxylase multienzyme complex (GDC) (see Fig.
1 and Table I, reaction 3) also contributes to the
one-carbon pool by generation of
5,10-CH2-H4folate (2), whereas formate
activation to 10-HCO-H4folate occurs via the synthetase activity of the C1-tetrahydrofolate synthase trifunctional enzymes (see
Fig. 1 and Table I, reactions 4c and 6c) (3).
Each of the one-carbon H4folate pools are interconverted by
the cyclohydrolase (see Fig. 1 and Table I, reactions 4b and
6b) and dehydrogenase (see Fig.
1 and Table
I, reactions 4a and
6a) activities of the C1-tetrahydrofolate synthase enzymes,
whereas flow between the mitochondrion and the cytoplasm is mediated by
the three one-carbon donor molecules. The result is a dynamic metabolic
system in which one-carbon units are interconverted between the
mitochondrial and cytoplasmic compartments as well as between oxidation
states. This flow has been the subject of extensive biochemical
analyses (4-9) that have shown that glycine, serine, and formate can
each act to supplement all of the different one-carbon pools. Much less, however, is known of how cells regulate these flows.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
Intercompartmental flow of one-carbon units
in Saccharomyces cerevisiae. In addition to gene
names and mutant strain phenotypes described below, the abbreviations
for metabolites of phospholipid biosynthesis are as follows:
PC, phosphatidyl choline; AdoHcy,
S-adenosylhomocysteine; PE,
phosphatidylethanolamine; PS, phosphatidylserine; and
AdoMet, S-adenosylmethionine. The figure is
adapted from West et al. (9).
Enzymes of one-carbon metabolism with the relevant gene designations
and phenotypes of mutants defective in each gene
In contrast to the other enzyme activities for the generation and interconversion of one-carbon units, glycine catabolism via the GDC is strictly mitochondrial. This enzyme is composed of four subunits each encoded separately. These are the P-protein (encoded by GCV2), the H-protein (encoded by GCV3), the T-protein (encoded by GCV1), and the L-protein (encoded by LPD1). Each of the GCV genes is unique to the GDC and on addition of exogenous glycine has been shown to be induced transcriptionally (10-12). For GCV1 and GCV2 expression this was due to a 17-base pair sequence with the hexanucleotide 5'-CTTCTT-3' at its core in their promoters (13), and similar sequences have been identified in the GCV3 promoter. This work also indicated that H4folate or one of its derivatives may play a role in signaling the need for increased GCV transcription when glycine is added to the medium. This possibility led us to the hypothesis that the glycine effect is only one component of a broader one-carbon metabolism regulatory system. This has been confirmed from genome-wide transcript analysis in which the addition of glycine to yeast cells growing in minimal medium led to the induction of other genes central to one-carbon metabolism (14).
Here, we have used a combination of genetics and biochemistry to
identify which derivative of H4folate acts to signal this response, and we propose a model for
5,10-CH2-H4folate-mediated regulation of
metabolism through transcription. Because of the large body of work on
the phenotypes of many one-carbon metabolic mutants (6, 15-17),
strains were available that were specifically altered with respect to
the different reduced H4folate pools. Gene expression
studies in these strains were combined with 13C NMR
experiments to monitor one-carbon flow. These data indicated that
control over cytoplasmic serine hydroxymethyltransferase activity is
responsible for determining how GCV gene transcription is
regulated. By monitoring the levels of the
5,10-CH2-H4folate pool (which is normally
derived from serine), cells can up-regulate glycine catabolism for
one-carbon generation when 5,10-CH2-H4folate is
limiting or spare the breakdown of serine when glycine is in surplus.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials
Amino acids, 2-mercaptoethanol, [2-13C]glycine, poly(dI-dC), and sodium H4folate were obtained from Sigma. 3-trimethylsilyl propionate-2,2,3,3-d4, sodium salt and [14C]formaldehyde were from ICN Pharmaceuticals, and DEAE-Sephadex was from Amersham Pharmacia Biotech. Deuterated dimethyl-d6-sulfoxide was obtained from MSD Isotopes (distributed by Merck). All other materials were of highest available quality and were obtained from various commercial vendors.
Strains and Media
The yeast strains relevant to this study are listed in Table
II. Yeast were grown in minimal
medium as described previously (10) with auxotrophic requirements added
at 40 mg liter
1. To elicit the glycine
response, glycine was added to a final concentration of 10 mM; a 2 M sodium formate stock (pH 7.0) was used to supplement media where indicated.
