Functional integration of mitochondrial and hydrogenosomal ADP/ATP
carriers in the
Escherichia coli
membrane reveals different biochemical
characteristics for plants, mammals and anaerobic chytrids
Ilka Haferkamp
1
, Johannes H. P. Hackstein
2
, Frank G. J. Voncken
2
, Guillaume Schmit
1
and Joachim Tjaden
1
1
Pflanzenphysiologie, Universita
¨
t Kaiserslautern, Germany;
2
Department of Evolutionary Microbiology, Faculty of Science,
Catholic University of Nijmegen, the Netherlands
The expression of mitochondrial and hydrogenosomal
ADP/ATP carriers (AACs) from plants, rat and the
anaerobic chytridiomycete fungus Neocallimastix spec. L2
in Escherichia coli allows a functional integration of the
recombinant proteins into the bacterial cytoplasmic
membrane. For AAC1 and AAC2 from rat, apparent
K
m
values of about 40 l

of ATP for all AACs tested. This is the first report of a
functional integration of proteins belonging to the mito-
chondrial carrier family (MCF) into a bacterial cytoplasmic
membrane. The technique described here provides a relat-
ively simple and highly reproducible method for functional
studies of individual mitochondrial-type carrier proteins
from organisms that do not allow the application of
sophisticated genetic techniques.
Keywords: ADP/ ATP carriers; mitochondria; hydrogeno-
somes; heterologous expression; Escherichia coli.
A high degree of compartmentalization is characteristic of
eukaryotic cells. The transport of metabolic intermediates
between organelles is necessary for complex metabolism and
is mediated by membrane proteins which function as
carriers or channels. Mitochondria and certain hydrogeno-
somes evolved peculiar ADP/ATP carriers (AACs) that
efficiently export ATP, whereas plastids acquired a different
type of nucleotide transporter that seems to be specialized in
ATP uptake [1–3].
In many multicellular animals and plants as well as in
yeast (Saccharomyces cerevisiae),twotofourAACisoforms
have been identified [4–8]. Mammalian AACs seem to have
a tissue specific expression [4–6,9], whereas in yeast, the
expression of the various isoforms is believed to be
characteristic for certain metabolic states, or to participate
in vacuolar metabolism [10,11]. However, the reasons for
the repeated evolution of AAC isoforms and the intrinsic
biochemical differences between the various isoforms
remained largely unknown until now.
Isolated mitochondria of plants and mammals are likely

¨
dinger-Str., D-67663 Kaiserslautern.
Fax: + 631 2052600, Tel.: + 631 2052505,
E-mail:
Abbreviations: AAC, ADP/ATP carrier; CCCP, m-chlorocarbonyl-
cyanide phenylhydrazone; IPTG, isopropyl thio-b-
D
-galactoside;
MCF, mitochondrial carrier family; PMF, proton motive force.
(Received 13 February 2002, revised 29 April 2002,
accepted 9 May 2002)
Eur. J. Biochem. 269, 3172–3181 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02991.x
might be a matter of choice also for other nucleotide
carriers, but recently published data suggested that mito-
chondrial-type AACs might be deposited preferentially as
inclusion bodies in E. coli; for a functional biochemical
analysis these inclusion bodies have to be solubilized and
reconstituted in vitro [17–19]. Here, we describe a technique
that allows the functional expression of a variety of single
mitochondrial-type AACs in E. coli. After IPTG-induction
we were able to obtain functional integration of the various
mitochondrial-type AACs into the cytoplasmic membrane
of E. coli. Measuring the uptake of the various adenylates
into intact E. coli cells expressing eukaryotic AACs, we were
able to determine the biochemical properties of the different
mitochondrial-type AACs. We studied two AAC isoforms
from rat (Rattus norwegicus) and a mitochondrial-type
AAC from the hydrogenosomes of the anaerobic chytrid
Neocallimastix. As the knowledge about plant mitochon-
drial nucleotide exchange is so far quite limited, we

