melanogaster mitochondrial thiamine pyrophosphate carrier protein DmTpc1p is described.. The closest relative of DmTpc1p and DmTpc2p in Saccharomy-ces cerevisiae is YPR011c whose functio
Trang 1pyrophosphate carrier from Drosophila melanogaster
Domenico Iacopetta1,*, Chiara Carrisi2,*, Giuseppina De Filippis2, Valeria M Calcagnile3, Anna R Cappello1, Adele Chimento1, Rosita Curcio1, Antonella Santoro1, Angelo Vozza3, Vincenza Dolce1, Ferdinando Palmieri3and Loredana Capobianco2
1 Department of Pharmaco-Biology, University of Calabria, Arcavacata di Rende, Cosenza, Italy
2 Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy
3 Department of Pharmaco-Biology, University of Bari, Italy
Introduction
Several cofactors (i.e coenzymes and prosthetic
groups) are essential for the functioning of important
metabolic processes occurring in mitochondria
Although most of these cofactors have to be imported
from the cytosol into mitochondria, very little is
known about the molecular basis of their transport across the mitochondrial membrane Thiamine pyro-phosphate (ThPP) is a fundamental coenzyme of vari-ous cytosolic and mitochondrial reactions It is synthesized in the cytosol [1,2], and is required in the
Keywords
CG2857 and CG6608;
Drosophila melanogaster; mitochondria;
proteomics; thiamine pyrophosphate carrier
Correspondence
L Capobianco, F Palmieri or V Dolce,
Department of Biological and Environmental
Sciences and Technologies, University of
Salento, 73100 Lecce, Italy; Department of
Pharmaco-Biology, University of Bari, 70125
Bari, Italy; Department of Pharmaco-Biology,
University of Calabria, Rende 87036 (CS),
Italy
Fax: +39 0 832 298 626;
+39 0 80 5442 770; +39 0 984 493 270
Tel: +39 0 832 298 864;
+39 0 80 5443 323; +39 0 984 493 177
E-mail: loredana.capobianco@unile.it;
fpalm@farmbiol.uniba.it; vdolce@unical.it
*These authors contributed equally to this
work.
(Received 28 July 2009, revised 15 December
2009, accepted 17 December 2009)
doi:10.1111/j.1742-4658.2009.07550.x
The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial brane The genome of Drosophila melanogaster encodes at least 46 mem-bers of this family Only five of these have been characterized, whereas the transport functions of the remainder cannot be assessed with certainty In the present study, we report the functional identification of two D mela-nogastergenes distantly related to the human and yeast thiamine pyrophos-phate carrier (TPC) genes as well as the corresponding expression pattern throughout development Furthermore, the functional characterization of the D melanogaster mitochondrial thiamine pyrophosphate carrier protein (DmTpc1p) is described DmTpc1p was over-expressed in bacteria, the puri-fied protein was reconstituted into liposomes, and its transport properties and kinetic parameters were characterized Reconstituted DmTpc1p trans-ports thiamine pyrophosphate and, to a lesser extent, pyrophosphate, ADP, ATP and other nucleotides The expression of DmTpc1p in Saccha-romyces cerevisiae TPC1 null mutant abolishes the growth defect on fer-mentable carbon sources The main role of DmTpc1p is to import thiamine pyrophosphate into mitochondria by exchange with intramitochondrial ATP and⁄ or ADP
Abbreviations
MCF, mitochondrial carrier family; NDP, nucleoside diphosphate; NMP, nucleoside monophosphate; NTP, nucleoside triphosphate;
Pi, phosphate; PPi, pyrophosphate; Th, thiamine; ThMP, thiamine monophosphate; ThPP, thiamine pyrophosphate; Tpc, thiamine
pyrophosphate carrier.
