(Received for publication, July 24, 1996, and in revised form, October 28, 1996)
From the § Department of Nutritional Sciences,
University of California, Berkeley, California 94720, the
Division of Nutritional Sciences, Cornell University,
Ithaca, New York 14853, and the ¶ Wadsworth Center,
Albany, New York 12201
The human mitochondrial serine
hydroxymethyltransferase (mSHMT) gene was isolated, sequenced, and
characterized. The 4.5-kilobase gene contains 10 introns and 11 exons,
with all splice junctions conforming to the GT/AG rule. The 5 promoter
region contains consensus motifs for several regulatory proteins
including PEA-3, Sp-1, AP-2, and a CCCTCCC motif common to many genes
expressed in liver. Consensus TATA or CAAT sequence motifs are not
present, and primer extension and 5
-rapid amplification of cDNA
ends studies suggest that transcription initiation occurs at multiple
sites. The mitochondrial leader sequence region of the deduced mRNA
contains two potential ATG start sites, which are encoded by separate
exons. The intervening 891-base pair intron contains consensus promoter elements suggesting that mSHMT may be transcribed from alternate promoters. 5
-Rapid amplification of cDNA ends analysis
demonstrated that the first ATG is transcribed in human MCF-7 cells.
However, transfection of Chinese hamster ovary cells deficient in mSHMT activity with the human mSHMT gene lacking exon 1 overcame the cell's
glycine auxotrophy and restored intracellular glycine concentrations to
that observed in wild-type cells, showing that exon 1 is not essential
for mSHMT localization or activity and that translation initiation from
the second ATG is sufficient for mSHMT import into the mitochondria.
Mitochondrial SHMT mRNA levels in MCF-7 cells did not vary during
the cell cycle and were not affected by the absence of glycine, serine,
folate, thymidylate, or purines from the media.
Folates function as a family of cofactors by carrying one-carbon units that are required for the synthesis of glycine, thymidylate, purines, methionine, and numerous methylation reactions in mammalian cells. Serine is the major source of one-carbon units that are generated in a reaction catalyzed by the enzyme serine hydroxymethyltransferase (SHMT).1 Alternatively, one-carbon units can also be generated from glycine in cells that contain a glycine cleavage activity. SHMT is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion of serine and H4PteGlu to glycine and 5,10-CH2-H4PteGlu. SHMT is present in both the mitochondria (mSHMT) and the cytoplasm (cSHMT) in mammalian cells. The human SHMT cDNAs encoding the two isozymes have been isolated and the genes localized to chromosomes 12q13 and 17p11.2, respectively (1). Currently, the metabolic role of the individual SHMT isozymes is not clearly understood. Chinese hamster ovary cells lacking mSHMT activity are auxotrophic for glycine, suggesting that the mitochondria are the primary site of glycine synthesis, whereas the enzymes responsible for thymidylate, purine, and methionine synthesis are present in the cytoplasm (2). The central role of SHMT isozymes in producing one-carbon-substituted folate cofactors has suggested that the regulation of these enzymes may influence cell growth and proliferation and that they may be targets for the development of antineoplastic agents.
Total SHMT activity and the concentration and metabolism of serine and
glycine varies among tissues, reflecting the different roles of these
amino acids in different organs (3). This effect is most pronounced in
the brain, where it has been demonstrated that there is a direct
correlation between SHMT activity and glycine concentration in
different regions of rat brain (4). SHMT activity may also be
developmentally regulated as SHMT-specific activity is 2-fold higher in
the the optical lobe of the rhesus monkey neonate and adult compared
with the fetus (5). There is evidence that SHMT is hormonally regulated
as SHMT activity is elevated in the uterus after injection of
17-estradiol to ovariectomized rats, with a 6-fold acceleration of
[3-14C]serine incorporation into purines, whereas
testosterone increases the specific activity of SHMT in the prostate
(6). SHMT may also be controlled by nutrient availability as SHMT
activity is increased 50% in folate-deficient versus
folate-supplemented chickens (7). Total SHMT enzyme activity has also
been demonstrated to be increased in tumor cells (8).
