Identification by bacterial expression and functional reconstitution of the yeast genomic sequence encoding the mitochondrial dicarboxylate carrier protein
ABSTRACT The inner membranes of mitochondria contain a family of transport proteins of related sequence and structure. The DNA sequence of the genome of Saccharomyces cerevisiae encodes at least 35 members of this family. Three of them can be recognised as known isoforms of the ADP-ATP translocase and two others as the phosphate and citrate carriers. The transport functions of the remainder cannot be identified with certainty. One of them, encoded on yeast chromosome xii, shows a fairly close sequence relationship to the known sequence of the bovine mitochondrial oxoglutarate-malate carrier. The yeast protein has been obtained by over-expression in Escherichia coli, reconstituted into phospholipid vesicles and shown to have transport properties characteristic of the mitochondrial carrier for dicarboxylate ions, such as malate, and also phosphate, previously biochemically characterised, but not sequenced, from both mammalian and yeast mitochondria. This is the first example of the biochemical identification of an unknown membrane protein encoded in the yeast genome since the completion of the genomic sequence.
EBS 17956 FEBS Letters 399 (1996) 299-302
Identification by bacterial expression and functional reconstitution of the
yeast genomic sequence encoding the mitochondrial dicarboxylate
Luigi Palmieri a,b, Ferdinando Palmieri b, Michael J. Runswick a, John E. Walker a,*
~The M.R.C. Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
t'Department of Pharmaco-Biology, Laboratory of Biochemistry and Molecular Biology, University of Bari, Via Orabona 4, 70125 Bari, Italy
Received 30 October 1996
Abstract The inner membranes of mitochondria contain a
family of transport proteins of related sequence and structure.
The DNA sequence of the genome of Saccharomyces cerevisiae
t.acodes at least 35 members of this family. Three of them can be
recognised as known isoforms of the ADP-ATP translocase and
two others as the phosphate and citrate carriers. The transport
functions of the remainder cannot be identified with certainty.
)ne of them, encoded on yeast chromosome xii, shows a fairly
close sequence relationship to the known sequence of the bovine
ndtochondrial oxoglutarate-malate carrier. The yeast protein has
heen obtained by over-expression in Escherichia coil, rcconsti-
luted into phospholipid vesicles and shown to have transport
properties characteristic of the mitochondrial carrier for
dicarboxylate ions, such as malate, and also phosphate,
previously biochemically characterised, but not sequenced, from
both mammalian and yeast mitochondria. This is the first
t.xample of the biochemical identification of an unknown
membrane protein encoded in the yeast genome since the
completion of the genomic sequence.
,£ey words: Dicarboxylate carrier; Transport; Mitochondria
The inner membranes of mitochondria contain a family of
~elated proteins that transport various substrates and prod-
acts through the membrane. Their sequences are charac-
!erised by three tandem related sequences of about 100 amino
,tcids, each of them probably being folded into two anti-par-
,dlel transmembrane c~-helices linked by an extensive hydro-
~hilic sequence. The three repeats are joined together by
.horter hydrophilic sequences. This arrangement was identi-
ied first  in the published sequence of the ADP/ATP trans-
ocase , and subsequently in the uncoupling protein from
~rown fat mitochondria , and in the mitochondrial phos-
~)hate , oxoglutarate-malate , citrate  and carnitine
:arriers . The various repeats are all related to each other
hroughout the family, and several characteristic sequence fea-
ures are conserved [8-11]. The sequences of proteins of un-
known function from various species also belong to the fam-
ly, whereas the biochemical properties of other carrier
)roteins from mitochondrial membranes, exemplified by the
ticarboxylate carrier, have been determined but their se-
quences are not known [8-11].
As described below, we have examined the proteins encoded
n the sequence of the genome of Saccharomyces cerevisiae for
~Corresponding author. Fax: (44) (1223) 412-178.
members of the mitochondrial carrier family, and we have
identified 35 members. They include three isoforms of the
ADP/ATP translocase [12-14] and the phosphate and citrate
carriers [15,16]. Otherwise the transport functions of the other
30 members of the family cannot be identified with certainty.