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Gene Expression Analyses
Constructs and Yeast Transformation-- Plasmids carrying the full-length GCV2 promoter::lacZ fusion (pRH2 and pRH1), full-length GCV1 promoter::lacZ fusion, and full-length GCV3 promoter::lacZ fusion have been described previously (13). These were integrated individually in appropriate strains as a single copy at the URA3 locus using the lithium acetate transformation method (22).
-Galactosidase Assays--
Yeast were grown to early log
phase in the media indicated and harvested for assays as described
previously (10).
NMR
Cell Growth, 13C-Labeling, and Extract
Preparation--
NMR studies followed the method described in Ref. 6.
A 1-liter culture of cells was grown to late log phase in yeast minimal medium with or without 100 mg liter1
[2-13C]-labeled glycine. Cells were harvested and
resuspended in 20 ml of 0.3 N HCl and warmed over a boiling
water bath for 1 h. This solution was centrifuged, the supernatant
was replaced with fresh HCl, and the process was repeated. The
supernatants from the two extractions were pooled, reduced using a
Rotavapor, and dried in a Speedivac. Samples were stored at this stage
at 
80 °C until analysis. Immediately prior to NMR, samples
were resuspended in 1 ml of deuterated dimethyl-d6-sulfoxide.
NMR Analysis-- NMR spectra were recorded on a Bruker DMX 600 spectrometer equipped with a TXI-XYZ probe at a temperature of 298 K. Chemical shifts are relative to internal 3-trimethylsilyl propionate-2,2,3,3-d4, sodium salt set to 0 ppm. (This results in our measured shifts being ~1.4 ppm lower in frequency than those reported relative to tetramethylsilicone.) Metabolites were identified by comparison with previously published data on chemical shifts (Table III) (6). Relative intensities of lines within "triplets" produced by mixtures of single 13C and double 13C-13C isotopomers were extracted with line shape deconvolution using the Bruker WINNMR program. 13C NMR spectra were acquired using power-gated decoupling. A total of 2400 scans with an acquisition time of 1.14 s and recycle delay of 5.0 s were acquired for each sample, resulting in an overall time of 4 h 7 min per sample. The spectral width was 190 ppm, and 64,000 points were collected. The pulse lengths were 9.0 µs, with a 78-degree flip angle. Spectra were processed with exponential multiplication and line broadening of 3 Hz.
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Chemical Synthesis of 5,10-CH2-H4folate
Labeled 5,10-CH2-H4folate was prepared
from H4folate and [14C]formaldehyde, based on
a previously published method (23). Equimolar proportions of
H4folate and formaldehyde (5%
[14C]formaldehyde) were combined in a stoppered oxygen-
and light-free container. The solution was brought to pH 5 by the
dropwise addition of 1 N NaOH, and the mixture was
incubated at room temperature for 15 min. The reaction mixture was left
at 4 °C for 60 min after which the pH was adjusted to 9.5 using 1 N NaOH. The 5,10-CH2-H4folate was
purified at 4 °C on a 10-ml DEAE-Sephadex column pre-equilibrated with 50 mM (NH4)2CO3
and washed immediately before loading with 100 ml of 50 mM
2-mercaptoethanol. After loading, the column was washed with 20 ml of
50 mM 2-mercaptoethanol and then eluted with a 100-ml
gradient of 10-500 mM
(NH4)2CO3 containing 50 mM 2-mercaptoethanol (progress was monitored by absorbance
at 295 nm). Fractions (2 ml) were collected, and those containing
5,10-CH2-H4folate were identified by absorbance
and scintillation counting. Concentration and purity were estimated by
correlating the pteridine ring concentration (
295 = 30,000 M1
cm1) with the amount of radioactivity (1 mmol = 1.3 × 1011 dpm) in a given fraction.
Maximum purity was estimated at greater than 90%.
Samples were freeze-dried and stored under argon at 80 °C away
from light. Prior to use, the powder was resuspended in 50 mM 2-mercaptoethanol.