Trang 2cytosol for the activity of transketolase, and in the
mitochondria for the activity of pyruvate-,
oxogluta-rate- and branched chain keto acid dehydrogenases
To our knowledge, in Drosophila melanogaster, only
the pyruvate dehydrogenase complex has been
charac-terized among the ThPP-dependent enzymes [3]
Exper-iments performed with intact rat liver mitochondria
have led to the proposal of the existence of different
transport systems for thiamine, ThPP⁄ thiamine (Th)
exchange, ThPP and thiamine monophosphate (ThMP)
transport, and ThMP uniport or ThMP⁄ phosphate
(Pi) exchange [4–6] To date, only the yeast ThPP
car-rier (Tpc1p) and the human Tpc have been identified
as being responsible for the mitochondrial transport of
ThPP and ThMP [7,8] In particular, the human Tpc
encoded by the SLC25A19 gene was previously
indi-cated as the deoxynucleotide carrier [9], and then
ascertained to be the human Tpc [8] Tpc1p and Tpc
belong to the mitochondrial carrier family (MCF) [10–
12] Family members have a tripartite structure
con-sisting of three tandemly repeated sequences of 100
amino acids in length Each repeat contains two
hydrophobic stretches that span the membrane as
a-helices and a characteristic sequence motif [10] An
analysis of the D melanogaster genome has led to the
identification of 46 possible MCF members [13] To
date, five D melanogaster mitochondrial carriers have
been identified by their high similarity with orthologs
in other organisms They are the two isoforms of the
ADP⁄ ATP translocase [14–16], the carnitine ⁄
acylcarni-tine [17,18], citrate [13] and mitoferrin carriers [19] In
the present study, we report the identification of two
D melanogaster genes, CG6608 and CG2857, which
are related to the human thiamine pyrophosphate
car-rier (TPC) and yeast TPC1 genes, as well as the
expression profile of the corresponding transcripts in
different developmental stages Moreover, in the
pres-ent study, we provide evidence that DmTpc1p
(encoded by CG6608) is the transporter of ThPP
DmTpc1p over-expressed in Escherichia coli and
recon-stituted into phospholipid vesicles transports ThPP
across liposomal membranes with high affinity
Fur-thermore, the expression of DmTpc1p in a yeast
mutant laking TPC restores the growth defect on
fermentable substrates
Results
Identification and characterization of DmTPC
cDNAs
The protein sequence of the human Tpc encoded by
the SLC25A19 gene [8,9] was used to search the
Fly-Base database (http://flybase.org) for homologous sequences Three putative transcripts corresponding to
D melanogastergenes CG6608 and CG2857 were iden-tified The CG6608 gene encodes for two transcripts (CG6608-RA and CG6608-RB), whereas CG2857 is an intronless gene coding for only one transcript The two transcripts of the CG6608 gene contained the same
999 bp ORF encoding a putative protein of 332 amino acid residues (henceforth named DmTpc1p) with a cal-culated molecular mass of 36.7 kDa (Fig 1) The CG2857 gene containing a 972 bp ORF encoded a putative protein of 323 amino acid residues (henceforth named DmTpc2p) with a calculated molecular mass of 36.4 kDa (Fig 1)
DmTpc1p and DmTpc2p share 39% of identical amino acids They have 33% and 31% sequence iden-tity and 53% and 51% sequence similarity to human Tpc The D melanogaster proteins were used to screen yeast databases for homologous sequences The closest relative of DmTpc1p and DmTpc2p in Saccharomy-ces cerevisiae is YPR011c whose function is not yet known (26% and 23% sequence identity, respectively), followed by yTpc1p encoded by the YGR096w gene (24% and 21% sequence identity, respectively), which has been demonstrated to be the transporter of ThPP [7] (Fig 1) DmTpc1p and DmTpc2p belong to the MCF because their amino acid sequences are composed
of three tandem repeats of 100 amino acids, each containing two transmembrane a-helices, linked by an extensive loop, and a conserved signature motif [10]
Expression of D melanogaster TPC transcripts in various developmental stages
To determine the expression levels of transcripts corre-sponding to the CG6608 and CG2857 genes, we per-formed a semi-quantitative RT-PCR analysis on total RNAs from wild-type embryos, larvae, pupae and adults, using primers based on sequence retrieved from FlyBase A PCR product of the predicted size was detected at high levels in embryos and adult flies, although a weaker but significant signal was found in larvae and pupae (Fig 2) for transcripts CG6608-RA and CG6608-RB The significance of these two tran-scripts, which have arisen from alternative splicing of the 5¢-UTR, is not yet known However, the 5¢-UTR
of eukaryotic mRNAs can play a role in the post-transcriptional regulation of gene expression through the modulation of translation efficiency and message stability [20]
No visible band of expression was found for the CG2857-RA transcript Furthermore, any attempt to amplify the coding sequence corresponding to the
Trang 3CG2857-RA transcript failed (data not shown) A
con-trol RT-PCR was carried out using specific primers for
Rp49 (Fig 2)
Bacterial expression of DmTpc1p
DmTpc1p, the only protein encoded by both
tran-scripts of the CG6608 gene, was expressed at high
lev-els in E coli BL21(DE3) (Fig 3, lane 4) to identify its
biochemical function It accumulated as inclusion bodies and was purified as described previously [9] (Fig 3, lane 5) The apparent molecular mass of the
CG6608 - RA
CG6608 - RB CG2857 - RA
RP 49
Fig 2 Expression of the DmTPC transcripts during development.
