Chen_et_al-2008-Genes_to_Cells

© 2008 The Authors Genes to Cells (2008) 13, 679–689
Journal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
679
DOI: 10.1111/j.1365-2443.2008.01197.x
Blackwell Publishing IncMalden, USAGTCGenes to Cells1356-95971365-2443© 2008The AuthorsJournal compilation © 2008 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.>XXXOriginal ArticlesHuman mitochondrialTMPKY-L Chen et al.
Identification of a putative human mitochondrial
thymidine monophosphate kinase associated with
monocytic/macrophage terminal differentiation
Yen-Ling Chen†
, Da-Wei Lin†
and Zee-Fen Chang*
Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, NationalTaiwan University, No. 1, Section 1, Jen-Ai Road,
Taipei 100,Taiwan
Mitochondrial DNA synthesis requires the supply of thymidine triphosphate (dTTP) independent
of nuclear DNA replication. In resting and differentiating cells that withdraw from the cell cycle,
mitochondrial thymidine kinase 2 (TK2) mediates thymidine monophosphate (dTMP) formation
for the dTTP biosynthesis in mitochondria.However,a thymidine monophosphate kinase (TMPK)
that phosphorylates dTMP to form thymidine diphosphate (dTDP) in mitochondria remains
undefined. Here, we identified an expressed sequence tag cDNA, which encodes a TMPK with a
mitochondrial import sequence at its N-terminus designated as TMPK2. HeLa cells expressing
TMPK2 fused to green fluorescent protein (GFP) displayed green fluorescence in mitochondria.
Over-expression of TMPK2 increased the steady-state level of cellular dTTP and promoted the
conversion of radioactive labeled-thymidine and -dTMP to dTDP and dTTP in mitochondria.
TMPK2 RNA was detected in several tissues and erythroblastoma cell lines. We also generated
TMPK2 antibody and used it for immunofluorescence staining to demonstrate endogenous
expression of TMPK2 in mitochondria of erythroblastoma cells. Finally, we showed that TMPK2
protein expression was upregulated in monocyte/macrophage differentiating cells, suggesting the
coordinated regulation of TMPK2 expression with the terminal differentiation program.
Introduction
In eukaryotic cells,DNA synthesis can occur in mitochon-
drial and nuclear compartments,separately (Bogenhagen
& Clayton 1977) .The supply of thymidine triphosphate
(dTTP) for DNA synthesis is dependent on the de novo
and salvage pathways.In the de novo pathway,thymidylate
synthase (TS) catalyzes the rate-limiting step of converting
dUMP to thymidine monophosphate (dTMP). In the
salvage pathway,thymidine kinase (TK) is the key enzyme
responsible for dTMP formation from thymidine.There
are twoTK isoforms in eukaryotic cells,one is cytoplasmic
TK1 and the other is mitochondrial TK2 (Johansson &
Karlsson 1997; Wang & Eriksson 2000). Thymidine
monophosphate kinase (TMPK),also known as thymidylate
kinase, phosphorylates dTMP either from TK-mediated
salvage pathway or fromTS-mediated de novo pathway in
all living cells to give thymidine diphosphate (dTDP),
which is subsequently converted to dTTP by nucleotide
diphosphate kinase for DNA synthesis. Therefore, the
function ofTMPK is essential for dTTP synthesis (Lee &
Cheng 1977; Van Rompay et al. 2000).
In proliferating cells, the expression levels of TS,
TMPK andTK1 in the cytoplasm are increased in the S
phase to coordinate with genomic DNA replication for
cell proliferation (Coppock & Pardee 1987; Huang et al.
1994;Ke & Chang 2004).Unlike nuclear DNA synthesis,
mitochondrial DNA replication is uncoupled with the
S phase progression (Bogenhagen & Clayton 1977). In
growing cells, dTDP synthesized in the cytoplasm is
transported into mitochondrial matrix and converted to
dTTP synthesis by mitochondrial NDP kinase (Pontarin
et al. 2003). In non-cycling cells, expression levels of
ribonucleotide reductase and cytoplasmic TK1 and TS
are significantly reduced due to withdrawal from the cell
cycle. Therefore, the dTTP supply for mitochondrial
DNA replication in differentiating and quiescent cells
is mainly dependent on a separate mitochondrial TK2
(Rampazzo et al.2004;Ferraro et al.2005).Genetic defect
Communicated by: Carl-Henrik Heldin
*Correspondence: Email: zfchang@ntu.edu.tw
†
These authors made equal contributions to the work.