Gel Mobility Shift Assay
Assays were performed as described previously (24) using
purified protein extracts obtained from heparin-Sepharose
chromatography (25). Proteins were extracted from yeast strain
BWG1-7A, purified, and incubated with 40-base pair DNA fragments
harboring the glycine regulatory region of GCV2. These were
prepared, separated by electrophoresis, and analyzed as described
previously (13). The relative amount of DNA present in the DNA/protein
complexes was estimated by PhosphorImager analysis. To reduce
the effects of loading inconsistencies, these values were normalized to
the total DNA present in each lane.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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One-carbon metabolism comprises a complex set of reactions that contribute to many central biosynthetic pathways. Several approaches can be taken to identify components of the signaling system regulating the glycine response, although direct biochemical analyses are difficult because of the ability of the reactants to interchangeably supplement the pools of one-carbon-derived compounds and the relative instability of the H4folate derivatives. By combining extensive genetic analysis with biochemical approaches, several groups have provided clear indications of the flow of metabolites and the consequences of specific metabolic blocks (6, 15-17). In particular, Appling and co-workers have effectively combined genetics with the use of 13C NMR to provide a wealth of information on the biochemistry of one-carbon metabolic flow. Here, we have combined genetic and biochemical approaches to extend our previous finding that a folate species plays a role in signaling the glycine response of the GCV genes (13). Because glycine catabolism via the glycine decarboxylase reaction directly contributes to the one-carbon pool, we first tested whether a mutant lacking GDC activity could still elicit a glycine response.
The Glycine Response Is Independent of the Ability to Metabolize
Glycine--
We previously proposed that changes in an intermediate or
product of one-carbon metabolism (e.g. a derivative of
H4folate) provide the signal for the glycine response. To
test whether these changes require glycine catabolism, we transformed a
strain mutant for glycine cleavage activity with a normally regulated
GCV2::lacZ fusion (pRH2) and compared reporter
gene expression in minimal medium with that in minimal medium
containing 10 mM glycine (Fig. 2). A 3- to 4-fold increase in gene
expression on the addition of glycine was observed, which is typical of
the wild-type glycine response previously reported (10). Because this
strain is mutant for the GCV1 gene product encoding the
T-protein of the glycine decarboxylase complex and has no detectable
GDC activity, it is unable to use glycine as a sole one-carbon and
nitrogen source (26). Thus glycine catabolism has no direct effect on
H4folate-mediated one-carbon metabolism. To reconcile this
result with the data indicating that GCV transcriptional
signaling involves a H4folate intermediate, we sought a
one-carbon metabolic mutant unable to elicit a normal glycine response.
We reasoned that this strain would reveal how glycine acts to control
one-carbon metabolism and the balance of cellular H4folate
molecules.
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Mutation of Cytosolic Serine Hydroxymethyltransferase Disrupts the
Glycine Response of the GCV Genes--
The cytoplasmic SHMT reaction
is normally the principle route for the synthesis of one-carbon charged
H4folate derivatives using H4folate and serine
as substrates (Fig. 1 and Table I, reaction 1). Fig.
3 shows the effect of a mutation
eliminating cytosolic SHMT activity (shm2) on glycine
induction of expression of all three GCV genes. Expression
of each of the GCV genes was constitutively high in minimal
medium with and without glycine. Interestingly, the elevated level of
expression in this strain in minimal medium was about the same as that
of the wild type (ADE3 SHM2) grown in minimal medium
containing glycine. The mutation in this strain therefore elicited the
same increase in gene expression as seen for the addition of glycine to
the wild type.
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To determine whether the effect of this mutation was due to a
disturbance of one-carbon metabolism and not simply loss of the SHMT
enzyme, we supplemented the cytosolic one-carbon pool with formate.
From Fig. 1 and Table I it can be seen that catabolism of formate in
the cytosol should compensate for any one-carbon imbalance of an
ADE3 shm2 strain via the activity of C1-tetrahydrofolate synthase (ADE3 gene product; Ref. 7). This enzyme first
activates the one-carbon units derived from formate to
10-HCO-H4folate before conversion to
5,10-CH2-H4folate. In the wild-type strain
(ADE3 SHM2) addition of excess formate caused a decrease in
GCV2 gene expression (Fig.
4A). This reduction was even
more evident in the ADE3 shm2 strain, occurring at a lower
formate concentration. This effect was eliminated by the introduction
of a mutation in the ade3 gene. The lack of response to
formate by the ade3-130 shm2 strain shows that assimilation
of cytoplasmic one-carbon units from formate is required for the
down-regulation of gene expression. Interestingly, in these experiments
the effect on control of adding glycine to the wild type was similar to
that of the shm2 mutation. That is, there was an
ADE3-dependent reduction in GCV gene
expression induced by glycine on the addition of formate (Fig.
4B). We propose, therefore, that the presence of excess glycine acts in a similar manner to the shm2 mutation by
causing a reduction in the level of one of the cytoplasmic one-carbon charged H4folates and that this decrease then acts as the
signal to increase GCV gene expression.