10
–AAC), the NdeI–XhoI
(NdeI–BamHI or NdeI–BglII) DNA inserts of the pBSK-
plasmids were introduced in-frame into the corresponding
restriction sites of the isopropyl thio-b-
D
-galactoside
(IPTG)-inducible T7-RNA polymerase bacterial expression
vector pet16b (Novagen, Heidelberg, Germany). Transfor-
mations of E. coli were carried out according to standard
protocols. The nucleotide sequences of the AACs reported
in this paper are available under the accession numbers
(EMBL database): AY042814 (aac1, A. thaliana),
AY050857 (aac2, A. thaliana), AL021749/gene ¼
ÔF20O9.60Õ (aac3, A. thaliana), X62123 (aac1, S. tubero-
sum), D12770 (aac1, R. norwegicus), D12771 (aac2,
R. norwegicus), AF340168 (hdgaac, Neocallimastix spec.
L2).
Heterologous expression of AACs in
E. coli
The E. coli strain BL21 (DE3) was used for heterologous
expression. The several cDNAs encoding the correspond-
ing AAC proteins under control of the T7-promoter were
transcribed after IPTG induction of the T7-RNA poly-
merase [21]. E. coli cells transformed with the AAC
expression plasmids (or control expression plasmid
pet16b) were inoculated with a fresh overnight culture
and grown at 37 °CeitherinYT
Amp/Clm
medium (YT:

M
pyruvate (rat and plant AACs) [20]. A D
600
value of
0.5–0.6 was required for the initiation of T7-RNA
polymerase expression by addition of IPTG (final con-
centration 0.012%). Cells were grown for 1 h after
Table 1. Oligonucleotides used for construction of the expression plasmids of plant, mammalian and chytridic AACs. Thelower-caseletterindicates
the introduced base exchange to create restriction sites (NdeIorBglII).
aac Oligonucleotide sequence
aac1 (A.t.) Sense 5¢-TGCAGAGTTCcAtATGGTTGATCAAG-3¢
Antisense 5¢-CGAAAAAAGGAGGAAGAAGCAATGC-3¢
aac2 (A.t.) Sense 5¢-TGTAGAGGTTcAtATGGTTGAACAGACTC-3¢
Antisense 5¢-CTTAATGACTGCGGGATTTGGTGGTAC-3¢
aac3 (A.t.) Sense 5¢-CTGATTTGTACAAcAtATGGATGGATC-3¢
Antisense 5¢-GGGCTATTCTTTCATCATCCTCATCG-3¢
aac1 (S.t.) Sense 5¢-TTAAACGTTcatATGGCAGATATGAACC-3¢
Antisense 5¢-GGAAGTTACGAGGCTGACTTAGGC-3¢
aac1 (R.n.) Sense 5¢-GCGCCCGCGTTTCcatATGGGGGATCAG-3¢
Antisense 5¢-CCACACAATGGATCTGTGAACCTGTG-3¢
aac2 (R.n.) Sense 5¢-CTTTTTTGCTTTCcAtATGACAGATGCCG-3¢
Antisense 5¢-TACAACATGCCAGAtCtCGGGGAGAAC-3¢
hdgaac (N.spec.) Sense 5¢-TTCCCCATATCCcAtATGGCCCAAAAG-3¢
Antisense 5¢-GCATTCGTTTAGTTCTTAATTCTCCAG-3¢
Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3173
induction and collected by centrifugation for 5 min at
5000 g (8 °C, Sorvall RC5B centrifuge, rotor type SS34;
Sorvall-Du Pont, Dreieich, Germany). The sediments were
resuspended to D
600

