Voncken2, Guillaume Schmit1and Joachim Tjaden1 1 Pflanzenphysiologie, Universita¨t Kaiserslautern, Germany;2Department of Evolutionary Microbiology, Faculty of Science, Catholic Universit
Trang 1Functional integration of mitochondrial and hydrogenosomal ADP/ATP
characteristics for plants, mammals and anaerobic chytrids
Ilka Haferkamp1, Johannes H P Hackstein2, Frank G J Voncken2, Guillaume Schmit1and Joachim Tjaden1
1
Pflanzenphysiologie, Universita¨t Kaiserslautern, Germany;2Department 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
Km values of about 40 lM for ADP, and 105 lM or
140 lM, respectively, for ATP have been determined,
similar to the data reported for isolated rat
mitochon-dria.The apparent Kmfor ATP decreased up to 10-fold
in the presence of the protonophore
m-chlorocarbonyl-cyanide phenylhydrazone (CCCP).The hydrogenosomal
AAC isolated from the chytrid fungus Neocallimastix
spec.L2 exhibited the same characteristics, but the
affinities for ADP (165 lM) and ATP (2 33 mM) were
significantly lower.Notably, AAC1-3 from Arabidopsis
thaliana and AAC1 from Solanum tuberosum (potato)
showed significantly higher external affinities for both nucleotides (10–22 lM); they were only slightly influenced
by CCCP
Studies on intact plant mitochondria confirmed these observations.Back exchange experiments with preloaded
E colicells expressing AACs indicate a preferential export
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), two to four AAC isoforms
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
to represent a mixture of mitochondrial variants that possess different isoforms of AACs.Consequently, a biochemical characterization of the various isoforms of AACs is difficult, if not impossible using conventional methods of cell fractionation.The function of the different AACs in S cerevisiae could be studied taking advantage of the sophisticated genetic tools available for this organism: deletion mutants for all three isoforms have been generated that allowed a detailed analysis of particular AACs in the absence of other isoforms [10,11].Trivially, comparable studies are only possible in organisms with a highly developed genetic tool kit (cf Drosophila [12]).Therefore, alternative methods are required to study particular iso-forms of mitochondrial-type AACs in organisms that do not allow the application of sophisticated genetic tech-niques.The aim of the present study is the development of a simple and reproducible approach to analyse the biochemi-cal properties [substrate affinities, proton motive force (PMF) dependency] of individual mitochondrial-type AACs
in order to identify differences in the function of the various isoforms and to understand the repeated evolution of AAC isoforms in a variety of organisms
We have shown earlier that it is possible to express adenine nucleotide transporters of plastids and certain parasites, i.e Chlamydia and Rickettsia, in Escherichia coli and to test their function in vivo [13–16].This technique
Correspondence to J.Tjaden, Pflanzenphysiologie, Universita¨t
Kaiserslautern, Erwin-Schro¨dinger-Str., D-67663 Kaiserslautern.
Fax: + 631 2052600, Tel.: + 631 2052505,
E-mail: tjaden@rhrk.uni-kl.de
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)
Trang 2might 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
investigated three AAC isoforms from Arabidopsis thaliana
and one AAC from potato (Solanum tuberosum).The
results obtained by expression in E coli were justified by
comparison with the adenylate uptake into intact plant
mitochondria isolated from Arabidopsis and Solanum leaves
or potato tubers, respectively
E X P E R I M E N T A L P R O C E D U R E S
DNA constructs for heterologous expression of AACs
inE coli
DNA manipulations were performed essentially as
des-cribed in Sambrook et al.[20].The expression plasmids
(pet16b, Novagen, Heidelberg, Germany) encoding the
recombinant AAC proteins with an additional N-terminal
tag of 10 histidine residues were constructed as follows: the
cDNA coding the entire AACs were generated by PCR
from first-strand cDNA of plants and mammals
(Arabid-opsis thaliana, Solanum tuberosum and Rattus norwegicus) or
from a full-length cDNA clone (Neocallimastix spec.L2)
with Pfx DNA polymerase (Gibco BRL, Eggenheim,
Germany), which possesses proof reading activity.Sense
primers incorporating an NdeI restriction site and antisense
primers were used for the PCR reaction (Table 1).The obtained PCR products were purified (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), subcloned into the EcoRV restriction site of the plasmid pBSK (Stratagene, Heidelberg, Germany) and checked by sequencing both strands by chain-termination reaction (MWG-Biotech, Ebersberg, Germany).For the construction of the E coli expression plasmids (encoding His10–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/gene¼
F20O9.60 (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 inE 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 37C either in YTAmp/Clm medium (YT:
5 gÆL)1 yeast extract, 8 gÆL)1 peptone, 2.5 gÆL)1 NaCl,
pH 7.0; for hydrogenosomal AAC) or in TBAmp/Clm medium (TB: 2.5 gÆL)1 KH2PO4, 12 5 gÆL)1 K2HPO4,
12 gÆL)1 peptone, 24 gÆL)1 yeast extract, 0.4% glycerin,
pH 7.0) supplemented with 10 mM malate and 10 mM
pyruvate (rat and plant AACs) [20].A D600 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 The lower-case letter indicates the introduced base exchange to create restriction sites (NdeI or BglII).