Y-L Chen et al.
Genes to Cells (2008) 13, 679–689 © 2008 The Authors
680
ofTK2 causes mitochondrial DNA depletion syndrome
in patients who developed severe myopathy, indicating
that TK2-mediated salvage pathway is required for
maintaining the integrity of mitochondrial DNA in
post-mitotic cells (Saada et al. 2001). Since expression of
cytoplasmicTMPK is also downregulated in a cell cycle-
dependent manner (Ke et al. 2005), a TMPK isoform
might exist in non-proliferating cells for dTTP synthesis
in mitochondria.However,a functional isoform of TMPK
localized in mitochondria remains undefined.
A recent study has used the mitochondria isolated
from mouse liver as an in vitro system to demonstrate that
dTMP at the nm range is imported to mitochondria and
becomesconcentratedatleast100-foldinthemitochondrial
matrix through a transport mechanism highly specific to
dTMP. Upon imported into mitochondria, dTMP is
converted into dTTP,indicating the presence of dTMPK
and dTDK kinase inside the mitochondria (Ferraro et al.
2006). In the present study, we reported a novel human
mitochondrial TMPK, designated TMPK2.We showed
its mitochondrial localization and its functional effect on
the steady-state level of cellular dTTP and metabolic
conversion of dTDP and dTTP in mitochondria. By
generating its specific antibody, we proved the endo-
genous expression of TMPK2 in erythroblastoma cells and
further showed its upregulation associated with monocyte/
macrophage differentiation.
Results
Cloning and expression of human mitochondrial
TMPK cDNA
By blast search, we found that the predicted amino acid
sequences of several human expressed sequence tag cDNA
clones deposited in GenBank contain the TMPK func-
tional domain. Among them, two expressed sequence
tag (EST) sequences,Loc129607and Hxm059368,encode
proteins with a mitochondrial targeting sequence located
in their N-terminal regions.The only difference of these
two EST sequences encoded proteins is their N-terminus
mitochondrial targeting motifs, in which an additional
N-terminal 26-amino acid is present in Loc129607 but
not Hxm059368 coding sequence. HeLa cells were
transfected with pLoc-GFP, pHxm-GFP and pGFP, and
the cell lysates were analyzed byWestern blot using GFP
antibody to detect the GFP-fusion proteins of Loc129607
(Loc-GFP), Hxm059368 (Hxm-GFP) and GFP, respec-
tively (Fig. 1A). By the MitoTracker mitochondrial dye
staining and fluorescent microscopic analysis, the results
showed that Loc-GFP was distributed in the cytosol in a
dotted green fluorescent pattern and co-localized with
red fluorescence of the MitoTracker dye. In contrast,
Hxm-GFP and GFP were distributed in the cytosol
and nucleus as well without colocalization signal with
MitoTracker staining (Fig.1B).This suggests that Loc129607
encodes a mitochondrial protein because of an intact
signal sequence for mitochondrial import (Fig. 1C).
Sequence analysis of N-terminal presequence ofTMPK2
by MitoPortII indicated the presence of amphipathic
α-helix at the position 4–21 amino acid residues required
for mitochondrial import receptorTom20 (Pfanner 2000)
(Fig. 1C). After translocation to mitochondria, the
mitochondrial import signal sequence of the imported
protein is processed to produce a mature mitochondrial
protein (Voos et al. 1999).A matrix processing peptidase
cleavage site required for this processing (Schneider et al.
1998)islocatedatthepositionofGly48-Ala49ofLoc129607
encoded protein.Because of a completeTMPK functional
domain composed of P-loop, catalytic site and lid motif
(Fig.1D), here we designated Loc129607 encoded protein
TMPK2.While this manuscript was in preparation, we
found that Hxm059368 was removed from GenBank
database.
Gene structure and expression of human TMPK2
RNA in tissues and cell lines
The TMPK2 gene is annotated on the Locus 129607 of
human genome with five exons and is located on chro-
mosome 2 (2p25.2) (Fig. 2A).Analysis of EST databases
by alignment with the corresponding vertebrate homo-
logues revealed that TMPK2 is conserved in higher
vertebrate species (Fig. 2B).