diameter, Orange Scientific, Waterloo, Belgium) under
vacuum previously moistened with potassium phosphate
buffer (50 m
M
, pH 7.0) [22]. Cells were further washed to
remove unimported radioactivity by addition of three
times 4-mL potassium phosphate buffer (50 m
M
,pH7.0).
The filter was subsequently transferred into a 20-mL
scintillation vessel and filled with 10 mL of water.
Radioactivity in the samples was quantified in a
Canberra–Packard Tricarb 2500 scintillation counter
(Canberra–Packard, Frankfurt, Germany). For back
exchange (efflux) experiments, the E. coli cells were
preincubated for 2 min at 30 °C with potassium phos-
phate buffer (50 m
M
, pH 7.0) containing [a-
32
P]ADP or
[a-
32
P]ATP (specific activity 100 lCiÆlmol
)1
). Preloading
was stopped by centrifugation (5000 g, 45 s, room tem-
perature). The washed sediment (four times with potas-
sium phosphate buffer) was subsequently resuspended in
incubation buffer containing the indicated additions and

radioactively labeled adenine nucleotides were determined
after autoradiography and correspond to R
f
values of
unlabeled nucleotides visualized under UV light [23].
Radiolabeling of AAC proteins synthesized in
E. coli
and enrichment of the histidine-tagged chimeric proteins
Ten milliliters of E. coli cells harbouring the indicated
plasmids were grown to exponential phase, collected by
centrifugation at D
600
¼ 0.5, and resuspended in 1 mL of a
methionine assay medium containing 42 m
M
Na
2
HPO
4
,
20 m
M
KH
2
PO
4
,18m
M
NH
4

M
Tris/HCl (pH 7.5), 1 m
M
EDTA, 0.1 m
M
pefabloc and 15% (v/v) glycerol, cells were
further disrupted by ultrasonication (250 W, 3 · 30 s, 4 °C)
and the suspension was centrifuged (10 min, 15 800 g,4°C)
to remove unbroken cells and inclusion bodies. Membranes
extracted in the supernatent were sedimented for 45 min at
100 000 g (TFT 80 rotor, Kontron Instruments, Munich,
Germany), resuspended in binding buffer A consisting of
10 m
M
imidazole, 300 m
M
NaCl, 100 m
M
Na
2
HPO
4
(pH 8.0, HCl), and 0.1% dodecylmaltoside. After addition
of dodecylmaltoside (3.3% final concentration) and incu-
bation on ice for 15 min, the detergent was 10 times diluted
with buffer A and centrifuged for 2 min (15 800 g,4°C).
The solubilized histidine-tagged AACs in the superna-
tent were purified by Ni-chelating chromatography accord-
ing to the supplier’s instructions (Novagen, Heidelberg,
Germany). Eluted proteins were precipitated by adding

M
NAD
+
during the isolation procedure as well as in the
3174 I. Haferkamp et al. (Eur. J. Biochem. 269) Ó FEBS 2002
storage medium. The mitochondrial fraction was washed
and resuspended in 20 m
M
Tris/HCl (pH 7.4), 2 m
M
MgCl
2
and 200 m
M
sucrose. Purity and intactness were analysed
using marker enzymes, as described previously [25–27].
Uptake of [a-
32
P]ADP and [a-
32
P]ATP was carried out
using a rapid filtration technique, as described by Winkler
et al. [28].
RESULTS
Heterologous expression of AACs in
E. coli
cells
Seven different mitochondrial-type AACs from plants, rat
and the anaerobic chytridiomycete fungus Neocallimastix
spec. L2 were cloned into the plasmid pet16b and expressed