Several studies have suggested that mSHMT is primarily responsible for glycine synthesis in the cell (2, 9). Revertants of mutant Chinese hamster ovary (CHO) cells that lack mSHMT activity showed a correlation between SHMT levels, intracellular glycine concentrations, and protein synthesis rates (2). While SHMT enzyme activity and one-carbon flux display both developmental, nutritional, and tissue-specific regulation, the metabolic significance of these changes has been difficult to interpret as most of these studies have been performed with crude homogenates that did not distinguish between mitochondrial and cytoplasmic SHMT activities. In order to better understand the differential metabolic roles of the SHMT isozymes, we have cloned and structurally characterized the human mSHMT gene and have commenced studies to determine the factors responsible for regulating its endogenous expression.
PteGlu, reduced folates, amino acids,
nucleosides, nucleotides, alcohol dehydrogenase, pyruvic acid, and NADH
were obtained from Sigma. [32P]dCTP (800 Ci/mmol) and
[2-3H]glycine (43.8 Ci/mmol) were obtained from DuPont
NEN. Restriction and modifying enzymes were obtained from Boehringer
Mannheim, Promega, or New England BioLabs. Taq polymerase
was from Perkin-Elmer. DMEM, a modification of -minimum essential
medium lacking serine, glycine, folic acid, thymidylate, and
hypoxanthine, was purchased from JHR Biosciences. All other materials
were of high quality and obtained from various commercial vendors.
MCF-7 human breast cancer cells (HTB22) were obtained from the American Type Culture Collection; wild-type CHO cells (WTT2) were obtained from Dr. Sharon Krag, Johns Hopkins University; GlyA, a CHO cell mutant lacking mSHMT activity, was obtained from Dr. Larry Thompson, Lawrence Livermore Labs. Cells were determined to be free of mycoplasm contamination by fluorescent DNA staining using 4,5-diamidino-2-phenylindole (Boehringer Mannheim) according to the manufacturer's instruction. Cells were cultured in 100-mm dishes containing 12 ml of media and incubated at 37 °C in a 5% CO2 atmosphere. Wild-type CHO and MCF-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum (Hyclone Inc.) and 20 nM folinic acid, whereas culture media for GlyA were supplemented with 200 µM glycine. For some experiments, the fetal bovine serum was dialyzed (dFBS) against 10 volumes of phosphate-buffered saline for 24 h with buffer changes every 4 h to deplete serum glycine and folate. Media were also supplemented with combinations of glycine (200 µM), hypoxanthine (20 µM), thymidine (20 µM), and serine (200 µM) for studies concerning effects of nutrient availability on mSHMT mRNA levels in MCF-7 cells. MCF-7 cells were synchronized using Lovastatin and mRNA isolated from the cells at 4-h intervals following release of the cell cycle block with mevalonic acid as described previously (10).
Library ScreeningA human genomic library (Lambda FIXTM II
vector (Stratagene)) generated from the lung fibroblast cell line W138
was screened (3 × 10 6 plaques) with
[32P]dCTP-labeled oligonucleotides generated using the
Random Primed DNA labeling kit (Boehringer Mannheim) and the human
mSHMT cDNA as the template. Following plaque purification, five
recombinants were obtained, two of which were identical and contained
the mSHMT gene as assessed by restriction enzyme mapping and Southern
analysis. The genomic insert (12 kb) was excised from the lambda vector using NotI and subcloned into pBluescript II KS+
vector (Stratagene) for sequence analysis. The fragment was digested further to obtain a HindIII-NotI fragment (4 kb)
that contained exons 2-11 of the mSHMT gene but lacked 121 nucleotides
from the 3-untranslated region reported in the mSHMT cDNA (1); and a HindIII-HindIII fragment (1 kb) that contained
exon 1 and 314 nucleotides of 5
-untranslated nucleotide sequence.