Ethidium bromide staining of the RT-PCR products obtained using
specific primers for D melanogaster transcript TPCs and cDNA
from Oregon R embryos (E), larvae (L), pupae (P) and adults (A) As
a control for the RNA integrity, the Rp49 was amplified.
97 kDa 66.2 kDa
45 kDa
31 kDa
21.5 kDa 14.4 kDa
M
Fig 3 Expression in E coli and purification of DmTpc1p Proteins were separated by SDS-PAGE and stained with Coomassie blue dye Lane M, markers (phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor and lysozyme); lanes 1–4,
E coli BL21(DE3) containing the expression vector, without (lanes
1 and 3) and with the coding sequence for DmTpc1p (lanes 2 and 4) Samples were taken at the time of induction (lanes 1 and 2) and
4 h later (lanes 3 and 4) The same number of bacteria was analy-sed in each sample Lane 5, purified DmTpc1p (5 lg) originating from bacteria shown in lane 4.
Fig 1 Comparison of predicted Tpc proteins from various species Alignment of D melanogaster, Homo sapiens and S cerevisiae proteins The accession numbers for the different sequences used in the alignment are NP_650034 (DmTpc1p); NP_611977 (DmTpc2p); NP_068380 (hTpc); NP_015336 (YPR011c); NP_011251 (yTpc1p) Dashes denote gaps Asterisks and dots indicate residues in all five sequences, which are identical and conserved, respectively.
Trang 4recombinant protein was 37 kDa (the calculated
value with initiator methionine was 37 499 Da) The
identity of the purified protein was confirmed by
N-terminal sequencing The protein was not detected in
bacteria harvested immediately before the induction of
expression (Fig 3, lane 2), nor in cells harvested after
induction but lacking the coding sequence in the
expression vector (Fig 3, lane 3) Approximately
90 mg of purified protein was obtained per litre of
culture
Functional characterization of recombinant
DmTpc1p
DmTpc1p was reconstituted into liposomes, and its
transport properties were tested in homo-exchange (i.e
same substrate inside and outside) experiments Using
external and internal substrate concentrations of 1 and
5 mm, respectively, the reconstituted protein catalyzed
an active dATPaS-[35S]⁄ dATP exchange but not
homo-exchanges for malate, oxoglutarate, citrate,
car-nitine, glutamate and aspartate (data not shown) No
dATPaS-[35S]⁄ dATP exchange was observed with
DmTpc1p that had been boiled before incorporation
into liposomes, nor by reconstitution of
sarcosyl-solu-bilized material from bacterial cells either lacking the
expression vector for DmTpc1p or harvested
immedi-ately before the induction of expression
The substrate specificity of recombinant DmTpc1p
was examined in detail by measuring the uptake of
dATPaS-[35S] into proteoliposomes preloaded with
various substrates As shown in Fig 4, the highest
activity was observed in the presence of internal ThPP
ADP and dADP were also transported at a
consider-able rate Significant activities were also observed with
internal pyrophosphate (PPi), nucleoside diphosphates
(NDPs), nucleoside triphosphates (NTPs), dNDPs and
dNTPs of the bases A, G, U or C Furthermore, no
significant exchange activity was found using Th,
ThMP, adenosine, Pi, nucleoside monophosphates
(NMPs) and dNMPs of the bases A, G, U or C No
activity was observed with guanosine, cytidine, uridine,
oxoglutarate, citrate, adenosine 3¢,5¢-diphosphate and
CoA (data not shown) The substrate that was best
transported comprised ThPP, followed by ADP and
dADP, which were transported with a slightly higher
efficiency than PPi and the remaining NDPs, NTPs,
dNDPs and dNTPs
Consistently, dATPaS-[35S] uptake in the presence
of 5 mm ADP inside the proteoliposomes was strongly
inhibited by the external addition of ThPP, ADP and
dADP (Fig 5A) A lower inhibition was found with
PPi, NDPs, NTPs, dNDPs and dNTPs of the bases A,
G, U or C Almost no effect was exerted by external
Th, ThMP, adenosine, Pi, NMPs and dNMPs of the base A, G, U or C
The reaction catalyzed by reconstituted DmTpc1p was completely inhibited by p-chloromercuribenzene sulfonate and bathophenanthroline (strong inhibitors
of several mitochondrial carriers) and, to a lesser extent, by pyridoxal 5¢-phosphate, mersalyl and mercu-ric chloride (other strong inhibitors of many mitochon-drial carriers) (Fig 5B) No significant inhibition was observed with N-ethylmaleimide The different inhibi-tory potency of SH reagents may be explained, at least
in part, by the different microenvironment surrounding the reactive cysteine(s) Carboxyatractyloside and bon-gkrekate, powerful inhibitors of the mitochondrial ADP⁄ ATP carrier [21,22], were partly effective on DmTpc1p (50% and 30% inhibition, respectively) A specific inhibitor of the mitochondrial citrate carrier, 1,2,3-benzenetricarboxylate, strongly reduced dATP⁄ ADP exchange No significant inhibition was observed with butylmalonate and phenylsuccinate (i.e inhibitors of other characterized mitochondrial carriers) (Fig 5B)
Adenosine Pi PPi Th ThMP ThPP AMP GMP CMP UMP ADP GDP CDP UDP ATP GTP CTP UTP dAMP dGMP dCMP dUMP dADP dGDP dCDP dATP dGTP dCTP dUTP
V (µmol·min –1 × mg protein)
Fig 4 Substrate specificity of DmTpc1p Liposomes reconstituted with DmTpc1p were preloaded internally with various substrates (concentration 5 m M ) Transport was started by addition of 125 l M dATPaS-[ 35 S] and terminated after 2 min Similar results were obtained in at least four independent experiments.