cells reached a stationary phase at a D
600
 1.0 after
approximately 1–2 h induction. This observation led to the
conclusion that E. coli cells did not possess, under these
conditions, a high energy state which is considered as a
prerequisite for the proper investigation of the AACs under
different PMF levels across the E. coli membrane. There-
fore, we optimized growth conditions (TB, see Experimental
procedures) for E. coli cells producing the higher expressed
plant and mammalian AAC proteins (Fig. 1). The unde-
fined TB medium and the addition of pyruvate and malate
stimulated the bacterial metabolism and led to the genera-
tion of a high proton motive force across the bacterial
membrane. Under these conditions, no retardation in cell
growth over a time span up to 24 h following IPTG-
induction was observed. The D
600
reached a value of about
12.
It was tempting to analyse whether the heterologously
expressedAACsintegratedintheE. coli membrane were
functional. As phosphatidylglycerol and cardiolipin in the
mitochondrial membranes are believed to be essential for
many mitochondrial functions [29], the deviating lipid
content of the E. coli membranes might hamper the
function of the transgenic AACs. In particular, cardiolipin
is well investigated and considered as very important for
mitochondrial AACs [30–32]. As revealed by high-resolu-
tion

ondrial nucleotide exchange bongkrekic acid and carboxya-
tractyloside [35] led to about 50% reduction of transport
activity in E. coli expressing the recombinant AACs (data
not shown). The treatment of E. coli cells with lysozyme was
crucial for this decrease to occur because the outer bacterial
membrane obviously prevents the penetration of the
mentioned inhibitors. Induced E. coli cells harbouring the
Fig. 1. Heterologous expression and membrane purification of
[
35
S]methionine-labeled His-tagged AAC proteins. E. coli cells har-
bouring plasmid encoding several AACs and E. coli control cells
(pet16b without any insert) were IPTG-induced for protein synthesis in
the presence of [
35
S]methionine. Details of induction, purification and
autoradiography are given in ÔExperimental proceduresÕ. Lane 1, Ecoli
controlcells;lane2,E. coli cells expressing the hydrogenosomal AAC
from Neocallimastix spec. L2; lane 3, E. coli cells expressing the AAC1
from Rattus norwegicus;lane4,E. coli cells expressing the AAC2 from
Rattus norwegicus;lane5,E. coli cells expressing the AAC1
from Arabidopsis thaliana;lane6,E. coli cells expressing the
AAC2 from Arabidopsis thaliana;lane7,E. coli cells expressing
the AAC3 from Arabidopsis thaliana;lane8,E. coli cells expressing the
AAC1 from Solanum tuberosum.
Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3175
pet16b control plasmid (without any insert) as well as E. coli
wildtype cells are not able to transport ADP or ATP [13].
Theimportof[a-
32

values range between 10 and 22 l
M
.
They are only slightly influenced by CCCP. On the other
hand, the kinetic data of the hydrogenosomal AAC from
Neocallimastix reveals apparent affinities that are four times
lower for ADP and about 20 times lower for ATP compared
to those of the rat AACs. Nevertheless, the influence of
CCCP on ADP and ATP affinities is similar to that
observed with the rat AACs.
In our approach, the maximal velocity (V
max
) of nucleo-
tide import into E. coli cells harbouring the AAC expressing
plasmids does not appear to be a direct function of the
amount of recombinant nucleotide carriers present in the
bacterial membrane (Fig. 1, Table 2). We believe that only a
certain fraction of the recombinant protein is integrated as a
functional homodimer into the bacterial membrane. More-
over, isoforms from different organisms are being compared
in these experiments.
Back exchange experiments with nucleotide preloaded
E. coli
cells
Mitochondrial AACs operate in a counter exchange mode
permitting influx or efflux of ADP and ATP in a 1 : 1
stoichiometry [38]. To investigate the internal affinities for
both nucleotides, we carried out several back exchange
experiments with E. coli cells preloaded with either ADP
or ATP. If a counter exchange mechanism is maintained in