Semi-quantitative PCR analysis was used to deter-
mine the expression levels ofTMPK2 andTMPK1 RNA
in different tissues.We used a panel of cDNAs synthesized
from polyA(+) RNA of different human tissues for PCR
reaction using a primer set specific to exons 2 and 3 of
TMPK2 (Fig. 3A) to give rise to a single DNA fragment
of 395 bp as expected.The results showed that TMPK2
is ubiquitously expressed in different tissues (Fig. 3B).
In accordance, TMPK2 sequence is found in the cDNA
sequences from various tissues deposited in GenBank.
PCR reaction using primer set specific to TMPK1 pro-
duced a specific DNA fragment of 268 bp.Using cDNA
synthesized from RNA of different human cell lines,
including K562, a chronic myeloid leukemia cell line,
D2,a GM-CSF-independent erythroblastoma line derived
fromTF-1,THP1,an acute monocytic leukemia cell line,
HEK 293T,an embryonic kidney cell line,HeLa,a cervical
cancer cell line,and SH-SY5Y,a neuroblastoma cell line,
as the templates for the PCR reaction, we found that
TMPK2 RNA was readily detectable in erythroblastoma

Human mitochondrial TMPK
681
Figure 1 Subcellular localization of GFP-fusion proteins that haveTMPK domain with putative mitochondrial targeting sequence.
(A) HeLa cells were transfected with pGFP-N1-hxm059368 (Hxm-GFP), pGFP-N1-Loc129607 (Loc-GFP) and pGFP-N1 (GFP)
expression plasmids.Western blot analysis of GFP-fusion protein expressed in HeLa cells using antibody against GFP.(B)After transfection
for 16 h, cells were stained with MitoTracker and examined by ﬂuorescent microscope. Green: GFP and GFP fusion protein; Red:
MitoTracker. (C) N-terminal presequence required for mitochondrial import. Arrow indicates the putative mitochondrial matrix
peptidase cleavage site of TMPK2.Amino acid residue in potential α-helix forming peptide is marked by “*”.A helical wheel view of
amphipathic α-helix peptide covering 4–21 amino acids of TMPK2. (D) Sequence alignment for the TMPK catalytic domain in
Loc129607, Hxm059368 and cytosolic TMPK. P-loop, Putative catalytic site and Lid motif were indicated by “*”. Multiple alignments
were performed using CLUSTALW.

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D2, K562 and THP1, but not in HeLa, SH-SY5Y and
HEK293T cells (Fig.3C).UnlikeTMPK2 RNA,TMPK1
RNA was detected in all these proliferating cell lines. In
contrast with THP1 monocytic leukemia cells that are
highly proliferative and express bothTMPK1 andTMPK2,
primary CD14+
monocytes isolated from fresh peripheral
blood expressedTMPK2 but notTMPK1.BecauseTMPK1
RNA expression is cell-cycle-dependent, lack ofTMPK1
in monocytes from peripheral blood is due to withdrawal
from the cell cycle of these differentiated cells.
Figure 2 Gene structure and vertebrate conservation of TMPK2. (A) The annotated sequence of TMPK2 on the Locus 129607 of
human genome, Exons are shown as boxes. (B) Sequence alignment for TMPK2 in higher vertebrate species. P-loop, Putative catalytic
site and Lid motif were indicated by “*”. Multiple alignments were performed using CLUSTALW.

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Functional effect of ectopic expression of TMPK2
on dTTP formation
In order to ascertain the in vivo function ofTMPK2, we
ectopically expressed different amounts of TMPK2 in
293T cells and measured the total cellular dNTP levels.
Increasing ectopic expression ofTMPK2-GFP expanded
the dTTP pool size up to 61% (Fig.4A,B),confirming that
TMPK2 is functionally involved in dTTP formation. In
the meanwhile,the levels of dGTP,dCTP and dATP were
slightly elevated by ectopic expression of TMPK2-GFP.