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The Principle Effect of Mutation of Cytosolic SHMT Is a Reduction in Cytosolic 5,10-CH2-H4folate-- Two principle lines of evidence support the model that the ADE3 shm2 strain is limited in its cytosolic one-carbon pool. First, when grown in minimal medium, it has a slightly slower growth rate than the wild type that can be restored by the addition of adenine to the medium (Table IV). This is also consistent with the observation made by West et al. (9) that a strain that was unable to oxidize cytoplasmic 5,10-CH2-H4folate could not generate sufficient one-carbon units in the mitochondrion for purine synthesis to support wild-type growth. This strain recovered when adenine was added to the medium.
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Secondly, we used 13C NMR to follow the incorporation of one-carbon units from the mitochondrion into molecules derived from the cytoplasmic one-carbon H4folate pools. When cells are grown in the presence of exogenous glycine it is incorporated into choline and adenine both directly as an intact molecule and also as a one-carbon unit from the cytoplasmic pool. Appling and co-workers (6) have shown that monitoring the incorporation of [2-13C]glycine into choline and adenine allows an insight into the flow of mitochondrial one-carbon metabolism to the cytoplasm.
Fig. 5 shows a comparison of the relevant
regions of the NMR spectra for extracts of three mutants used for the
gene expression studies (ADE3 SHM2, ade3-130
SHM2, and ADE3 shm2). Fig. 5A shows that
there was an approximate 4-fold reduction in the incorporation of label
into choline in the ADE3 shm2 strain when compared with either the wild-type or the ade3-130 SHM2 strain. This
demonstrated that synthesis of choline was principally derived from
one-carbon units formed as a result of the cytoplasmic SHMT reaction.
Residual labeled choline (notably at the C-4 position) in this
strain was generated by the export of labeled formate from the
mitochondrion and its activation and interconversion by the
ADE3 gene product (Fig. 1 and Table I, reactions
4a-4c). In contrast, the mitochondrially derived one-carbon
metabolites contributed much more extensively to adenine biosynthesis
because the ade3-130 mutation virtually eliminates the
incorporation into all labeled positions (C-2, C-5, and C-8) of
adenine, whereas the shm2 mutation had a much lesser effect
(Fig. 5B). These data indicate that the majority of
one-carbon units from glycine catabolism in the mitochondrion are used
for purine synthesis, which is consistent with the requirement for
adenine for optimal growth of the ADE3 shm2 mutant outlined above. Because for purine synthesis the major drain on the one-carbon pool is at the level of 10-HCO-H4folate, very little enters
the 5,10-CH2-H4folate pool, which explains the
significant decrease in the amount of labeled choline in this strain.
These data indicated that the principle metabolic effect on folate
intermediates of the shm2 mutation was the reduction of
cytoplasmic 5,10-CH2-H4folate. The data also
show that 10-HCO-H4folate levels were not as greatly affected in the ADE3 shm2 mutant.
5,10-CH2-H4folate is therefore a prime
candidate as mediator of the glycine response; however other molecules
derived from it (e.g.
5-CH3-H4folate) could be candidates, and
10-HCO-H4folate remains a possible but unlikely one.
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Adenine and 10-HCO-H4folate Levels Do Not Influence the
Glycine Response--
The shm2 strain has a requirement for
adenine for optimal growth, and hence it is possible that reduction in
the cellular purine pool is responsible for the transcriptional effect.
To test this, we monitored the expression of the
GCV2::lacZ fusion in an ade3-130 SHM2
strain that through lack of cytoplasmic C1-tetrahydrofolate synthase
activity is unable to synthesize purine metabolites de novo
(27). Fig. 5B shows the NMR data for the ade3-130
SHM2 strain illustrating that there was no synthesis of adenine
from [2-13C]glycine, as occurred in the wild-type strain.
However, Fig. 6 illustrates that the
effect of this mutation on the glycine response was to augment the
extent of gene repression seen in the absence of glycine; the
fully induced level of expression was unaffected. The interpretation of
the data from this strain is complicated by the need to add exogenous
adenine to the medium; however, expression of the GCV genes
in the ade3-130 shm2 mutant was constitutively derepressed
even with the addition of exogenous adenine at the same concentration
(data not shown). This rules out purine nucleotides as the mediator(s)
of the glycine response because the regulation would be expected to be
similar between these mutants. A likely explanation for the observed
difference is that in the ade3-130 SHM2 strain
5,10-CH2-H4folate would be elevated because of
reduction in flow to purine synthesis, whereas in the ade3-130
shm2 strain it would be depleted. Hence
5,10-CH2-H4folate is a prime candidate as the
mediator.