alone. After subjecting the cell extracts to thin layer
chromatography, both components ATP and ADP could
be detected with a similar ATP/ADP ratio of about 0.5
(Fig. 3A–C, lane 1; for ATP-preloading data not shown).
Figure 3 shows representative back exchange experiments
for AAC2 from Arabidopsis (Fig. 3A), AAC1 from rat
(Fig. 3B) and hydrogenosomal AAC (Fig. 3C) expressed
in E. coli. As indicated in lane 2 (Fig. 3A–C), no
significant release of radoactivity after incubation of
preloaded E. coli cells in potassium buffer (without any
substrates) was noticed. The radioactive components
exported after addition of ATP (Fig. 3A–C, lane 3) or
ADP (Fig. 3A–C, lane 4) exhibit a radioactive pattern
with a similar ATP/ADP ratio of about 2 for the rat and
the hydrogenosomal AACs and about 4 for the plant
AAC. This indicates that E. coli cells expressing the plant,
mammalian or hydrogenosomal mitochondrial-type AACs
exhibit a preferential export of ATP under energized
conditions independent of the given counter exchange
molecule (ATP or ADP). Interestingly, the plant AAC
seems to possess a much higher internal affinity for ATP
compared to the hydrogenosomal and mammalian AACs.
Moreover, the change of the ratio of exported ATP to
ADP in the presence of the uncoupler CCCP is remark-
able (Fig. 3A–C, lane 5 and 6). This holds true particularly
for the plant AAC (Fig. 3A, lane 5 and 6). The significant
decrease of the export affinity for ATP compared to ADP
indicates that this transport is strongly dependend on the
PMF across the bacterial cytoplasmic membrane.
Mitochondria isolation and nucleotide uptake

potato tuber mitochondria (Fig. 4A) whereas the difference
between ADP and ATP uptake for the Arabidopsis leaf
mitochondria was less pronounced (Fig. 4B). These results
are in close agreement with data observed in rat liver and
bovine heart mitochondria [28,41].
The adenine nucleotide uptake measured under these
conditions was almost totally inhibited by carboxyatrac-
tyloside and bongkrekic acid (Fig. 4). For the determin-
ation of the K
m
values, the mitochondria were incubated
with radioactively labeled nucleotides for 7 s. Interest-
ingly, the apparent affinities of mitochondria from
different plant tissues of potato and Arabidopsis were
about 1–2 l
M
(Table 3) for both, ADP and ATP, and
thus significantly higher than those described for rat
mitochondria [28,36].
DISCUSSION
The functional expression of recombinant AACs in E. coli
provides a unique possibility to study the biochemical
properties of a homogeneous population of AACs in vivo.
Although the unfavourable codon usage of E. coli had been
assumed to hamper the expression of recombinant AACs
[19], the AACs from plants, mammals and anaerobic
chytrid fungus studied here, were expressed without any
significant retardation of E. coli cell growth. Under these
conditions, we could obtain functional integration of several
AACs into the E. coli cell membrane which allowed to

)1
), E. coli cells were preincubated with 100 l
M
CCCP
for 2 min for uncoupling.
ADP ATP ADP + CCCP ATP + CCCP
AAC K
m
V
max
K
m
V
max
K
m
V
max
K
m
V
max
AAC1 (Rattus norwegicus) 38 0.51 105 0.31 35 0.63 30 0.58
AAC2 (Rattus norwegicus) 40 0.55 140 0.65 26 0.92 12 0.45
AAC1 (Arabidopsis thaliana) 10 0.18 15 0.1 10 0.42 10 0.35
AAC2 (Arabidopsis thaliana) 14 4.41 22 1.34 8.5 2.23 6.5 0.94
AAC3 (Arabidopsis thaliana) 10 0.18 12 0.23 5 0.08 5 0.12
AAC1 (Solanum tuberosum) 14 0.29 18 0.12 10 0.15 8 0.14
AAC1 (Neocallimastix spec. L2) 165 7.11 2325 6.9 155 2.58 226 3.87
Ó FEBS 2002 Functional integration of AACs in E. coli (Eur. J. Biochem. 269) 3177