The mSHMT gene was sequenced from double-stranded template by dideoxynucleotide chain termination methods using 35S-dATP and the Sequenase II kit (U.S. Biochemical Corp.) or using an Applied Biosciences Model 373A automated DNA sequence analyzer located at the Biotechnology Center, Cornell University. The entire SHMT gene sequence was determined and verified by sequencing both DNA strands.
Transfection of GlyA CellsA 4.0-kb HindIII-NotI fragment containing the human mSHMT gene lacking exon 1 was subcloned into the mammalian expression vector pCep 4 (Invitrogen) which contains the hygromyocin resistance gene. The construct was linearized with NarI and introduced into GlyA cells (2 × 107 log phase cells) by electroporation using a Gene-Pulser I (Bio-Rad) (500 V, 25 microfarads). The electroporation solution contained 20 µg of the construct in 0.5 ml of 7 mM sodium phosphate buffer, pH 7.5, containing 272 mM sucrose and 1 mM MgCl2 in 0.4-cm cuvettes. Following electroporation, the cells were incubated for 10 min on ice and cultured in six 100-mm plates per cuvette. The cells were incubated as described above in DMEM supplemented with 100 µM glycine for 40 h. Stable transfectants were selected in DMEM containing 500 µg/ml hygromyocin and 20 nM folinate. Resistant colonies were isolated and maintained in DMEM containing 50 µg/ml hygromyocin and 20 nM folinate.
Determination of SHMT mRNA Levels by RT-PCRTotal
cellular RNA was isolated from MCF-7 and CHO cells using the guanidine
hydrochloride method (11) and converted to cDNA using the First
Strand Synthesis kit (Clonetech). A competitive reverse
transcriptase-polymerase chain reaction (RT-PCR) method, based on the
mimic strategy (Clonetech), was used to measure mSHMT mRNA levels.
A competitive internal standard with identical primer binding sites
used to amplify 300 bp of the mSHMT cDNA was generated by
amplifying 500 bp of v-erbB with two composite primers
(40-mer) (5-TGTTCCGGGAGTACTCCCTGCGCAAGTGAAATCTCCTCCG-3
) and
(5
-GGAACTGTCGAGAAGTTAAGTTGAGTCCATGGGGAGCTTT-3
). The first 20 nucleotides of the 40-mer were complementary to mSHMT, whereas the
following 20 nucleotides were complementary to v-erb-B. The
540-bp internal standard was synthesized and purified following protocols described by the manufacturer. The primers used to amplify mSHMT cDNA and the internal standard were
(5
-TGTTCCGGGAGTACTCCCTG-3
) and (5
-GGAACTGTCGAGAAGTT-3
). The primer
binding sites are located in exons 8 and 9 thereby avoiding possible
amplification of genomic DNA. The primers used to create the
glyceraldehyde-3-phosphate dehydrogenase internal standard were
(5
-ACCACAGTCCATGCCATCACCGCAAGTGAAATCTCCTCCG-3
) and
(5
-TCCACCACCCTGTTGCTGTATTGAGTCCATGGGGAGCTTT-3
), and the primers
used to amplify glyceraldehyde-3-phosphate dehydrogenase and its
internal standard were (5
-TCCACCACCCTGTTGCTGTA-3
) and (5
-ACCACAGTCCATGCCATCAC-3
). RT-PCR experiments were performed by
converting total RNA (2 µg) to cDNA using the first strand synthesis kit (Clonetech) and protocols suggested by the manufacturer. Mimic internal standard was added to 0.4 µg of cDNA at several concentrations until a target/standard ratio of 1.0 was achieved (1 × 10
2 to 1 × 10
5
amol/reaction). Target cDNA and the internal standard were
amplified with Taq polymerase using
32P-end-labeled primers. The cycling parameters were
94 °C for 45 s, 65 °C for 45 s, and 72 °C for 1 min
for a total of 28 cycles. Internal standard and target-amplified DNA
fragments were separated on a 1.8% agarose gel, transferred to Zeta
Probe membrane (Bio-Rad), and quantified using a Molecular Dynamics
PhosphorImaging system. The mSHMT levels were reported relative to
mimic concentration required to achieve a mSHMT/mimic ratio of 1.0. Glyceraldehyde-3-phosphate dehydrogenase mRNA levels were also
quantified for each sample in control experiments.