Trang 5Kinetic characteristics of recombinant DmTpc1p
The uptake of 0.5 mm dATPaS-[35S] into
proteolipo-somes was measured either as uniport (in the absence
of internal substrate) or as exchange (in the presence
of internal 5 mm ADP) (Fig 6A) The uptake of
dATP by exchange followed a first-order kinetics (rate constant 0.016 min)1; initial rate 1.47 lmolÆmin)1Æmg protein)1) with isotopic equilibrium being approached exponentially (Fig 6A) By contrast, no dATPaS-[35S] uptake was observed without an internal substrate, indicating that DmTpc1p does not catalyze the unidi-rectional transport (uniport) of dATP, but only the exchange reaction The uniport mode of transport was further investigated by measuring the efflux of dATPaS-[35S] from prelabeled active proteoliposomes because it provides a more convenient assay for unidi-rectional transport [23] In the absence of external sub-strate, no efflux was observed even after incubation for
60 min (Fig 6B), whereas extensive efflux occurred upon addition of external ThPP A significant efflux
of dATPaS-[35S] from prelabeled proteoliposomes was observed after the addition of external UTP or ATP These results demonstrate that reconstituted DmTpc1p catalyzes an obligatory exchange reaction of
Adenosine
Pi
PP
Th
ThMP
ThPP
AMP
GMP
CMP
UMP
ADP
GDP
CDP
UDP
ATP
GTP
CTP
UTP
dAMP
dGMP
dCMP
dUMP
dADP
dGDP
dCDP
dATP
dGTP
dCTP
dUTP
Inhibition (%)
HgCl2
pCMBS
Mersalyl
N-ethylmaleimide
PLP
BAT
BTA
PHS
BMA
CAT
BKA
Inhibition (%)
A
B
Fig 5 Effect of inhibitors on the dATPaS-[ 35 S] ⁄ ADP exchange
mediated by DmTpc1p Proteoliposomes were preloaded internally
with 5 m M ADP; transport was initiated by adding 125 l M
dATPaS-[ 35 S] and terminated after 2 min (A) Effect of external substrates.
The external substrates (concentration 0.5 m M ) were added
together with dATPaS-[35S] (B) Effect of mitochondrial carrier
inhib-itors Thiol reagents were added 2 min before the labeled
sub-strate; the other inhibitors were added together with dATPaS-[ 35 S].
The final concentrations of the inhibitors were 10 l M
carboxyatract-yloside (CAT) and bongkrekic acid (BKA); 0.1 m M
p-chloromercuri-benzene sulfonate (pCMBS), mersalyl and mercuric chloride
(HgCl 2 ); 2 m M N-ethylmaleimide (NEM),
benzene-1,2,3-tricarboxy-late (BTA), butylmalonate (BMA) and phenylsuccinate (PHS); 10 m M
pyridoxal 5¢-phosphate (PLP) and bathophenanthroline (BAT) The
extent of inhibition (%) from a representative experiment is
reported Similar results were obtained in at least five experiments.