up the required proton motive force across the membrane.
Thus, the expression in E. coli allows an easy and
reproducible method to assess the influence of PMF on
mitochondrial or hydrogenosomal AACs from different
organisms.
In contrast to mammalian AACs, the heterologous
expression in E. coli of the three AAC isoforms from
Arabidopsis thaliana and one AAC from potato shows
significantly higher affinities for ADP and ATP that are less
influenced by the protonophore CCCP (Table 2). Uptake
experiments with intact plant mitochondria strengthen these
observations. The apparent affinities of different plant
mitochondria were about 1–2 l
M
for both ADP and ATP
Fig. 3. Thin-layer chromatography of exported radioactively labeled
adenine nucleotides. E. coli cells expressing several AACs were pre-
loaded with radioactively-labeled [a-
32
P]ADP at following concentra-
tions: (A) 15 l
M
(AAC2, Arabidopsis thaliana); (B) 25 l
M
(AAC1,
Rattus norwegicus); (C) 100 l
M
(hdgAAC, Neocallimastix spec. L2).
Preloaded cells were used for back exchange under indicated condi-
tions: Lane 1 (A–C), separation of radioactive compounds of disrupted

P]ADP, [a-
32
P]ATP uptake into iso-
lated mitochondria. Isolated potato tuber mitochondria (A) and Ara-
bidopsis leaf mitochondria (B) were incubated at 0 °Cwith1l
M
radioactively labeled ADP (j)or1l
M
radioactively labeled ATP (d)
for the indicated time periods. For inhibition of ADP (h)orATP(s)
uptake, the mitochondria were preincubated with 50 l
M
bongkrekic
acid and 200 l
M
carboxyatractyloside. Data is the mean of three
independent experiments. SE <8% of the mean values.
3178 I. Haferkamp et al. (Eur. J. Biochem. 269) Ó FEBS 2002
and thus, about 10 times higher compared to the apparent
affinities of the single plant AACs in the E. coli system
(Table 3). A major problem for studies with intact plant
mitochondria is the potential loss of the PMF during the
isolation procedure. As pointed out by Walker et al. [42],
glycine uptake into isolated mitochondria is PMF-depend-
ent. The isolated mitochondria used in our experiments for
nucleotide uptake showed a strong CCCP inhibition of
glycine uptake down to 8% (data not shown). However, it
remains unclear whether the PMF of isolated plant
mitochondria is sufficient to allow measurements of external
ADP/ATP affinities of energized mitochondria in vivo.

proteoliposome system could not be considered; (c)
unfortunately, the mitochondrial-type AAC did not
exhibit the unidirectional insertion in proteoliposomes
[50]. The orientation of the reconstituted pea AACs were
not examined so that the determination of ADP and ATP
affinities is a mixture of external and internal K
m
values.
In reconstitution experiments it is difficult to control the
orientation of mitochondrial-type AACs; a mutation of a
single amino acid could lead to a change of orientation
from about 50 : 50 (right side-out/inside-out) for the wild-
type to a ratio of 80 : 20 for some mutants [19].
Another approach was the reconstitution of a chroma-
tography-purified AAC from maize mitochondria [51]. The
deduced K
m
values for ADP (26 l
M
)andATP(17l
M
)are
close to our data obtained in the E. coli system. The choice
of phospholipids, detergents and buffer concentrations as
well as the presence of PMF dramatically influence the
nucleotide exchange rates [19].
In conclusion, the E. coli expression system reveals
similar biochemical properties for the three AAC isoforms
from Arabidopsis thaliana. Therefore, it would be interesting
to find out whether the expression of these isoforms is tissue

various mitochondrial-type AACs and the easy determin-
ation of biochemical features that are comparable to native
properties enables an interesting array of structure–function
studies based on site-directed mutagenesis to commence.
ACKNOWLEDGEMENTS
This work was financially supported by the Deutsche Forschungs-
gemeinschaft (TJ 5/1-1, TJ 5/1-2). We thank Dipl. Ing. Zeina Mezher
(Pflanzenphysiologie, Universita
¨
t Kaiserslautern, Germany) for dis-
cussing and critically reading the manuscript. We also thank Michaela
Leroch for the help with the nucleotide uptake experiments. The
support and helpful discussions of Prof H. E. Neuhaus (Pflanzenphys-
iologie, Universita
¨
t-Kaiserslautern) are gratefully acknowledged.
Table 3. K
m
and V
max
values for ATP and ADP of isolated plant mitochondria under different energy conditions. K
m
isgivenin[l
M
], V
max
is given in
(nmolÆmg protein
)1
Æh