We further isolated mitochondria from cells expressing
TMPK2-GFP or GFP.The mitochondria freshly prepared
from cells were incubated with H3
-thymidine and H3
-TMP,
separately.After incubation for 10 min,mitochondria were
extensively washed for nucleotide extraction.UsingTLC
to separate thymidine,dTMP,dTDP and dTTP,we deter-
mined the amounts of radiolabeled thymidine, dTMP,
dTDP and dTTP,and calculated the relative conversion to
dTTP and dTDP.The results showed that cells expressing
TMPK2-GFP increased conversion of dTMP or thymidine
to dTTP and dTDP by twofold, further indicating the
functional role ofTMPK2 in mitochondria (Fig. 4C).
Figure 3 Expression of TMPK2 RNA
in human tissues and various cell lines.
(A) Location of PCR primer set for
detecting TMPK2 RNA expression.
Oligonucleotide sequences in exons 2 and
3 of TMPK2 as indicated by the arrow were
synthesized and specificity of each primer
was confirmed with blast analysis. (B)
Human multi-tissue cDNAs were subjected
to PCR reaction using primer set as
described above to have a product size at
395 bp for TMPK2; 268 bp for TMPK1
and 245 bp for GAPDH.N.C.:no template
control; P.C. : plasmid control. Asterisk
indicates a nonspecific band. (C) RT-PCR
reactions were performed with RNAs
isolated from the indicated cell lines. Top
panel:expression ofTMPK2;middle panel:
expression of TMPK1 and bottom panel:
expression of GAPDH.
Figure 4 Over-expression ofTMPK2 increases the total cellular
dTTP pool and mitochondrial dTDP/dTTP conversion.
HEK293T cells were transfected with pGFPN1 (GFP) and
different amounts of pGFPN1-TMPK2 (TMPK2-GFP) as
indicated for 48 h.Cells were harvested for dNTP determination.
The results shown represented the average ± SD from three
separate experiments. Statistics were conducted as student t-test;
*P < 0.1 and **P < 0.05 denote significant differences from
control or pEGFP-TMPK2 transfected cells.(B) Cell lysates were
analyzed by Western blot using GFP Ab. (C) Mitochondria
prepared from HEK293T cells transfected with control vector or
pGFPN1-TMPK2 were incubated with 10 µCi of each [H3
]-
thymidine or [H3
]-TMP for 10 min as indicated. Nucleotides
were extracted and separated as described in Methods section.
Relative conversion of thymidine and dTMP to dTDP and dTTP
in mitochondria was calculated by dividing radioactive counts of
dTDP and dTTP by total counts of thymidine,dTMP,dTDP and
dTTP. The results represented the average from two separate
experiments.

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Upregulation of TMPK2 during monocyte/
macrophage differentiation
Next, we purified the recombinant TMPK2 protein for
generating antibody specifically against human TMPK2
to verify the existence of endogenousTMPK2 (Fig. 5A).
We used this antibody to perform immunostaining of
D2 cells, which were plated onto a fibronectin-coated
dish to allow cell adhesion and spreading (Fig. 5B). In
agreement with the results from the ecotopic expression
Figure 5 Reciprocal expression ofTMPK1 andTMPK2 during monocytic differentiation.(A) GST-TMPK2 was expressed in BL21
E. coli and purified by Glutathione Sepharose 4B.After purification, GST-TMPK2 was separated by SDS-PAGE and the gel was stained
with Coomassie Blue.The gel-purified GST-TMPK2 was then used to generate antiserum against hTMPK2 by immunizing rabbit. (B)
Immunostaining of endogenous TMPK2. D2 cells adhering to fibronectin-coated cover slip were immunostained using TMPK2
antibodies with or without GST-TMPK (∆N48) protein neutralization together with MitoTracker for mitochondrial staining. (C)
Western blot analysis usingTMPK2 antibody.D2 cells treated with 32 nm PMA for 3 days were harvested forWestern blot analysis using
TMPK2 antibody that had been neutralized with or without purified GST-TMPK2 (∆N48) protein or GST-TMPK1 protein.The same
blot was probed with β-tubulin antibody. (D) Differential expression ofTMPK1 andTMPK2 during differentiation. RNAs and proteins
were prepared from D2 cells after treatment with 32 nm PMA for the indicated time for RT-PCR reaction (upper panel) and Western
blot analysis (lower panel), respectively.