Trang 3induction and collected by centrifugation for 5 min at
5000 g (8C, Sorvall RC5B centrifuge, rotor type SS34;
Sorvall-Du Pont, Dreieich, Germany).The sediments were
resuspended to D600¼ 5 using potassium phosphate
buffer (50 mM, pH 7.0) [16] and stored at 8C until use
Uptake of radioactively labeled ATP and ADP
IPTG-induced E coli cells (100 lL) harbouring the AAC
expressing plasmids (or the given controls) were added to
100 lL potassium phosphate buffer (50 mM, pH 7 0)
containing radioactively labeled ATP or ADP.[a-32P]
adenine nucleotides were used at specific activities between
100 and 3000 lCiÆlmol)1 [a-32P]ADP was synthesized
enzymatically from [a-32P]ATP (NEN, Bad Homburg,
Germany), as described by Tjaden et al [13], and the
purity of the [a-32P]ADP preparation was confirmed by a
thin-layer chromatography [13].Uptake of nucleotides
was carried out at 30C in an Eppendorf reaction vessel
incubator and terminated after the indicated time periods
by transferring the cells to a 0.45-lm filter (25 mm
diameter, Orange Scientific, Waterloo, Belgium) under
vacuum previously moistened with potassium phosphate
buffer (50 mM, pH 7.0) [22] Cells were further washed to
remove unimported radioactivity by addition of three
times 4-mL potassium phosphate buffer (50 mM, pH 7 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 30C with potassium
phos-phate buffer (50 mM, pH 7.0) containing [a-32P]ADP or
[a-32P]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
incubated for 1.5 min at room temperature The cells were
sedimented for 2 min at room temperature in an
eppen-dorf centrifuge (5000 g).The supernatents were
trans-ferred to new reaction vessels and heated to 95C for
10 min to prevent any further reaction.An aliquot of the
exported radioactive solution was used for separation by
thin-layer chromatography [23]
Thin-layer chromatography of radioactively labeled
adenine nucleotides
To identify the type of adenine nucleotide exported from
[a-32P]ADP or [a-32P]ATP preloaded E coli cells, we
employed a thin-layer chromatography system according
to the method of Mangold [23].Radioactively labeled
samples were loaded onto a 0.5-mm poly(ethylene amine)
cellulose thin-layer chromatography plate and dried with a
fan Separation was carried out for 0.5 min using 0.5M
sodium formiate (pH 3.4), for 2 min using 2M sodium
formiate (pH 3.4) and the front was allowed to run for
15 cm with 4M sodium formiate (pH 3.4) Rf values of
radioactively labeled adenine nucleotides were determined
after autoradiography and correspond to Rf values of
unlabeled nucleotides visualized under UV light [23]
Radiolabeling of AAC proteins synthesized inE 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 D600¼ 0.5, and resuspended in 1 mL of a methionine assay medium containing 42 mM Na2HPO4,
20 mM KH2PO4, 18 mM NH4Cl, 8.5 mM NaCl, 1 mM
MgSO4, 0 1 mMCaCl2, 20 mMglucose, and 0.1 mgÆmL)1 thiamine (Difco Laboratories, Heidelberg, Germany).T7-RNA polymerase synthesis was induced by adding 1 mM
IPTG.After shaking the culture for 15 min at 37C, 20 lL rifampicin (stock 20 mgÆmL)1, dissolved in methanol) was added to inhibit the E coli RNA polymerase E coli cells were shaken for additional 15 min after which 5 lL [35S]methionine (50 lCi) were added to label newly syn-thesized proteins for 20 min at room temperature.Cells were sedimented by centrifugation and transferred to liquid nitrogen to destroy cell intactness.After resuspension in a medium consisting of 10 mM Tris/HCl (pH 7.5), 1 mM
EDTA, 0.1 mMpefabloc 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, 4C)
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 mM imidazole, 300 mM NaCl, 100 mM Na2HPO4
(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, 4C) 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 acetone (80% f.c.), incubated at )70 C for 2 h and sedimented by centrifugation (15 800 g, 10 min, 4C) For SDS/PAGE, the air dried protein sediments were resuspended in 40 lL of double concentrated SDS/PAGE sample buffer medium and incubated on ice for 60 min Finally, the preparation was applied to a polyacryl-amide gel (3% stacking gel, 15% running gel) for electro-phoresis in the presence of 0.1% sodium dodecyl sulfate (SDS/PAGE).After drying, the gels were autoradiographed for 3 days