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Interestingly, the purine auxotrophy of the ade3-130 SHM2 mutant is not thought to be a product of 10-HCO-H4folate deficiency (despite the lack of 5,10-CH+-H4folate cyclohydrolase activity of the C1-tetrahydrofolate synthase enzyme) (Fig. 1 and Table I, reaction 4b). West et al. (9) showed that an ade3-130 SHM2 strain does produce 10-HCO-H4folate by the action of an alternate (NAD-dependent) 5,10-CH2-H4folate dehydrogenase activity (Fig. 1 and Table I, reaction 5) coupled with spontaneous oxidation of 5,10-CH+-H4folate. The purine auxotrophy of the ade3 strain may therefore be due to a structural requirement for the C1-tetrahydrofolate synthase protein. This work also described a strain whose 10-HCO-H4folate (DAY4) levels were low because of its consumption for purine biosynthesis. There was no alteration of glycine control of GCV2 expression in this strain (see Fig. 8). Changes in 10-HCO-H4folate are therefore unlikely to be responsible for the changes in GCV gene expression. Whereas 5,10-CH2-H4folate remains the primary candidate, 5-CH3-H4folate derived from it has yet to be ruled out.
Decreased 5-CH3-H4folate Does Not Eliminate the Glycine Response-- 5,10-CH2-H4folate can be converted to 5-CH3-H4folate by the action of methylene-H4folate reductase (MTHFR) (Fig. 1 and Table I, reaction 7). In yeast this activity is encoded by the MET13 gene product that supplies one-carbon units for the synthesis of methionine (28). This pathway transfers the methyl moiety via S-adenosylmethionine to the C-4 position of choline (resonating at ~55.3 ppm in 13C NMR spectra; Table III). Fig. 5A shows that there was a significant reduction in labeling of this carbon in the ADE3 shm2 strain, indicating lowered levels of 5-CH3-H4folate in the cell. The possibility that changes in the 5-CH3-H4folate pool mediate the glycine response was therefore analyzed in a met13 strain lacking MTHFR activity.
The met13 strain retained the ability to elicit the glycine
response (Fig. 7). Furthermore, in
contrast to the constitutively high expression in the ADE3
shm2 mutant, the level of GCV2 expression in minimal
medium was markedly lower than in the wild-type strain (less
than 5%). This was not an artifact of methionine addition to the
growth medium because the ADE3 shm2 strain retained
constitutively high levels of expression under the same culture
conditions (data not shown). This result was not surprising because we
have previously noted that excess methionine caused a slight increase
in GCV2 expression (13). This experiment excludes the
possibility that low 5-CH3-H4folate is the
cause of increased GCV2 expression in the ADE3
shm2 strain. Moreover under all four conditions described above in
which the cytoplasmic 5,10-CH2-H4folate pool
would be in surplus, the level of GCV gene expression was
down-regulated. This effect was most dramatic in the met13
mutant when the cytoplasmic pool of
5,10-CH2-H4folate would be greatest because of
the block to synthesis of 5-CH3-H4folate and
because one-carbon metabolism via the SHMT reaction is directed more
toward the generation of methyl groups (methionine, choline, and dTMP)
than of purines (see "The Principle Effect of Mutation of Cytosolic
SHMT Is a Reduction in Cytosolic 5,10-CH2-H4folate"
above and "How Is 5,10-CH2-H4-folate Used in the Cell
to Maintain a One-carbon Metabolic Balance").
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How Could the Addition of Glycine to Cells Affect 5,10-CH2-H4folate Levels?-- The evidence above indicates that 5,10-CH2-H4folate is the molecule mediating the regulation of the GCV genes. Because metabolism of glycine via the GDC is not essential to elicit a glycine response, we sought to explain how the addition of glycine to cells could modulate the cytoplasmic concentration of 5,10-CH2-H4folate. In a detailed analysis of the regulation and catalytic mechanism of SHMT from rabbit liver and Escherichia coli, Schirch et al. (29) have shown that glycine in the range of 4-37.5 mM inhibited the conversion of serine to glycine and 5,10-CH2-H4folate. Hence end-product inhibition of the SHMT reaction is one explanation. More significantly, however, was the discovery that 5-CH3-H4folate or 5-HCO-H4folate binds SHMT in conjunction with glycine to form a dead-end complex that dramatically inhibits the activity of the enzyme (30). The 20-fold reduction of GCV gene expression in the met13 strain (Fig. 7) when grown in minimal medium may thus reflect the consequences of additive metabolic disturbances leading to a build up of cytoplasmic 5,10-CH2-H4folate levels: disruption of the use of 5,10-CH2-H4folate for 5-CH3-H4folate synthesis and loss of 5-CH3-H4folate inhibition of SHMT activity.