0 20 40 60 80
100
A
B
Time (min)
0 2000 4000 6000 8000
10 000
12 000
Time (min)
dATP efflux (cpm x 10
3 )
Fig 6 Kinetics of dATPaS-[ 35 S] transport in proteoliposomes recon-stituted with DmTpc1p (A) Uptake of dATP A concentration of
500 l M dATPaS-[35S] was added to proteoliposomes containing
5 m M ADP (exchange, ) or 5 m M NaCl and no substrate (uniport, ) Similar results were obtained in three independent experiments (B) Efflux of dATPaS-[35S] from proteoliposomes reconstituted in the presence of 5 m M ADP The internal substrate pool was labeled with dATPaS-[ 35 S] by carrier-mediated exchange equilibration Then the proteoliposomes were passed through Sephadex G-75 dATP aS-[ 35 S] efflux was initiated by adding Hepes 10 m M (pH 6.9), without (•) or with 0.5 m M dithioerythritol ( ), 2 m M ThPP with 0.5 m M dithioerythritol ( ), 2 m M UTP with 0.5 m M dithioerythritol (.) or 2 m M ATP with 0.5 m M dithioerythritol (¤) Similar results were obtained in five independent experiments.
Trang 6substrates In another set of experiments, the addition
of 5 mm ThPP following a 60 min incubation, during
which dATPaS-[35S] uptake by proteoliposomes had
almost reached equilibrium, caused an extensive efflux
of radioactive compound This efflux shows that the
dATPaS-[35S] taken up by proteoliposomes is released
in exchange for externally added ThPP Therefore,
ThPP is transported by reconstituted DmTpc1p not
only when it is inside liposomes, but also when added
externally
The kinetic constants of the recombinant purified
DmTpc1p were determined by measuring the initial
transport rate at various external dATPaS-[35S]
con-centrations in the presence of a constant saturating
internal concentration (5 mm) of ADP The Km and
Vmax values (measured at 25C) were 107.6 ± 0.4 lm
and 1.73 ± 0.12 lmolÆmin)1Æmg protein)1, respectively
(means of 30 experiments) The activity was calculated
by taking into account the amount of DmTpc1p
recov-ered in the proteoliposomes after reconstitution
Sev-eral external substrates were competitive inhibitors of
dATPaS-[35S] uptake (Table 1) because they increased
the apparent Km without changing Vmax (not shown)
These results confirm that the affinity of DmTpc1p for
ThPP is higher than that for dADP, UTP and ATP
Furthermore, the Ki value of ThPP is more than
200-fold lower than that of AMP
DmTpc1p functions as a ThPP transporter in
S cerevisiae
The yeast TPC1 null mutant does not grow on
thia-mine-less synthetic minimal medium supplemented
with fermentable carbon sources [7] This phenotype is
explained by the ability of Tpc1p to import ThPP into
mitochondria Thus, the expression of a mitochondrial
carrier protein that recognizes ThPP as a substrate
should mitigate or abolish the growth defect of the tpc1D knockout The DmTpc1p expressed in tpc1D cells via the yeast vector pYES2 fully restored growth
of the tpc1D strain on galactose (Fig 7), indicating that DmTpc1p imports ThPP into yeast mitochondria
By contrast, when the tpc1D cells were transformed with the empty vector, no growth restoration was observed
Discussion
In the present study, DmTpc1p (encoded by the CG6608 gene) was shown, by direct transport assays,
to transport ThPP after expression in E coli and reconstitution into liposomes This approach, which has previously been used for the identification of mito-chondrial carriers from high eukaryotes [10], yeast [24] and plants [25], revealed that DmTpc1p is different from any previously described mitochondrial carrier protein On the basis of the transport properties and kinetic characteristics of DmTpc1p reported in the present study, this protein is the D melanogaster mito-chondrial transporter for ThPP Furthermore, comple-mentation of the yeast TPC null mutant by the expression of DmTpc1p clearly indicates that DmTpc1p is able to transport ThPP into mitochondria The related sequence DmTpc2p (encoded by the CG2857 gene) could not be functionally characterized because no corresponding cDNA was generated by RT-PCR in any developmental stage analysed The absence of transcripts of the intronless CG2857 gene was not unexpected because its structure clearly indi-cates that it is a paralogous gene, produced by retro-transposition, of the pre-existing ‘parent’ gene CG6608 [26,27] Indeed, a virtual screening of the expressed sequence tag databases showed that CG2857, similar
to the OXPHOS paralogous genes [27], is expressed (at very low levels) only in testis [27,28]
Table 1 Competitive inhibition by various substrates of
dATPaS-[35S] uptake in proteoliposomes containing recombinant DmTpc1p.