Primer extension was performed at 70 °C
using Tth DNA polymerase in the presence of Mn2+
using protocols described by the manufacturer (Promega) with 5 ng of
the primer (5-GCCGCCCAAAACAAAGAGAAGTACAGCATCGCAACTCGG-3
) that
corresponds to bases 29 through
10 of the mSHMT gene. 5
-RACE products were generated, and 15 products were cloned and sequenced using a kit from Life Technologies, Inc. following protocols
recommended by the manufacturer.
A sensitive radioassay was used to measure relative changes in mSHMT and cSHMT enzyme activity in cell extracts from as few as 1 × 106 cultured cells as described previously (12). The assay is based on the observation that H4PteGlu accelerates the SHMT-catalyzed exchange of the pro-2S proton of glycine, and 5-CHO-H4PteGlu inhibits this reaction (13). In a typical experiment, 5 × 106 CHO cells in 1.0 ml of 10 mM potassium phosphate buffer, pH 7.5, containing 300 mM sucrose were disrupted by 50 strokes of a Wheaton pestle A (tight fitting), and the cytoplasmic and mitochondrial fractions were prepared as described previously (10). Lactate dehydrogenase and glutamate dehydrogenase activities were determined in each fraction to correct for mitochondrial breakage and cytoplasmic fraction contamination (14). The isolated mitochondria were lysed in 200 µl of 20 mM sodium phosphate buffer, pH 7.2, 10 mM 2-mercaptoethanol, 0.5% Triton X-100. mSHMT activity was measured by diluting 40 µl of the mitochondrial fraction to 500 µl with 10 mM potassium phosphate buffer, pH 7.5, 10 mM 2-mercaptoethanol, and glycine such that the final glycine concentration was 1 mM with a specific activity of 2 × 106 dpm/µmol. The reaction was initiated by the addition of 100 pmol of H4PteGlu and incubated at 37 °C for 30-120 min. Control reactions were performed to correct for background exchange by the addition of 100 pmol of 5-CHO-H4PteGlu in lieu of H4PteGlu. The reaction was terminated by the addition of 3 ml of 50 mM HCl (4 °C), and the solution was passed through a column containing 0.8 ml of Dowex 50 AG (Bio-Rad) to remove radiolabeled glycine. The column was washed with an additional 2 ml of 50 mM HCl, and the tritiated water was collected and quantified by scintillation counting. Control reactions containing 5-CHO-H4PteGlu exhibited less than 4% proton exchange compared with the H4PteGlu-catalyzed exchange reaction. All assays were performed in duplicate, and all experiments were repeated at least twice. Mitochondrial fractions were determined to be free of lactate dehydrogenase activity and were not corrected for cSHMT contamination. Protein concentrations were determined by the method of Lowry as described previously using bovine serum albumin as a standard (15).
Amino Acid AnalysisIntracellular free amino acids were
isolated from cultured cells by a modification of the procedure
described previously (16). Cells were cultured in 100-mm plates
containing 10 ml of -minimum essential medium/10% dFBS lacking
glycine for 36 h. The media was removed by aspiration, and the
cells were washed five times with 10 ml of phosphate-buffered saline.
The cells were harvested using a cell scraper after the addition of 300 µl of 5% trichloroacetic acid. Cell extracts were transferred to
microcentrifuge tubes, vortexed for 30 s, and centrifuged at
10,000 × g for 10 min. The trichloroacetic acid was
removed from the supernatant by extracting three times with an equal
volume of water-saturated diethyl ether. The aqueous solution
containing the free amino acids was vacuum-dried. The free amino acids
were quantified following o-phthaldialdehyde derivatization
at the Cornell Biotechnology Analytical/Synthesis Facility.