685
of TMPK2-GFP observed in HeLa cells, immuno-
fluorescence staining of D2 cells withTMPK2 antibody
detected endogenousTMPK2 in mitochondria.TMPK2
antibody which had been neutralized by recombinant
TMPK2 (∆N48) protein, a deleted form at the putative
mitochrondrial processing site, was unable to elicit the
immunostaining signal in mitochondria, indicating the
specific detection of endogenous TMPK2 by this
antibody (Fig. 5B). This antibody was also used for
Western blot analysis.As expected,a protein at molecular
weight of 44 kDa in extracts of D2 cells was specifically
detected byTMPK2 antibody (Fig. 5C).Despite sharing
partial homology in sequence within theTMPK domain,
GST-TMPK2 but not GST-TMPK1 protein was able to
neutralize this antibody in specific detection of endo-
genousTMPK2. In order to establish the relationship
betweenTMPK1 andTMPK2 expression during differ-
entiation, we treated D2 cells with PMA to induce
monocyte/macrophage differentiation for RT-PCR
reaction andWestern blot analysis. Expression ofTMPK1
at the RNA and protein level was decreased with differ-
entiation induction.In contrast,TMPK2 protein expression
was significantly increased with differentiating time even
though only a slight increase of RNA expression was
seen in one day induction. These results indicated a
reciprocal relationship in protein expression pattern of
TMPK1 and TMPK2 during monocytic/macrophage
differentiation (Fig. 5D). Taken together, our data sug-
gest that upregulation of TMPK2 during differentiation
may substitute for cytosolicTMPK1 for dTTP synthesis
in mitochondria biogenesis.
Discussion
In this study, we described a mitochondrial TMPK,
designated TMPK2. The cDNA of human TMPK2
codes for a 49 kDa polypeptide consisting of a typical
mitochondrial targeting sequence and a TMPK func-
tional domain. The subcellular localization of TMPK2
suggests its biological function in mitochondrial dTTP
biosynthesis.We also found thatTMPK2 protein expres-
sion is markedly increased in erythroblastoma cells after
PMA-induced monocyte/macrophage differentiation,
where TMPK1 expression becomes diminished.Accord-
ingly,we proposed that regulation ofTMPK2 expression
is closely associated with cellular terminal differentiation,
by which dTTP is produced for DNA synthesis in mito-
chondrial compartment in the non-cycling cells.
The importance of mitochondrial dTTP supply has
been highlighted by the studies reporting that genetic
diseases characterized by depletion of mitochondrial
DNA are associated with abnormalities in dTTP meta-
bolism (Elpeleg et al. 2002;Elpeleg 2003).As mentioned
earlier,malfunction ofTK2 causes deficiency of thymidine
salvage, resulting in defect in mitochondrial DNA repli-
cation (Saada et al. 2001).Genetic deficiency of thymidine
phosphorylase, which catalyzes the reversible phospho-
rolysis of thymidine, leads to accumulation of thymidine
in body fluids,resulting in mitochondrial neurogastroin-
testinal encephalomyopathy (MNGIE) (Nishino et al. 1999,
2000).This is because too much dTTP is produced to
perturb the balance of dNTP pools, leading to patho-
genic multiple mtDNA depletion in skeletal muscle.
These studies indicate that both lack and overproduc-
tion of thymidine phosphates impair replication or
maintenance of mitochondrial DNA.The identification
of TMPK2 adds another nuclear gene participating in
mitochondrial biogenesis for future investigating genetic
diseases related to mitochondrial defects.
Unlike cytosolic TK1, which phosphorylates only
thymidine, mitochondrial TK2 phosphorylates thymidine
and deoxycytidine (Wang & Eriksson 2000). In non-
cycling cells, the mitochondrial dNTP synthesis is
dependent on the salvage pathway by the mitochondrial
dGK andTK2 that phosphorylate all four deoxyribonu-
cleotides (Van Rompay et al. 2000). It is known that
human tissues contain a cytosolic TMPK1, a uridylate-
cytidylatekinase(UMP-CMPK)(VanRompayet al.1999b),
five isozymes of adenylate kinase (AK) (Yamada et al. 1989;
Xu et al. 1992; Yoneda et al. 1998; Van Rompay et al.
1999a),and several guanylate kinases (GUK) (Jamil et al.