The second important control molecule, 5-HCO-H4folate, is produced from 5,10-CH+-H4folate by the SHMT from rabbit liver and E. coli in the presence of excess glycine (31). This reaction appears in E. coli to be the sole source of 5-HCO-H4folate, a metabolite that is also present in yeast but whose function until now was not known. Hence both end-product inhibition and the effect of glycine in conjunction with folate derivatives in dead-end complex formation would lead to a reduction in 5,10-CH2-H4folate synthesis on addition of glycine to the cell. This situation mimics that observed above in the ADE3 shm2 strain.
To What Extent Does the Above Dead-end Complex Formation Contribute
to the in Vivo Transcription Response to Glycine?--
GCV
gene expression was assayed in strain WHY1, mutant for the putative
5,10-CH+-H4folate synthetase gene
(YER183c). This mutant cannot convert 5-HCO-H4folate to
5,10-CH+-H4folate.2 Blocking
this reaction (Fig. 1 and Table I,
reaction 8) would lead to an
elevated intracellular concentration of 5-HCO-H4folate and
should therefore result in increased inhibition of SHMT, decreased 5,10-CH2-H4folate, and altered control of
GCV gene transcription. If this reaction were important in
the control system, then in the absence of exogenous glycine the mutant
should show increased GCV expression because of the very
high affinity of SHMT for intracellular glycine in the presence of
5-HCO-H4folate (30). The mutant should also show a further
response on addition of exogenous glycine because this is the third
component of the ternary complex. Both outcomes were observed as shown
in Fig. 8.
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5,10-CH2-H4folate Affects the Binding to
DNA of a Protein That Binds the Control Motif in the Promoter of
Glycine-responsive Genes--
We have previously identified by gel
mobility shift analysis a protein that binds to the glycine response
region mediating the transcriptional control of the GCV
genes. This binding was increased by the addition of
H4folate in vitro (13). Because the above data
implicated 5,10-CH2-H4folate in the control of GCV2 transcription, we synthesized the compound and used it
in gel mobility shift assays. Fig. 9
shows that addition of up to 2 µM
5,10-CH2-H4folate led to a decrease in the
binding of the protein to the specific DNA; this decrease, however, was
only to 60% of the initial level. Our previous work using
H4folate showed an increase in DNA/protein complex
formation but at a higher concentration than that measured here for
5,10-CH2-H4folate (50 µM-1
mM). Other commercially available folates (folic acid,
folinic acid, and 5-CH3-H4folate) had no effect
on complex formation over the range up to 1 mM (data not
shown).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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It is understood how metabolites flow through the various one-carbon metabolic pathways in the cell (4-9), but less is known on mechanisms controlling this flow. It has been shown here that there is an inverse relationship between cytoplasmic 5,10-CH2-H4folate levels and GCV gene expression. We propose that this is a control mechanism whereby the cell regulates the balance of serine, glycine, and one-carbon metabolism to meet conflicting demands. This is achieved by altering the balance of one-carbon metabolism between the cytoplasm and the mitochondrion to ensure a constant supply of one-carbon units to the important biosynthetic pathways such as those concerned with purine and pyrimidine biosynthesis. At the center of this control loop is regulation of the levels of cytoplasmic 5,10-CH2-H4folate.
How is 5,10-CH2-H4folate Used in the Cell to Maintain a One-carbon Metabolic Balance?-- Fig. 1 and Table I show the many ways in which 5,10-CH2-H4folate is involved in metabolism in the cytoplasm. Our data and those of many others show that the SHMT-catalyzed reaction serves as the principle source of this molecule, which is the primary contributor to the one-carbon pool (1). Pathways that are involved in the consumption of 5,10-CH2-H4folate include methionine synthesis via the action of the MTHFR enzyme and 5,10-CH+-H4folate synthesis via the methylene-H4folate dehydrogenase function of the C1-tetrahydrofolate synthase enzyme. When each of these functions was disturbed, alterations in the level of 5,10-CH2-H4folate signaled the need for the cell to synthesize one-carbon units from the alternate donor molecule glycine and thus shift the balance of one-carbon metabolism into the mitochondrion. From these data, we have been able to gain an insight into the regulation and flow of one-carbon metabolites in the cell under normal metabolic conditions.