One of them, named xii-C1 (the only mitochondrial carrier
encoded on chromosome xii), is quite closely related in se-
quence to the bovine oxoglutarate-malate carrier protein. Its
coding sequence has been cloned into a bacterial expression
vector. The over-expressed yeast protein has been reconsti-
tuted into phospholipid vesicles and identified from its trans-
port characteristics as the carrier for dicarboxylate ions. From
earlier work, this dicarboxylate carrier is known to catalyse an
electroneutral exchange of dicarboxylates, such as malate, and
also inorganic phosphate [17,18] by a simultaneous antiport
reaction mechanism [19,9]. It also transports sulphate and
thiosulphate [20,21]. It can be inhibited by the impermeant
dicarboxylate analogue butylmalonate , by bathophenan-
throline and by sulphydryl reagents (excluding N-ethylmalei-
mide) [23,24]. The protein has been isolated previously, but
not sequenced from the mitochondria of rat liver  and
yeast , and has an apparent molecular mass of 28 kDa.
In liver, it plays an important role in gluconeogenesis, urea
synthesis and sulphur metabolism, and much less activity is
present in cardiac muscle .
2. Materials and methods
Amersham Radiochemicals (Amersham, UK) supplied L-[l,4(2,3)-
14C]malic acid and [33P]phosphate. Egg-yolk phospholipids (egg le-
cithin) were obtained from Fluka, Dorset, UK, and sarkosyl (N-lau-
roylsarcosine) from Sigma Chemical Company, Poole, UK.
2.2. Computer search for family members
The proteins encoded in the yeast genome were compared with the
sequences of the mitochondrial carriers whose function is also known
with the aid of the program BLAST .
2.3. Amplification of the coding sequence from yeast genomic DNA
Oligonucleotide primers were synthesised with the sequences 5'-
consist of the sequences at the extremities of the coding sequence
for xii-C1 (nucleotides 827 869-826 973 on the negative strand of chro-
mosome xii; Genbank accession number U19028), with additional
NdeI and HindllI sites, respectively. The sequence of interest was
amplified from S. cerevisiae genomic DNA (10 ng; kindly donated
by Dr. R. Arkowitz) by 30 cycles of PCR. The amplified DNA con-
tained a 0.9 kb product visible by ethidium bromide staining after
electrophoresis in a 1% agarose gel. The product was cloned into
the expression vector pMW172, which was transformed into E. coli
DH5c~ cells. Transformants selected on 2 x TY plates containing am-
)014-5793/96l$12.00 © 1996 Federation of European Biochemical Societies. All rights reserved.
DH S00 14-5793(96)01 350-6
M 1 2 3 4 5 M
Fig. 1. Expression in E. coil and purification of the yeast xii-C1
protein. Proteins were separated by SDS-PAGE and stained with
Coomassie blue dye. Lanes M, markers (bovine serum albumin, car-
bonic anhydrase and cytochrome c); lanes 14, E. coil C41(DE3)
containing the expression vector, without (lanes 1 and 3), and with
the coding sequence for xii-C1 (lanes 2 and 4). Samples were taken
at the time of induction (lanes 1 and 2) and 5 h later (lanes 3 and
4). The same number of bacteria was analyzed in each sample.
Lane 5, purified yeast xii-Cl protein (3.5 gg) originating from bac-
teria shown in lane 4.
picillin (100 gg/ml) were screened by direct colony PCR, and by re-
striction digestion of the purified plasmid DNA. The sequence of the
insert coding for xii-C1 was verified by the modified dideoxy chain
termination method .