1975; Brady et al. 1996) for the step of conversion of
dNMP to dNDP.Among them, only AK2, 3 and 4 are
suggested be located in mitochondria for dADP forma-
tion,but the enzyme phosphorylating dTMP within the
mitochondria remains to be identified. Since a mito-
chondrial NDPK that catalyzes dTTP formation from
dTDP has been identified (Milon et al. 1997, 2000), the
TMPK2 in this study might fill the missing gap for the
second phosphorylation step that forms dTDP in mito-
chondria. However, attempt to define its substrate
specificity and kinetic properties has not been successful,
since we were unable to use the purified recombinant
TMPK2 protein to detect appreciable level of enzyme
activity in vitro. Nor did we detect its enzymatic activity
using cell extracts over-expressingTMPK2. Perhaps,this
enzyme requires a specific cofactor for its in vitro reaction
activity assay.Nonetheless,ectopic expression ofTMPK2
does increase four dNTP pools, raising a possibility that
it might have broad substrate specificity asTK2.
The detection of TMPK2 RNA in different human
tissues and its sequence conservation in higher eukaryotic
species indicate its ubiquitous function.Although we did
find several EST sequences containing a completeTMPK

Y-L Chen et al.
686
functional domain by blast search, only TMPK2 has an
intact mitochondrial import signal sequence. Here, we
do not exclude the possibility that there are several
isoforms of TMPK capable of providing dTDP formation
in post-mitotic cells because of the availability of mito-
chondrial dNDP transporter (Dolce et al. 2001).Never-
theless,TMPK2 located in mitochondria would be more
directly and efficiently coupled withTK2 and NDPK in
dTTP synthesis in a spatiotemporal manner.The physi-
ological significance ofTMPK2 would be quite limiting
in proliferating cells that contain high level of TMPK1
but very littleTMPK2.However,the function of TMPK2
might become particularly important in the terminal
differentiating cells whereTMPK1 expression is decreased
and the dTTP supply is still needed for mitochondrial
DNA synthesis. To support this notion, we found that
monocytes from peripheral blood still retainTMPK2 but
not TMPK1 RNA. In addition, the expression level of
TMPK2 is significantly increased in PMA-induced dif-
ferentiating D2 erythroblastoma cells, in whichTMPK1
expression is reciprocally decreased.Indeed,gene sequence
identified as a thymidylate kinase family lipopolysaccha-
ride (LPS)-inducible member (Lee & O’Brien 1995;
Kimura et al. 2006) is identical to that of TMPK2.There-
fore, upregulation of TMPK2 expression with terminal
differentiation might represent a mechanism for main-
taining mitochondrial biogenesis in non-dividing cells.
Experimental procedures
Materials
Phorbol-12-myristate-13-acetate (PMA) was purchased from
Sigma Chemicals (St. Louis, MO) and was dissolved in DMSO.
Anti-hTMPK1 polyclonal antibody was prepared as described
previously (Ke et al. 2005). Antiserum against hTMPK2 was
obtained by immunizing rabbit with purified TMPK2∆N48
protein and was affinity-purified. Anti-β-tubulin and anti-β-
actin were purchased from Sigma Chemicals (St Louis, MO).
[3
H]-labeled dTMP (49.4 Ci/mmole) was purchased from
Moravek Biochemicals (Brea, CA). [3
H]-labeled thymidine
(25 Ci/mmole) was purchased from GE Healthcare (Little
Chalfont, UK).
Cell culture
HeLa, 293T, and cells were maintained in Dulbecco’s modified
Eagle’s medium (DMEM, Invitrogen Life technologies, Carlsbad,
CA) supplemented with 10% fetal bovine serum plus 100 µg/mL
streptomycin and 100 U/mL penicillin (Invitrogen Life Technol-
ogies) at 37 °C under 5% CO2. K562 and D2 cells were main-
tained in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal bovine serum plus 100 µg/mL streptomycin
and 100 U/mL penicillin (Lai et al. 2001). THP1 cells were
maintained in RPMI-1640 medium supplemented with 10%
heat-inactivated fetal bovine serum plus 100 µg/mL streptomycin,
100 U/mL penicillin and 2.5 g/L glucose.
SH-SY5Y cells were maintained in a 1 : 1 mixture of Eagle’s
Minimum Essential Medium (EMEM) and Ham’s F12 medium
supplemented with 10% fetal bovine serum.
cDNA cloning, expression and subcellular
localization of human TMPK2
The GenBank EST database at the National Center for Biotech-
nology Information was searched with the Basic Local Alignment
Search Tool (BLAST) to identify human EST cDNA sequences
that encode proteins containing functional domain of TMPK.