(1989) Two bovine genes for mitochondrial ADP/ATP
translocase expressed differently in various tissues. Biochemistry
28, 866–873.
5. Klingenberg, M. (1989) Molecular aspects of the adenine
nucleotide carrier from mitochondria. Arch. Biochem. Biophys.
270, 1–14.
6.Hatanaka,T.,Takemoto,Y.,Hashimoto,M.,Majima,E.,
Shinohara, Y. & Terada, H. (2001) Significant expression of
functional human type 1 mitochondrial ADP/ATP carrier in yeast
mitochondria. Biol. Pharm. Bull. 24, 595–599.
7. Kolarov, J., Kolarova, N. & Nelson, N. (1990) A third ADP/ATP
translocator gene in yeast. J. Biol. Chem. 265, 12711–12716.
8. Lo
¨
ytynoja, A. & Milinkovitch, M.C. (2001) Molecular phyloge-
netic analyses of the mitochondrial ADP-ATP carriers: the Plan-
tae/Fungi/Metazoa trichotomy revisited. Proc. Natl Acad. Sci.
USA 98, 10202–10207.
9. Shinohara, Y., Kamida, M., Yamazaki, N. & Terada, H. (1993)
Isolation and characterization of cDNA clones and a genomic
clone encoding rat mitochondrial adenine nucleotide translocator.
Biochim. Biophys. Acta 1152, 192–196.
10. Drgon, T., Sabova, L., Nelson, N. & Kolarov, J. (1991) ADP/
ATP translocator is essential only for anaerobic growth of yeast
Saccharomyces cerevisiae. FEBS Lett. 289, 159–162.
11. Drgon, T., Sabova, L., Gavurnikova, G. & Kolarov, J. (1992)
Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic
properties but their deletion produces different phenotypes. FEBS
Lett. 304, 277–280.
12. Zhang, Y.Q., Roote, J., Brogna, S., Davis, A.W., Barbash, D.A.,

J. Bacteriol. 181, 1196–1202.
16. Krause, D.C., Winkler, H.H. & Wood, D.O. (1985) Cloning and
expression of the Rickettsia prowazekii ADP/ATP translocator in
Escherichia coli. Proc. Natl Acad. Sci. USA 82, 3015–3019.
17. Fiermonte, G., Walker, J.E. & Palmieri, F. (1993) Abundant
bacterial expression and reconstitution of an intrinsic membrane-
transport protein from bovine mitochondria. Biochem. J. 294,
293–299.
18. Fiermonte,G.,Palmieri,L.,Dolce,V.,Lasorsa,F.M.,Palmieri,
F., Runswick, M.J. & Walker, J.E. (1998) The sequence, bacterial
expression, and functional reconstitution of the rat mitochondrial
dicarboxylate transporter cloned via distant homologs in yeast and
Caenorhabditis elegans. J. Biol. Chem. 273, 24754–24759.
19. Heimpel, S., Basset, G., Odoy, S. & Klingenberg, M. (2001)
Expression of the mitochondrial ADP/ATP carrier in Escherichia
coli. J. Biol. Chem. 276, 11499–11506.
20. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular
Cloning: a Laboratory Manual. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, New York, USA.
21. Studier, F.W., Rosenberg, A.H., Dunn, J.J. & Dubendorff, J.W.
(1990) The use of T7 polymerase to direct expression of cloned
genes. Methods Enzymol. 185, 60–89.
22. Winkler, H.H. (1986) Membrane transport in Rickettsia. Methods
Enzymol. 125, 253–259.
23. Mangold, H.K. (1967) Nukleinsa
¨
uren und Nukleotide. In
Du
¨
nnschicht-Chromatographie. Ein Laboratoriumshandbuch