2.4. Bacterial expression of the yeast xii-Cl protein
The protein was over-expressed at 37°C in E. coli C41(DE3) ,
and inclusion bodies were obtained as described previously for the
oxoglutarate-malate carrier . Control cultures containing the
empty pMW172 vector were processed in parallel. The crude inclusion
body pellet was resuspended in TE buffer (10 mM Tris-HC1, pH 8.0,
0.1 mM EDTA; 2 ml) and fractionated by centrifugation at
131000xg for 4.5 h at 4°C, on a step gradient made of 10 ml of
40%, 15 ml of 53% and 4 ml of 70% (w/v) solutions of sucrose dis-
solved in the buffer. The xii-C1 protein collected in a grey-gold band
at the interface between the 53% and 70% sucrose layers . The
inclusion bodies were washed at 4°C first with TE buffer, then twice
with a buffer containing 10 mM PIPES, pH 7.0, Triton X-114 (3%, w/
v) and 1 mM EDTA, and then once again with TE buffer. The xii-C1
protein was solubilised from the inclusion bodies in buffer containing
10 mM Tris-HC1, pH 7.0, sarkosyl (2.5%, w/v) and 0.1 mM EDTA.
Insoluble material was removed by centrifugation (258 000 x g for 1 h
Samples taken from cultures at various points of growth and pur-
ified inclusion bodies were examined by SDS-PAGE in 17.5% gels
. The proteins were either stained with Coomassie blue dye or
transferred to poly(vinylidene difluoride) membranes, stained with
Coomassie blue dye, and their N-terminal sequences determined
with a pulsed liquid protein sequencer (Applied Biosystems 477A).
The yield of purified yeast protein per litre of bacterial culture was
estimated from a Coomassie blue-stained SDS-PAGE gel with an
LKB 2202 Ultroscan laser densitometer, with carbonic anhydrase as
2.5. Reconstitution of the yeast xii-C1 protein into liposomes
The solubilised protein was diluted 5-fold with a buffer containing
2 mM PIPES, pH 7.0, 0.6% (w/v) Triton X-114 and 0.2 mM EDTA,
and then reconstituted into liposomes in the presence of appropriate
substrates, as described before for the bovine mitochondrial oxoglu-
tarate-malate . Similar protocols have been used subsequently for
the citrate and phosphate carriers [15,16].
2.6. Activity assays
External substrate was removed from proteoliposomes on a Sepha-
dex G-75 column. Transport at 25°C was started by adding
[14C]malate or [33P]phosphate to the proteoliposomes, and was termi-
nated after 60 s (unless indicated otherwise) by addition of either 30
mM butylmalonate or a mixture containing 30 mM pyridoxal 5'-
phosphate and 10 mM bathophenanthroline . In control samples,
the inhibitor was added at the beginning together with the labelled
L. Palmieri et al./FEBS Letters 399 (1996) 299-302
substrate, Extraliposomal labelled substrate was removed , and the
radioactivity in the liposomes was measured . Transport activity
was calculated by subtracting the respective control from the experi-
mental values. Transport of other substrates by the reconstituted yeast
protein was assayed in a similar way. Effects of inhibitors and exter-
nally added substrates on the [14C]malate/phosphate exchange were
studied in liposomes containing xii-C1, and loaded with 20 mM phos-
phate. The exchange reaction was started by adding 1 mM
[14C]malate, and was stopped after 1 min. Thiol reagents, pyridoxal
5'-phosphate, carboxyatractyloside and c~-cyanocinnamate were
added 3 min before the radioactive substrate; other inhibitors and
external substrates were added together with [L4C]malate. All inhibi-
tors and substrates were used at a concentration of 20 raM, except for
N-ethylmaleimide which was used at 2 mM, and organic mercurials,
carboxyatractyloside and a-cyanocinnamate at 0.15 mM. The control
transport values of uninhibited malate/phosphate exchange in three
independent experiments ranged between 3244 and 3628 lamol/min/g
3. Results and discussion
3.1. Expression of the yeast xii-C1 protein in bacteria
The yeast xii-C1 protein was highly expressed in E. coli
C41(DE3) (see Fig. 1, lane 4). Its apparent molecular mass
was about 31 kDa (the calculated molecular mass for xii-C1 is
32 991, including the initiator methionine residue), and the N-
terminal sequence determined for residues 1-10 (STNAKE-
SAGK) corresponds exactly to the anticipated sequence.