Intracellular serine and glycine concentrations were normalized to
intracellular valine or isoleucine concentrations as internal
standards.
A gt11 Fix II
library was screened as described under "Experimental Procedures,"
and five recombinants were obtained, two of which were found to be
identical by restriction enzyme mapping and Southern analysis. The
identical recombinants were restriction mapped, and the gene was
localized to a 4.0-kb HindIII-NotI fragment and a
1.0-kb HindIII-HindIII fragment. Both fragments
were sequenced. The 1-kb fragment contained exon 1 and 314 nucleotides
of 5
-flanking sequence. The 4.0-kb fragment contained the remainder of
the coding region of the mSHMT gene but lacked the terminal 121 nucleotides of the 3
-untranslated region present in the cDNA. The
introns were sequenced, and the mSHMT gene nucleotide sequence has been deposited in the EMBL/Genbank Data Libraries (accession number U23143[GenBank]).
Sequence analysis and restriction mapping of the remaining three clones
suggested that they were not the mSHMT or cSHMT genes and are currently
being investigated. The coding sequence of the gene was in agreement
with the previously published cDNA sequence (1) with two
exceptions. Codon 281 in the cDNA contains the nucleotide C in the
number 1 position, whereas the gene contains the nucleotide T resulting
in a Phe to Leu change. Both the human and rabbit cSHMT enzymes contain
a Leu in this position, whereas the rabbit mitochondrial protein
contains a Phe at this position. The third position of codon 293 coding
for Leu in the cDNA contains a T, whereas the gene contains the
nucleotide G in this position; however, both codons code for Leu.
The gene contains 10 introns and 11 exons spanning about 4.5 kb (Table
I). The entire nucleotide sequence was determined, and
all intron/exon splice junctions conform to the gt-ag rule (17). All
introns are relatively small ranging from 86 to 891 bp. The 3 splice
sites do not show any preference for thymidine at the
4 position as
has been found for mitochondrial aspartate aminotransferase and other
nuclear-encoded mitochondrial proteins (18). The gene contains a 2-fold
higher occurrence of type 2 intron splice junctions than the average
for mammalian genes (19), with 50% type 0 introns, 20% type 1 introns, and 30% type 2 introns.
|
Previous
studies have suggested that mSHMT is located in the mitochondrial
matrix (20), and the primary sequence of the rabbit liver mSHMT has
been determined by amino acid sequencing (21). Amino-terminal
sequencing of the rabbit liver mSHMT enzyme did not yield a start
methionine suggesting either that the mitochondrial import presequence
had been cleaved or the enzyme may have been subjected to proteolysis
at its amino terminus during purification (21). Fig. 1 shows the
amino-terminal residues of the human mSHMT primary sequence, deduced
from the nucleotide sequence obtained by 5-RACE analysis, aligned with
the rabbit liver mSHMT protein sequence. Amino-terminal residues 1-8
in the rabbit liver primary sequence align to residues 30-37 in the
human mSHMT (Fig. 1), suggesting that residues 1-29 in
the human mSHMT primary sequence represent a mitochondrial import
presequence. The presequence is rich in the amino acids Arg, Leu, and
Ser which are favorable for mitochondrial import. Analysis of the mSHMT
presequence suggests that it can form an amphipathic
-helix with the
positively charged Arg residues and hydrophobic residues residing on
opposite sides of the helix, typical of classical mitochondrial import
presequences (22). The translational initiation site contains a near
consensus translation initiation sequence (23), with a G in the
3 and
6 positions. However, the mitochondrial leader peptide contains an
internal methionine residue at position 22. The two methionine codons
are separated by an 891-bp intron that could potentially serve as an
alternate promoter as seen in the rat glucokinase gene (24). The second
AUG codon is also contained within a near consensus translation
initiation sequence, with the nucleotide A in the
3 position and a G
in the +4 position and
6 positions.