199–232.
30. Kra
¨
mer, R. & Klingenberg, M. (1977) Reconstitution of adenine
nucleotide transport with purified ADP/ATP-carrier protein.
FEBS Lett. 82, 363–367.
31.Brandolin,G.,Doussiere,J.,Gulik,A.,Gulik-Krzywicki,T.,
Lauquin, G.J. & Vignais, P.V. (1980) Kinetic, binding and ultra-
structural properties of the beef heart adenine nucleotide carrier
protein after incorporation into phospholipid vesicles. Biochim.
Biophys. Acta 592, 592–614.
32. Hoffmann,B.,Stockl,A.,Schlame,M.,Beyer,K.&Klingenberg,
M. (1994) The reconstituted ADP/ATP carrier activity has an
absolute requirement for cardiolipin as shown in cysteine mutants.
J. Biol. Chem. 269, 1940–1944.
33. Beyer, K. & Klingenberg, M. (1985) ADP/ATP carrier protein
from beef heart mitochondria has high amounts of tightly bound
cardiolipin, as revealed by
31
P nuclear magnetic resonance. Bio-
chemistry 24, 3821–3826.
34. vanderDoes,C.,Swaving,J.,vanKlompenburg,W.&Driessen,
J.M. (2000) Non-bilayer lipids stimulate the activity of the
reconstituted bacterial protein translocase. J. Biol. Chem. 275,
2472–2478.
35. Stubbs, M. (1981) Inhibitors of the adenine nucleotide translocase.
Int. Encycl. Pharm. Ther. 107, 283–304.
36. Souverijn, J.H.M., Huisman, L.A., Rosing, J. & Kemp, A. (1973)
Comparison of ADP and ATP as substrates for the adenine
nucleotide translocator in rat-liver mitochondria. Biochim.

particulate components and soluble cytoplasm of isolated rat-liver
cells. Biochim. Biophys. Acta 333, 393–399.
46. Akerboom, T.P.M., Bookelman, H., Zuurendonk, P.F., van der
Meer, R. & Tager, J.M. (2001) Intramitochondrial and extra-
mitochondrial concentrations of adenine nucleotides and in-
organic phosphate in isolated hepatocytes from fasted rats. Eur. J.
Biochem. 84, 413–420.
47. Stitt, M.McC., Lilley, R. & Heldt, H.W. (1982) Adenine nucleo-
tide levels in the cytosol, chloroplasts and mitochondria of wheat
leaf protoplasts. Plant Physiol. 70, 971–977.
48. Stitt, M.McC., Lilley, R., Gerhardt, R. & Heldt, H.W. (1989)
Metabolite levels in specific cells and subcellular compartments of
plant leaves. Methods Enzymol. 174, 518–552.
49. Schu
¨
nemann,D.,Borchert,S.,Flu
¨
gge, U.I. & Heldt, H.W. (1993)
ATP/ADP translocator from pea root plastids. Comparison with
translocators from spinach chloroplasts and pea leaf mitochon-
dria. Plant Physiol. 103, 131–137.
50. Palmieri, F. (1994) Mitochondrial carrier proteins. FEBS Lett.
346, 48–54.
51. Genchi,G.,Ponzone,C.,Bisaccia,F.,DeSantis,A.,Stefanizzi,L.
& Palmieri, F. (1996) Purification and characterization of the
reconstitutively active adenine nucleotide carrier from maize
mitochondria. Plant Physiol. 112, 845–851.
52. Marvin-Sikkema,F.D.,Driessen,A.J.M.,Gottschal,J.C.&Prins,
R.A. (1994) Metabolic energy generation in hydrogenosomes of
the anaerobic fungus Neocallimastix: Evidence for a functional