The over-expressed protein accumulated in the bacterial cyto-
sol as inclusion bodies, and the protein was readily purified by
centrifugation and washing steps (see Fig. 1, lane 5). A 1 litre
culture of E. coli C41(DE3) yielded about 25 mg of the pur-
ified yeast protein.
3.2. Substrate specificity and inhibitor sensitivity
In the search for the function of xii-C1, the substrates for
known mitochondrial transporters were tested, but the yeast
xii-C1 protein did not catalyse the homo-exchange of oxoglu-
tarate, adenosine diphosphate, pyruvate, citrate, glutamate,
aspartate, carnitine, ornithine and glutamine (external concen-
Transport properties of proteoliposomes containing bacterially ex-
pressed yeast xii-C1 protein
Internal substrate Substrate transported (gmol/10 min/g
None (CI- present)
Proteoliposomes containing xii-C1 were loaded internally with the
substrate (concentration 20 mM). Transport was started by the ex-
ternal addition of 0.1 mM [~4C]malate or 0.1 mM [33P]phosphate, and
stopped after 10 min. Similar results were obtained in four experi-
ments; a representative experiment is shown.
L Palmieri et al./FEBS Letters 399 (1996) 299-302
-2 -1 0
!/[-S'] (mM -11
tig. 2. Lineweaver-Burk plots of the [14C]malate/malate and
[ ~P]phosphate/malate exchanges in liposomes reconstituted with the
bacterially expressed yeast xii-C1 protein. Radioactive substrates
v.ere added at the concentrations indicated to proteoliposomes con-
t dning 20 mM malate; O, phosphate: ©, malate.
t'ation 0.5 mM for 10 min), nor did it catalyse a phosphate/
i, hosphate exchange inhibitable by N-ethylmaleimide (charac-
l,~'ristic of the phosphate carrier). However, the reconstituted
x east protein did catalyse a [14C]malate/phosphate exchange
i ruction that could be inhibited by butylmalonate, character-
i.tic reactions of the dicarboxylate carrier protein established
Freviously in rat and yeast mitochondria [17,18,20,25,26]. No
s ach exchange activity was detected either in material from
I acterial cells lacking the expression vector for xii-C1, or in
tells harvested immediately before induction of expression of
The substrate specificity of the yeast xii-C1 protein was
~amined in greater detail by measuring the uptake of
[ 4C]malate and of [33p]phosphate into proteoliposomes which
lad been pre-loaded with various substrates (see Table 1). The
t ighest activities were observed in the presence of internal L-
~ relate, malonate, phosphate and succinate. To a lesser extent,
,, alphate and thiosulphate were exchanged for both external
J lalate and phosphate. A very low activity was also found in
l ae presence of internal oxoglutarate (about 10-15% of the
,,ample with internal malate). No significant exchange was
~.bserved with internal fumarate, aspartate, glutamate, citrate,
• tDP, pyruvate, carnitine or ornithine. The residual activity in
l lae presence of these substrates was approximately the same
,s the activity observed in the presence of sodium chloride.