The values were calculated from Lineweaver–Burk plots of the rate
of dATPaS-[ 35 S] versus substrate concentrations The competing
substrates at appropriate constant concentrations were added
together with 0.005–1.25 m M dATPaS-[35S] to proteoliposomes
containing 5 m M ADP The data represent the mean ± SD of at
least three different experiments.
Fig 7 The yeast tpc1D strain is fully complemented by the gene for DmTpc1 Four-fold serial dilutions of wild-type, tpc1D, DmTPC1-pYES2 tpc1D and DmTPC1-pYES2 tpc1D cells were plated on solid thiamine-less synthetic minimal medium supplemented with 2% galactose The plates were incubated at 30 C for 4 days.
Trang 7DmTpc1p and DmTpc2p, which share 39% identity,
have a higher degree of sequence identity with the
unknown yeast protein YPR011c (26% and 23%,
respectively) than with the yeast Tpc1p encoded by the
YGR096w gene [7] However, a phylogenetic analysis
(Fig 8) carried out using several Tpc sequences, as
well as other mitochondrial carriers, revealed that
DmTpc1p, DmTpc2p, yeast Tpc1p [7] and human Tpc
[8–9] are monophyletic, whereas the yeast protein
YPR011c clusters with the Grave’s disease carrier (and
its yeast homologue leu5p) and SLC25A42 [29–31]
The biochemical properties of the recombinant reconstituted DmTpc1p are different from the human and yeast Tpc proteins in several respects: DmTpc1p catalyzes an obligatory counter-exchange; the substrate that is more efficiently transported is ThPP; the affinity
of DmTcp1 for this substrate is very high (Ki for ThPP, 10 lm), a value that is 20-fold lower than that measured in yeast (no data is available in humans); the
D melanogaster protein is unable to transport ThMP; effective counter-substrates for ThPP probably are ATP (NTPs), ADP (NDPs) and PPi; the Ki for dATP
is similar to that determined for the human carrier encoded by SLC25A19 [9], whereas it is five-fold lower than that determined for yeast [7]; and 1,2,3-benzene-tricarboxylate, a known inhibitor of the citrate carrier, strongly reduces the dATP⁄ ADP exchange rate to 15%
Because ThPP is produced in the cytosol by thia-mine pyrophosphokinase [1,2], the primary function of DmTpc1p is to catalyze the uptake of ThPP into mito-chondria However, given that DmTpc1p functions by
a counter-exchange mechanism, the carrier-mediated uptake of ThPP requires the efflux of a counter-substrate The internal counter-ion for exchange could
be either ADP or most likely ATP Thus, in the resting state, the intramitochondrial ATP⁄ ADP ratio is 4 [32] and the rate of exchange of external ThPP for internal ATP is favored by the high amount of ATP generated by oxidative phosphorylation Therefore, the physiological role of the DmTpc1p is probably to cata-lyze the uptake of ThPP into the mitochondrial matrix
in exchange for internal ATP
DmTpc1p is crucial for mitochondrial metabolism because ThPP is an essential coenzyme for the E1 components of pyruvate dehydrogenase and oxogluta-rate dehydrogenase, which are located in the mito-chondrial matrix In agreement with its importance in mitochondrial metabolism, DmTpc1p is localized in the mitochondria, as revealed by immunofluorescence analysis (V Dolce & L Capobianco, unpublished data) and is expressed during all stages of develop-ment Mutations of SLC25A19 cause lethal Amish microcephaly, which is characterized by severe congen-ital microcephaly, elevated levels of a-ketoglutarate in urine, almost no orientation to sight or sound and no motor development Studies using TPC1 null mutants
of D melanogaster could help to gain insight into the molecular and cellular pathogenetic mechanisms of Amish microcephaly Indeed, although the investiga-tion of rodent models is sometimes of significant impact, invertebrate models offer several advantages (i.e short life span, large number of offspring and numerous genetic techniques, amongst others) that can
0.1
yNdt2p
yNdt1p yAnt1p
yTpc1p
DmTpc2p DmTpc1p
YPR011c yLeu5p
hSLC25A42 hGDC
yAAC3 yAAC2 yAAC1 hAAC4 hAAC3 hAAC2
hAAC1 hACP3 hACP2
hACP1
yPTP
hPICB
hPiCA
yGgc1p
hSAMC
yRim2p
ySam5p
hANC
hTPC
Fig 8 Phylogenic tree of amino acid sequences of mitochondrial
transporters from various organisms The unrooted dendogram
orig-inated from an alignment performed by CLUSTALW (http://www.ebi.