Among them, Loc129607 (accession number NM_207315) and
Hxm059368 (accession number XM_059368.6) EST cDNAs
were found to have mitochondrial targeting sequence by using the
MitoPortII search. Complementary DNAs synthesized by reverse
transcriptase using total RNA of K562 cells were subjected to PCR
with two pairs of primers flanking the open reading frame of these
two cDNAs for amplification.The RT-PCR products, 1350 bp
for Loc129607 and 1272 bp for Hxm059368, were subsequently
cloned into pGEM-T-easy cloning vector (Promega),followed by
subcloning into pEGFP-N1 at the EcoRI and BamHI sites.
Monocyte isolation for RT-PCR
Fresh, whole blood was drawn with informed consent from
healthy donors into vacutainer tubes containing EDTA.Peripheral
blood mononuclear cells (PBMC) were isolated by Ficoll density
gradient separation (Amersham Biosciences, Piscataway, NJ).
CD14+
monocytes were isolated from PBMC by positive selec-
tion using a MACS system (Miltenyi Biotech,Bergisch Gladbach,
Germany), according to the manufacturer’s protocol.
Recombinant protein purification and antibody
generation
The DNA fragment corresponding to human TMPK2 was
inserted into the pGEX-2T vector to express a GST-fused
TMPK2 in Escherichia coli. GST-TMPK2 expression was induced
in E. coli with 0.2 mm IPTG for 14 h at 20 °C.The recombinant
protein was enriched from crude bacterial extracts using
glutathione-4B Sepharose (Amersham Pharmacia Biotech,Uppsala,
Sweden). The gel-purified recombinant protein was used to
generate antiserum against hTMPK2 by immunizing rabbit, after
which polyclonal antibody was affinity-purified.
RNA isolation and RT-PCR analysis
Total RNA was isolated from various cell lines, and was reverse
transcribed with reverse transcriptase (Promega, Madison, WI).
Complementary DNAs of 12 human tissues purchased from
Clontech (Human multiple tissue cDNA Panel) and prepared

687
from cell lines were used for semi-quantitative PCR amplification.
The semi-quantitative PCR forTMPK2 was performed under the
following cycling conditions: 95 °C for 5 min, 30 cycles of 30 s
at 95 °C, 30 s at 58 °C and 1 min at 72 °C. Semi-quantitative
PCRs for TMPK1 and GAPDH were performed under the
following cycling conditions: 95 °C for 5 min, 25 cycles of 30 s
at 95 °C, 30 s at 55 °C and 30 s at 72 °C.The primer sequences
were as follows: human TMPK2 sense primer, 5′-CAGCGCC
TCTGGGAGGTGCAAGACGGCA-3′ and antisense primer,
5′-GATCTTCCTCCACTGGCCAATGCAAGAGGGTGGT
GACT-3′; human TMPK1 sense primer, 5′-CGCAAGCTTCG
GTTCCCGGAAGATCAACT-3′ and antisense primer, 5′-
CGCAAGCTTTCACACGTCTGGCTGTTACACCAGTCTAG-
3′ and human GAPDH sense primer, 5′-CATGGCACCGT
CAAGG-3′ and antisense primer, 5′-CACCATGGGGGCAT
CAGC-3′.
dNTP pools determination
Cells (1 × 106
) were washed twice with 10 mL of cold PBS and
extracted with 1 mL ice-cold 60% methanol at −20 °C for 1 h,
followed by centrifugation for 30 min at 16 000 g.The supernatant
was transferred to a fresh tube and dried under vacuum.The
residue was dissolved in sterile water and store at −20 °C for later
analysis.Determination of the dNTP pool size in each extract was
based on DNA polymerase-catalyzed incorporation of radioactive
dATP or dTTP into the synthetic oligonucleotide template
method described by Sherman and Fyfe (1989).