Our results indicate that when cells are growing normally cytoplasmic 5,10-CH2-H4folate is used primarily for the production of methyl groups via methionine biosynthesis and secondly for the production of more oxidized C1-H4folate derivatives and metabolites such as purines. For purine synthesis there is therefore a balance between one-carbon metabolites derived from mitochondrial metabolism of glycine and the cytoplasmic conversion of serine to 5,10-CH2-H4folate. This model is supported by the fact that a ser1 allele (ade9, defective in its ability to produce serine from glycolytic intermediates) was originally identified as an adenine auxotroph when serine was limiting (32). At low serine levels, adenine limited growth of this strain, indicating that serine can be channelled preferentially into fulfilling the requirements of the cytoplasmic 5,10-CH2-H4folate pool for 5-CH3-H4folate and subsequently methionine biosynthesis at the expense of the more oxidized C1-H4folates required for purine biosynthesis. However, whereas flow from the cytoplasmic 5,10-CH2-H4folate pool for synthesis of purines may have lower priority, it does still play a role in normal cell growth.
Exogenous adenine was required for the shm2 strain to grow at an optimal rate, indicating that the mitochondrial one-carbon pathway cannot fully maintain the requirements for purine biosynthesis. However, Pasternack et al. (6) have shown that at least 25% of one-carbon units for purine synthesis are mitochondrially derived. They concluded that the normal pathway for glycine-derived one-carbon units is through the synthesis in the mitochondrion of formate, which is exported to the cytosol for purine synthesis. Thus the normal balance for flow of one-carbon units derived from serine is toward methyl group biogenesis, whereas glycine derived from serine in the mitochondrion supplements the supply of one-carbon units for purine biosynthesis.
Under What Circumstances Is This Flow Altered?--
Under
physiological conditions the cell needs to adjust the synthesis and
degradation of serine and glycine to fulfil the requirements for
protein synthesis as well as one-carbon metabolism and hence
needs to regulate the balance of one-carbon flow. We explored
two situations in which the use of glycine for one-carbon metabolism
was enhanced (shown by an increase in GCV gene expression): when cells were grown with exogenous glycine and when the
SHM2 gene was disrupted. These two situations resulted in a
similar reduction of the levels of cytoplasmic
5,10-CH2-H4folate, which forms the basis for
the signal to increase GCV gene transcription. Interestingly, in a recent study on the global transcriptional effects
of the DNA-damaging reagent methyl methanesulfonate, the transcription
of GCV2, GCV1, and GCV3 was increased
12.5-, 7.5-, and 2.6-fold, respectively (33). According to the
regulatory mechanism proposed above, a decrease in
5,10-CH2-H4folate levels due to an increased
demand on cytoplasmic one-carbon metabolism for nucleotide biosynthesis
would signal a need to increase GCV gene transcription.
Because the principle route for one-carbon unit synthesis is through
the cytoplasmic SHMT, it was not surprising that loss of its function
resulted in a decrease of 5,10-CH2-H4folate levels. It was less apparent, however, how addition of glycine also
affected the levels. We have shown that the side reaction of the
cytoplasmic SHMT described by Schirch and co-workers (29-31), which
results in its inhibition by excess glycine, has an important physiological role. We therefore propose a model of how cells control
the flow of metabolites through the one-carbon pathways (Fig.
10).
|
Inhibition of the activity of cytoplasmic SHMT alters the cytoplasmic 5,10-CH2-H4folate level, which modulates expression of genes involved in one-carbon metabolism, including those in the glycine cleavage complex. Transcription of the GCV genes is repressed by high levels of cytoplasmic 5,10-CH2-H4folate, a situation that signals that alternate sources of one-carbon units are not required. When cytoplasmic 5,10-CH2-H4folate levels are low, however (either through one-carbon "starvation" or the presence of excess glycine, causing production of 5-HCO-H4folate, which inhibits SHMT activity), the cell calls upon glycine catabolism in the mitochondrion to supplement its one-carbon requirements.