ac.uk/clustalw) using the default options Branch lengths are drawn
proportional to the amount of sequence change The bar indicates
the number of substitutions per residue, with 0.1 corresponding to
a distance of ten substitutions per 100 residues The tree was
visu-alized using DENDROSCOPE software [38] The proteins have the
accession numbers: DmTpc1p, NP_650034; DmTpc2p, NP_611977;
hAAC1, NP_001142; hAAC2, NP_001143; hAAC3, NP_001627;
hAAC4, NP_112581; hACP1, NP_998816; hACP2, NP_077008;
hACP3, NP_001006643; hANC, NP_006349; hGDC, NP_689920;
hPiCA, NP_005879; hPiCB, NP_002626; hSAMC, NP_775742;
hSLC25A42, NP_848621; hTpc, NP_068380; yAAC1, NP_013772;
yAAC2, NP_009523; yAAC3, NP_009642; yAnt1p, NP_015453;
yGgc1p, NP_010083; yLeu5p, NP_011865; yPTP, NP_012611;
yNdt1p, NP_012260; yNdt2p, NP_010910; YPR011c, NP_015336;
yRim2p, NP_009751; ySam5p, NP_014395; yTpc1p, NP_011251.
Dm, D melanogaster; h, human; y, yeast; AAC, ADP ⁄ ATP carrier;
ACP, ATP-Mg ⁄ Pi carrier; ANC, peroxisomal adenine nucleotide
carrier; GDC, Graves’ disease carrier; PiC, phosphate carrier;
SAMC, S-adenosylmethionine carrier; SLC25A42, CoA and
adeno-sine 3¢,5¢-diphosphate carrier; Ant, peroxisomal adenine nucleotide
transporter; Ggc, GTP ⁄ GDP carrier; Leu5, accumulation of CoA in
the matrix; yPTP, phosphate transport carrier; Ndt, NAD+ carrier
protein; Rim, pyrimidine nucleotides carrier; YPR011c.
Trang 8address some important issues underlying neurological
disease [33]
Experimental procedures
Computer search for DmTPC genes
The D melanogaster genome annotated in the FlyBase
(http://flybase.org) was screened with the human sequence
of the mitochondrial Tpc also known as deoxynucleotide
carrier [8,9] with the aid of tblastn (http://blast.ncbi
nlm.nih.gov/blast.cgi) Amino acid sequences were aligned
with clustalw (http://www.ebi.ac.uk/tools/clustalw2/index
html)
Construction of the expression plasmid coding
for DmTpc1p
Total RNA was extracted from Oregon R adult flies using
RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and reverse
transcribed as described previously [13] The coding region
for DmTpc1p was amplified from first strand cDNA
(100 ng) by PCR with 5¢-GCGGTAACCACAGGCTC-3¢
(sense primer) and 5¢-CTAATGATGATGATGATGGAA
GCGCACCTGCTTGAGCT-3¢ (antisense primer) of the
D melanogaster transcript CG6608-RA The forward and
reverse primers carried NdeI and HindIII restriction sites,
respectively, as linkers The reverse primer also carried a
DNA sequence coding for six histidines followed by a stop
codon The reaction product was recovered from agarose
gel, cloned in the expression vector pMW7 [34] and
trans-formed into E coli TG1 cells Transformants, selected on
LB plates containing ampicillin (100 lgÆmL)1), were
screened by direct colony PCR, and by restriction digestion
of purified plasmids The sequences of the inserts were
verified
Expression analysis by semiquantitative RT-PCR
Total RNA was extracted from Oregon R embryos, larvae,
pupae and adult flies using RNeasy Mini Kit (Qiagen) and
reverse transcribed as described previously [13] The
constit-utive ribosomal gene (Rp49) was used as an internal
con-trol The sense and antisense gene-specific primers were:
CG6608-RA, sense 5¢-AGGCATGATACTAAATGCCAT
TGAA-3¢ and antisense 5¢-TCCAGAACTGACAAATGC
CGTAC-3¢; CG6608-RB, sense 5¢-GTGGAGCATGATAC
TTAAATGCCA-3¢, and antisense 5¢-TCCAGAACTGACA
AATGCCGTAC-3¢; CG2857-RA, sense 5¢-CTCTTCTACA
AGTACCTCAACGCGG-3¢ and antisense 5¢-TTCTCCCA
AGATACTAATGCTTGCC-3¢; Rp49, sense 5¢-ATGACC
ATCCGCCCAGCATACA-3¢ and antisense 5¢-TTGGTG
AGGCGGACCGACAG-3¢ The PCR products were
analy-sed by 1% agarose gel electrophoresis Band intensities
were quantified using quantity one 1-D Analysis Software (Bio-Rad, Hercules, CA, USA)
Bacterial expression and purification of DmTpc1p
The over-expression of DmTpc1p as inclusion bodies in the cytosol of E coli was accomplished as described previously [35] Control cultures with the empty vector were processed