The effects of inhibitors on the [14C]malate/phosphate ex-
t hange reaction catalysed by the reconstituted yeast xii-C1
]rotein were also examined (see Section 2). The exchange
~eaction was inhibited by the substrate analogues butylmalo-
~ate (86% inhibition), benzylmalonate (82%), phenylsuccinate
~O'Vo) and phthalate (78%), as well as by the sulphydryl re-
,gents mersalyl (97%) and p-chloromercuriphenylsulphonate
~95%), and by the inhibitors bathophenanthroline (99%) and
pyridoxal 5'-phosphate (96%). Butylmalonate was a more ef-
lective inhibitor of the malate/phosphate exchange than phen-
~lsuccinate, as is the dicarboxylate carrier in mitochondria
. The oxoglutarate carrier is more sensitive to the latter
inhibitor than to the former . Inhibitors of other charac-
terised mitochondrial carriers, such as N-ethylmaleimide, car-
boxyatractyloside, 1,2,3-benzenetricarboxylate and c~-cyano-
cinnamate, had no effect on the activity of xii-C1. In
addition, the reconstituted malate/phosphate exchange was
inhibited by the external addition of the well-known sub-
strates of the dicarboxylate carrier, namely malate (93% in-
hibition), phosphate (85%), malonate (93%), succinate (76%),
sulphate (54°/,,) and thiosulphate (61%). It was also inhibited
to a much lesser extent by oxoglutarate (25%), and it was
virtually unaffected by fumarate, citrate, ADP, aspartate, glu-
tamate, pyruvate, carnitine, glutamine and ornithine. The
same results were obtained in three independent experiments.
The transport characteristics of the yeast xii-C1 protein and
the effects of inhibitors on transport are the same as those
determined previously for the dicarboxylate carrier in mito-
chondria [17,18,20-24], and after its purification from mito-
chondria and reconstitution into liposomes [25,26].
3.3. Kinetic' characteristics of the reconstituted yeast xii-C1
The dependence of the exchange rate on substrate concen-
tration was studied by changing the concentration of external
[14C]malate at a constant internal concentration of 20 mM
malate or 20 mM phosphate. The data from a typical experi-
ment are shown in Fig. 2. With both external substrates, lin-
ear functions were obtained that intersected the ordinate close
to a common point. Therefore, the V, ..... value is independent
of the type of substrate, as observed previously with the di-
carboxylate carrier in mitochondria  and after purification
. However, the different slopes indicate that the Km for
phosphate is more than three times higher than for malate
(the mean values from four experiments are 1.65+0.19 and
0.56+0. 09 mM for phosphate and malate, respectively).
Thus, the yeast xii-C1 protein is also similar in these respects
to the rat liver dicarboxylate carrier, where its Km values for
phosphate and malate are 1.5 mM and 0.26 mM, respectively,
in mitochondria , and 1.41 mM and 0.49 mM, respec-
tively, after purification . From the inhibition constants
(Ki values) of different substrates for the [14C]malate/phos-
phate exchange, the order of affinity was: malate ~ malonate
> phosphate ~ succinate > thiosulphate > sulphate >> oxoglu-
tarate, in close agreement with affinities measured with the rat
liver dicarboxylate carrier [18,34~36]. The V, ..... value for re-
constituted xii-C1 (5.6 +0.7 gmol/min/mg protein at 25°C) is
higher than the value reported for the rat liver dicarboxylate
carrier in mitochondria (70 nmol/min/mg protein at 9°C) ,
and it is virtually the same as the value reported for the
purified and reconstituted rat liver carrier .
From its transport properties and kinetic characteristics,
there is no doubt that the yeast xii-C1 protein is a dicarbox-
ylate carrier protein• As yet, there is no definitive proof that
this protein is the same as the dicarboxylate carrier detected in
the inner membranes of yeast mitochondria, which has not
been analysed by protein sequencing. However, it is likely to
be so. With the exception of the phosphate and citrate carriers
in mammals [4,6], but not in yeast [15,16], the mitochondrial
carrier proteins do not have N-terminal extensions that are
removed during entry to the organelle, and none is associated
with xii-C1. However, with the possible exceptions of the
L. Palmieri et al./FEBS Letters 399 (1996) 299-302
maize brittlel protein and the PMP-47 protein from Can-
dida boidinii , the characterised members of this family of
carriers have been found exclusively in mitochondrial mem-
branes. Therefore, this is the most likely sub-cellular localisa-
tion of xii-C1.
Acknowledgements." This work was supported in part by the CNR
target project 'Ingegneria genetica' and by the 'Collaborazione Ita-
lo-Britannica per la Ricerca e l'Istruzione Superiore, 1996'.
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