To determine if intron 1 serves as an alternate promoter, 5-RACE
products were generated from human MCF-7 cell mRNA. Fifteen RACE
products were sequenced, and all included the initiation codon present
in exon 1. If intron 1 served as an alternate promoter in these cells,
primers complementary to the 3
-untranslated region of intron 1 and to
internal cDNA sequences would be expected to generate PCR products
using MCF-7 cell cDNA as a template. However, no such amplification
products were detected. Intron 1 does not serve as a promoter region in
human MCF-7 cells. However, these studies do not preclude the
possibility that this region may serve as a promoter in other cell
types.
To determine
if translation initiation could occur from the second ATG in the mSHMT
mRNA and result in the synthesis of a functional mSHMT protein,
GlyA cells were transfected with the human mSHMT gene
lacking exon 1 and under the control of the cytomegalovirus immediate
early enhancer/promoter in the mammalian expression vector pCep4
(Invitrogen). Five stable transfectants (GlyA-human-mSHMT) were obtained by selection in media containing hygromycin, and colonies
were maintained for over 12 months in the selection media. Fig.
2 shows RT-PCR analysis of total RNA isolated from WTT2, GlyA, and GlyA-human-mSHMT cells using primers
designed to amplify 300 bp of the human mSHMT cDNA as described
under "Experimental Procedures." A fragment of the expected size
was amplified from GlyA-human-mSHMT, WTT2, and
GlyA cDNA. This suggests that CHO mSHMT mRNA
contains sufficient nucleotide identity to the human mRNA to permit
amplification by the human-specific primers and that the human gene is
correctly spliced in CHO cells. All five stable
GlyA-human-mSHMT transfectants were enriched with
mSHMT mRNA compared with WTT2 cells as determined by RT-PCR. The
GlyA-human-mSHMT transfectants contained 10-20 amol of
human mSHMT mRNA/mg of total RNA. In comparison, MCF-7 cells
contain 1 attomole mSHMT mRNA/mg total RNA.
Expression of the mSHMT gene lacking exon 1 in GlyA cells eliminated the glycine auxotrophy in all transfectants. All CHO cell lines were characterized by measuring SHMT activity and by analyzing intracellular amino acid concentrations (Table II) as described under "Experimental Procedures." Intracellular serine is elevated 9-fold in GlyA cells compared with WTT2 cells, whereas glycine is nearly undetectable in GlyA cells, consistent with the absence of mSHMT activity. GlyA-human-mSHMT glycine levels were elevated about 10-fold compared with GlyA cells and were similar to WTT2 cells, whereas intracellular serine levels in the transfectants were decreased 50%. These changes in intracellular amino acid concentrations occurred despite only minor increases in mSHMT activity (Table II). WTT2 cells displayed 2- and 50-fold higher total and mitochondrial SHMT activities, respectively, compared with GlyA cells when assayed using the [3H]glycine exchange assay. Expression of the partial human mSHMT gene lacking exon 1 in GlyA cells resulted in less than a 10% increase in total SHMT activity but nearly a 3-fold increase in mSHMT activity. These results suggest that expression of the human mSHMT gene lacking exon 1 in GlyA cells is sufficient for mSHMT expression and mitochondrial import. These results also suggest that only a 2-3-fold increase in mSHMT activity is required to alleviate the glycine auxotrophy of GlyA and that mSHMT activity can be reduced greater than 90% without compromising intracellular glycine concentrations. It is also apparent from whole cell SHMT activity measurements that expression of the partial human mSHMT gene did not result in measurable increased SHMT activity in the cytoplasm. Expression of mSHMT in the cytoplasm would not be expected to overcome the glycine auxotrophy as overexpression of cSHMT in GlyA cells does not overcome the glycine requirement.2
|
The transcriptional
initiation sites of the mSHMT gene were determined by primer extension
analysis of total cellular RNA isolated from MCF-7 cells. Multiple
transcription initiation sites of equal intensities were observed (Fig.