in parallel Inclusion bodies were purified on sucrose den-sity gradient and washed at 4C, first with TE buffer (10 mm Tris⁄ HCl, pH 8), then twice with a buffer contain-ing Triton X-114 (2%, w⁄ v) and 10 mm Hepes (pH 6.9) and, finally, with Hepes 10 mm (pH 6.9) Proteins were sol-ubilized in 2.5% sarkosyl (w⁄ v) and DmTpc1p was purified
by centrifugation and Ni+-NTA-agarose affinity chroma-tography, as described previously [9]
Reconstitution into liposomes and transport assays
The recombinant protein in sarkosyl was reconstituted into liposomes in the presence or absence of substrates [23] The reconstitution mixture contained purified proteins (150 lL with 0.8–1 lg of protein), 10% Triton X-114 (90 lL), 10% phospholipids as sonicated liposomes (90 lL), 5 mm ADP (except where indicated otherwise), 10 mm Hepes (pH 6.9) and water to a final volume of 700 lL These components were mixed thoroughly, and the mixture was recycled 13 times through the same Amberlite column (Bio-Rad) The external substrate was removed from proteolipo-somes on a Sephadex G-75 columns pre-equilibrated with
50 mm NaCl and 10 mm Hepes (pH 6.9) [23] Transport at
25C was started by adding dATPaS-[35S] (Perkin Elmer, Boston, MA, USA) at the indicated concentrations The carrier-mediated transport was terminated by addition of
30 mm pyridoxal 5¢-phosphate and 10 mm bathophenanthr-oline In control samples, the inhibitors were added at time
0 according to the inhibitor stop method [23] All transport measurements were carried out at the same internal and external pH values Finally, the external substrate was removed, and the radioactivity in the liposomes was mea-sured [23] The experimental values were corrected by sub-tracting control values The initial transport rate was calculated from the radioactivity taken up by proteolipo-somes after 1 min (in the initial linear range of substrate uptake) For efflux measurements, proteoliposomes contain-ing 5 mm ADP were labeled with 20 lm dATPaS-[35S] by carrier-mediated exchange equilibration [23] After 60 min, external substrate was removed by exclusion chromato-graphy in the presence of a reversible inhibitor (0.1 mm p-chloromercuribenzene sulfonate) to avoid efflux of internal substrate Efflux was started by adding Hepes 10 mm (pH 6.9) without or with 0.5 mm dithioerythritol or unlabeled external substrate in the presence of 0.5 mm dithioerythritol
Trang 9In all cases, the transport was terminated by adding the
inhibitors indicated above
Complementation of a yeast mutant lacking
TPC1 by DmTPC1
BY4741 (wild-type) and tpc1D yeast strains were provided
by the EUROFAN resource center EUROSCARF
(Frank-furt, Germany) In the tpc1D mutant, the tpc1 (YGR096w)
locus of S cerevisiae strain BY4741 (MATa; his3D1;
leu2D0; lys2D0; ura3D0) was replaced by kanMX4
The coding sequence of DmTpc1p was cloned into the
BamHI-EcoRI sites of the expression vector pYES2 that
had been previously modified by cloning a DNA sequence
coding for the V5 epitope and six histidines into XhoI-XbaI
sites (DmTPC1-pYES2) This plasmid was introduced into
the tpc1D yeast strain, and trasformants were selected for
uracil auxotrophy Wild-type, tpc1D, DmTPC1-pYes2 tpc1D
and pYes2 tpc1D strains were grown in rich medium
con-taining 2% bactopeptone and 1% yeast extract, synthetic
complete medium or thiamine-less synthetic minimal
med-ium [36] All media were supplemented with 2% glucose or
2% galactose
Other methods
Proteins were analysed by SDS-PAGE and stained with
Coomassie blue dye The N-termini were sequenced, and
the amount of pure DmTpc1p was estimated by laser
densi-tometry of stained samples using carbonic anhydrase as a
protein standard The amount of protein incorporated into
liposomes was measured as described previously [37]
Approximately 20% of DmTpc1p was reconstituted
Acknowledgements
This work was supported by grants from the Ministero
dell’Universita` e della Ricerca (MIUR) and Apulia
Region Neurobiotech (Progetto Strategico 124) We
gratefully thank Dr Daniela Fiore for helpful
discussion
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