Preparation of mitochondria and transport
experiments
Mitochondria from HEK293T cells were prepared using
Qproteome Mitochondria Isolation Kit (Qiagen, Venlo, the
Netherlands). The mitochondrial pellet from 5 × 106
cells were
suspended in 40 µL of mitochondrial reaction buffer (220 mm
mannitol/70 mm sucrose/5 mm MOPS, pH 7.4/0.2 mm EGTA/
0.2 mg/mL BSA/1 mm MgCl2/1 mm ATP).After adding 10 µCi
of [3
H]dTMP or [3
H] thymidine,mitochondria were incubated at
37 °C for 10 min and then 5 µL of 1 mm cold dTDP was added
to the reaction mixtures prior to centrifugation at 4 °C for 10 min
at 6000 g. The intact mitochondria in the pellets were washed
twice with 500 µL of mitochondrial reaction buffer, after which
500 µL of ice-cold 60% methanol was added to the mitochondrial
pellets and stored at −80 °C for 1 h.The methanol extracts were
collected by centrifugation at 4 °C for 30 min at 16 000 g and
transferred to a fresh tube for drying under vacuum. 5 µL of
nuclease-free water and 2 µL of marker mixture containing 1 µg
of dTMP,dTDP,dTTP,and thymidine were added to dissolve the
residue, which was then spotted onto PEI-cellulose-F TLC plate
(Merck, Whitehouse Station, NJ) and developed in a solution
containing 2 m acetic acid and 0.5 m LiCl.After development and
drying, the spots corresponding to thymidine, dTMP, dTDP, and
dTTP were detected by 254 nm using UV detector and were cut
for radioactivity measurement by a liquid scintillation counter
(Beckman Coulter, Fullerton, CA). The conversion ratio was
calculated by dividing cpm of (dTDP + dTTP) by that of
(Thd + dTMP + dTDP + dTTP).
Immunostaining of endogenous TMPK2
D2 cells were plated on a fibronectin-coated coverslip. Before
immunostaining, cells were incubated with a medium containing
MitoTracker Red580 (Molecular Probe, Eugene, OR) for
15 min. Cells were washed twice with PBS and fixed with 3%
paraformaldehyde/PBS for 30 min. After fixation, cells were
permeablized with 0.3% Triton X-100/TBST (50 mm Tris-HCl,
pH7.4,150 mm NaCl,0.1%Triton X-100) for 5 min and blocked
with 5.5% normal goat serum inTBST for 1 h at room temperature,
followed by incubation with purified anti-hTMPK2 antibody in
TBST-3%BSA for 2 h at room temperature.AfterTBST washing,
cells were incubated with FITC-conjugated goat anti-rabbit
IgG antibody (Sigma) at a 1 : 200 dilution in TBST-3% BSA for
1 h at room temperature. The coverslip was then washed with
TBST three times and mounted for analysis with Leica TCS SP2
confocal spectral microscope and Zeiss Axioskop2 microscope.
Transient transfection and immunoblotting
293T cells (1 × 106
) were plated on a 100-mm-diameter dish and
transiently transfected with a mixture of 30 µg of LipofectAMINE
(Life Technologies, Inc., Gaithersburg, MD) and 5 µg of plasmid
DNA.After transfection for 48 h, cells were extracted for dNTP
pool size measurement and Western blot analysis.Thirty micro-
grams of cell lysates were resolved on SDS-PAGE [10% (w/v) gel],
followed by electrophoretic transfer to polyvinylidene fluoride
(PVDF) membranes (Millipore, Bedford, MA). After blocking
with 5% (w/v) powdered non-fat milk, the membrane was incu-
bated with antiserum against GFP (1 : 2000) for 16 h, and treated
for 1 h with horseradish peroxidase-conjugated goat anti-rabbit
IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA). ECL
detection for the horseradish peroxidase reaction was performed
according to the manufacturer’s instructions.
Acknowledgements
We thank Chun-Mei Hu and Yi-Chang Chang for technical
assistance and Peter Reichard (Department of Biology,University
of Padova, Italy) for critical reading of the manuscript.This study
was supported by grants NSC96-2628-B-002-079-MY2 from
National Science Council and NHRI-EX-97-9701BI from
National Health Research Institute,Taiwan (R.O.C.).
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Received: 16 October 2007
Accepted: 31 March 2008

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