3) which is consistent with the absence of TATA or
CAAT-like sequences (Fig. 4). The 5 promoter region of
the mSHMT gene contains consensus DNA recognition sequence for Sp-1
(
290 to
284), AP-2 (
249 to
241), HC3 (
150 to
144), and PEA3
(
239 to
245). A zeste-white sequence (
184 to
190) is present
and may account for some of the observed changes in mSHMT expression
during development. Additionally, a CCCTCCC motif (
128) is present
that is common to a number of genes that are expressed predominantly in
the liver (25).
The sequence 5 to the second translational initiation site (intron 1 region) also does not contain any TATA or CAAT-like sequences. A
consensus core sequence for CTF/NF1 is located on the antisense strand
(757-762) (20). Consensus sequence motifs were also found for PuF-1,
PEA3, Sp-1, UBP1, octB2, Adh1, TRE, GRE, FSE2, HC3, and IE1. The
possible role of these sequences in regulating the endogenous
expression of the mSHMT is currently under investigation.
The expression of many genes
involved in DNA synthesis, including those encoding
folate-dependent enzymes such as dihydrofolate reductase
and thymidylate synthase, is enhanced upon entry into the S phase of
the mammalian cell cycle. Both dihydrofolate reductase and thymidylate
synthase contain a cis-acting element that binds the transcription
factor E2F, and E2F binding is sufficient for growth-regulated promoter
activity at the G1/S phase boundary (26). MCF-7 mSHMT
mRNA levels were measured by RT-PCR through the cell cycle to
determine if the mSHMT gene is co-regulated with other genes involved
in DNA synthesis. Cells were synchronized with Lovastatin, an inhibitor
of 3-hydroxy-3-methylglutaryl-coenzyme A reductase. Lovastatin blocks
the cell cycle reversibly in G1, and the block can be
released by mevalonic acid, the product of the reductase reaction.
Lovastatin is preferable to other cell cycle blocking agents as DNA
synthesis is inhibited over 96% and cells remain synchronized for at
least three cycles after release of the block. The percentage of cells
in S-phase was determined by [3H]thymidine incorporation
and by histone H4 expression as described (10). No variations in
mitochondrial SHMT or control glyceraldehyde-3-phosphate dehydrogenase
mRNA levels were observed (Fig. 5) suggesting that mSHMT mRNA levels are not cell cycle-regulated.
Nutrient Control of SHMT Expression in MCF-7 Cells
A previous study showed that SHMT activity in chicks was increased in folate deficiency (7), suggesting that folate status and perhaps other nutrients may play a role in mSHMT expression. The hydroxymethyl group of serine is incorporated into purines, methionine, and thymidylate, and the availability of these products of one-carbon metabolism may regulate mSHMT expression. MCF-7 cells were cultured in DMEM, 10% dFBS with and without purines, thymidine, methionine, glycine, or serine as described under "Experimental Procedures." The omission of purines, thymidine, methionine, glycine, serine, or combinations thereof did not change SHMT mRNA levels after maintenance in this media for 72 h (data not shown) as determined by competitive RT-PCR. Likewise, mSHMT mRNA levels were unchanged in folate-deficient MCF-7 cells passaged for over 7 weeks in media lacking folic acid. These results suggest that mSHMT gene transcription is not influenced by nutrient status associated with folate-mediated one-carbon metabolism in MCF-7 cells.