These conclusions also explain the unexpected observation by Pasternack et al. (6) of an apparent interdependence of two physically compartmentalized enzyme activities. Yeast ade3-30 mutants lacking the synthetase activity of cytoplasmic C1-tetrahydrofolate synthase are unable to activate formate to 10-HCO-H4folate for purine synthesis. In a particular genetic background, ade3-30 cells could not use glycine to replace serine to supplement their one-carbon pool. Under normal conditions, glycine metabolized by the GDC and mitochondrial SHMT produces serine. This is exported to the cytoplasm, where cytoplasmic SHMT acts to fulfil cytoplasmic one-carbon metabolic requirements. We propose that when glycine accumulates, flow through cytoplasmic SHMT is reduced (by glycine inhibition), cytoplasmic 5,10-CH2-H4folate levels drop, and one-carbon metabolism is channelled to the mitochondrion. If reductive synthesis of 5,10-CH2-H4folate is blocked by the ade3-30 mutation and glycine inhibits production in the oxidative direction, the cells are effectively starved for cytoplasmic one-carbon-loaded H4folate molecules. Lack of mitochondrial C1-tetrahydrofolate synthase (encoded by MIS1) activity could reverse this phenotype. The mis1 mutation may act to increase production of serine in the mitochondrion (because of an inability to oxidize 5,10-CH2-H4folate) and thus increase serine export to the cytoplasm. Excess serine may partially overcome the SHMT block, thus supplying one-carbon-loaded H4folate molecules to the cytoplasm.
By What Mechanism Does 5,10-CH2-H4folate Effect Transcriptional Change?-- Our previous work demonstrated that H4folate could directly interact with a protein binding to the DNA sequence harboring a glycine response control sequence (GRR) (13). This interaction altered its ability to bind but only occurred at H4folate levels above those found in the cell. Here, we describe an interaction with 5,10-CH2-H4folate that also altered the ability of the protein to bind the GRR, but at lower and physiologically significant concentrations. Addition of high levels of 5,10-CH2-H4folate did not, however, completely inhibit binding of the protein to the GRR.
Because glycine induction of the GCV genes has been shown to be coordinately regulated, it is interesting to note that control of GCV2 and GCV3 transcription is regulated by repression, whereas that of GCV1 is regulated by activation (13).3 These data indicate that signaling may be mediated by 5,10-CH2-H4folate altering the activity of the DNA-binding protein rather than by affecting its ability to bind DNA and that changes in in vitro binding reactions were the results of a conformational change in the protein due to binding of the folate. In vivo, the signaling mechanism may be further complicated by a very low concentration of free folates present in cells because of substrate channelling of folates between enzymes (reviewed in Ref. 34). The most likely explanation involves the existence of a protein that can detect 5,10-CH2-H4folate levels, possibly by association with the cytoplasmic SHMT enzyme and dictated by the presence of the competing folates that regulate SHMT activity. By dissociation and subsequent nuclear localization, the protein may function to modulate gene transcription. Purification and identification of the transcription factor that binds the GRR and effects transcriptional activity will help answer these questions. Work on identification of the DNA-binding protein is under way in our laboratory.
Furthermore, because 5,10-CH2-H4folate is
central to one-carbon metabolism, we predict that this control system
forms the basis for a new metabolic transcriptional regulon. We are
currently investigating the genome-wide transcriptional consequences of alterations to the cytoplasmic
5,10-CH2-H4folate pool.
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ACKNOWLEDGEMENTS |
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We thank Dr. Dean Appling (Biochemical Institute, Univ. of Texas, Austin) for the generous provision of yeast strains WHY1 and DAY4 and yeast strains used to generate new strains for this study. We also thank Dr. Leonard Guarente (Department of Biology, Massachusetts Institute of Technology) for providing strain BWG1-7a.
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FOOTNOTES |
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* This work was supported by Australian Research Council Grant A10007007.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by an Australian Postgraduate Award.
§ Present address: Dept. of Microbiology, Columbia University, New York, NY 10027-6902.
To whom correspondence should be addressed. Tel.:
61-2-9385-2089; Fax: 61-2-9385-1050; E-mail:
i.dawes@unsw.edu.au.
Published, JBC Papers in Press, June 27, 2000, DOI 10.1074/jbc.M004248200
2 W. Holmes and D. Appling, personal communication.
3 M. D. Piper, S. P. Hong, and I. W. Dawes, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: H4folate, tetrahydrofolate; SHMT, serine hydroxymethyltransferase; GDC, glycine decarboxylase multienzyme complex; MTHFR, 5,10-methylene-tetrahydrofolate reductase; GRR, glycine regulatory region of DNA required for the glycine response.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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