The differential metabolic role of the two SHMT isozymes in folate-dependent anabolic pathways is not understood, but mSHMT activity has been suggested to be the primary source of intracellular glycine (2, 9). In order to understand the differential metabolic roles of the SHMT isozymes, we have cloned the mSHMT gene and initiated studies to determine the factors and mechanisms that cells use to regulate the endogenous expression of the gene. Recent studies have demonstrated that elimination of mitochondrial folate pools in CHO cells results in a glycine auxotrophy (27, 28) and that CHO cell glycine auxotrophs lacking mSHMT activity have an elevated intracellular serine concentration but are not able to accumulate intracellular glycine (9). These data suggest that mSHMT is primarily responsible for glycine synthesis and that cSHMT is not effective in catalyzing the formation of glycine from serine even in the presence of elevated serine concentrations (9). However, the possibility that the cell lines had additional mutations that may have been responsible for the observed metabolic disturbances could not be eliminated. Our results confirm that GlyA cells also contain very low levels of intracellular glycine despite the high accumulation of intracellular serine and the presence of cSHMT activity. In addition, we have shown that increasing the mSHMT activity in GlyA cells overcomes the glycine auxotrophy, thereby demonstrating that mSHMT deficiency alone is responsible for the glycine auxotrophy associated with GlyA cells.
Expression of the partial human mSHMT gene in GlyA cells resulted in only modest increases in mSHMT activity, representing less than 5% of total WTT2 cell mSHMT activity, despite expression of high levels of human mSHMT mRNA in the GlyA-human-mSHMT cells. We conclude that exon 1 of the mSHMT gene is not essential for mSHMT activity or mitochondrial import. These data also suggest that mSHMT activity can be inhibited greater than 95% in CHO cells without inducing a glycine auxotrophy or affecting intracellular glycine levels, although serine levels were still elevated.
It has been suggested that the mitochondrial and cytoplasmic SHMT
isozymes function cooperatively in shuttling one-carbon units between
the cytoplasm and mitochondria analogous to the shuttling of reducing
equivalents that occurs in the malate-aspartate shuttle (29). Studies
of the regulatory regions of the aspartate aminotransferase genes
demonstrated that the mitochondrial and cytoplasmic isozymes do not
share common promoter elements (30), and the two genes are regulated
independently. Previous studies have demonstrated that the relative
ratio of SHMT in the cytoplasm and mitochondria varies among cell and
tissues, suggesting that the SHMT genes are also regulated
independently. While mSHMT is found in all cell types, it is enriched
in certain tissues, including the kidney and liver. Analysis of the
human mSHMT 5 promoter region suggests that it is a housekeeping gene
with multiple transcriptional initiation sites, but it also contains a
CCCTCCC element that is present in many genes that are expressed
predominantly in the liver (24) including the phenylalanine
hydroxylase gene. It is also of interest that the 5
promoter
region contains a zeste-white element that may be responsible for the
observed developmental variations in SHMT activity and serine
metabolism (3). We have also demonstrated that mSHMT message levels do
not change throughout the cell cycle in MCF-7 cells, consistent with
similar observation for the yeast mSHMT message levels (31).
Additionally, no evidence for changes in mSHMT mRNA levels in MCF-7
cells were observed during folate, purine, thymidine, or methionine
deprivation. Continuing studies will elucidate the contribution of the
mSHMT promoter elements to mSHMT expression and their influence on
glycine and folic acid metabolism.
Intron 1 appears to contain consensus regulatory elements capable of expressing the mSHMT gene. Analysis of the mitochondrial import peptide suggests that exon 1 contains most of the hydrophobic and basic amino acid residues that may be required for efficient mitochondrial protein import but are not essential. Expression of the human mSHMT lacking exon 1 did not result in measurable increases in SHMT activity in the cytoplasm. The low level of mSHMT activity resulting from the expression of the human mSHMT gene lacking exon 1 in the GlyA transfectants is most probably due to either inefficient translation from the translation initiation site located in exon 2 or rapid turnover of the mSHMT protein in the cytoplasm prior to mitochondrial import due to its shortened import presequence. In light of these observations, we are currently investigating the stability of the mSHMT protein in the cytoplasm and the amino acid residues that are essential for mitochondrial import, and we are determining whether the putative promoter elements present in intron 1 are capable of expressing reporter genes in human cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U23143[GenBank].