Preparation and uptake experiments of plant mitochondria
Mitochondria were isolated from potato and Arabidopsis leaves and from potato tubers by isopycnic centrifugation in density gradients of Percoll following the procedure of Neuburger et al.[24].The plant material was ground in prechilled extraction medium.The homogenate obtained after the mincing process was squeezed through eight layers
of muslin.The flow-through was subject to several centrif-ugation steps.The resulting pellet was layered on Percoll gradient.To obtain a discrete fraction, we increased the Percoll concentration up to 32% dependent on the plant material.To obtain energized mitochondria, we added
5 mM glycine, 10 mMmalate, 10 mMpyruvate and 2 mM
NAD+ during the isolation procedure as well as in the
Trang 4storage medium.The mitochondrial fraction was washed
and resuspended in 20 mMTris/HCl (pH 7.4), 2 mMMgCl2
and 200 mMsucrose.Purity and intactness were analysed
using marker enzymes, as described previously [25–27]
Uptake of [a-32P]ADP and [a-32P]ATP was carried out
using a rapid filtration technique, as described by Winkler
et al.[28]
R E S U L T S
Heterologous expression of AACs inE 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
in E coli (BL21, DE3).Induction by IPTG was carried out
in the presence of radioactive [35S]methionine in order to
detect even low amounts of newly synthesized proteins
The membrane fractions of the induced E coli cells were
isolated and the recombinant AAC proteins were further
purified by Ni-nitrilotriacetic acid chromatograpy.The
autoradiography of the corresponding SDS/PAGE
pre-sented in Fig.1 confirmed that all AACs studied had been
inserted into the bacterial membrane.The overexpressed
AAC proteins migrate according to their calculated
apparent molecular masses (Fig.1, lanes 2–8) Notably,
the expression levels of AAC2 from rat, AAC1 from
potato and AAC3 from Arabidopsis are much higher than
those of the AAC1 and AAC2 isoforms from Arabidopsis
and AAC1 from rat.The hydrogenosomal AAC from
Neocallimastix spec.L2 exhibited the lowest expression
level.Furthermore, Western blot analyses of purified
membranes of the induced E coli cells using an anti-His
Ig have corroborated the integration of the AACs into
the bacterial membrane (data not shown).Not even traces
of the recombinant proteins were detectable in the
corresponding membrane fraction isolated from induced
E colicells harbouring the empty plasmid pet16b (Fig.1,
lane 1)
Interestingly, when E coli cells hosting plant and mam-malian AACs are induced for protein expression in normal growth medium (YT, see Experimental procedures), the growth was retarded with respect to control.These E coli cells reached a stationary phase at a D600 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 D600reached a value of about 12
It was tempting to analyse whether the heterologously expressed AACs integrated in the E 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-tion31P-NMR, the AAC from beef heart mitochondria has high amounts of tightly bound cardiolipin and the removal
of these lipids renders the carrier inactive [33].The phospholipid composition of the E coli inner membrane
is clearly different from the mitochondrial membranes.It corresponds to about 30% of the acidic phospholipids phosphatidylglycerol and cardiolipin (phosphatidylglycerol, 20–25%; cardiolipin, 5–10% [34]), which might be crucial for the functional integration of the AACs.Notwithstand-ing, we were able to demonstrate a time dependent nucleotide uptake for all seven investigated AACs (Fig.2, Table 2)
Figure 2 shows representative diagrams for the plant (Fig.2A; AAC2, A.t.), mammalian (Fig.2B; AAC2, R.n.) and hydrogenosomal (Fig.2C; hdgAAC, N spec.L2) AACs expressed in E coli.The import of32P-labeled ADP
or ATP into intact E coli cells expressing the plant and mammalian AACs was linear with time for about 30 min, when the cells were harvested under optimal growth conditions.Interestingly, the weakly expressed hydrogeno-somal AAC (Fig.1) showed much higher specific uptake rates.However, the rates are linear only for about 10 min The ratios of ADP to ATP uptake into E coli cells expressing the plant AACs were significantly lower (Fig.2A) than the ratios of ADP to ATP uptake of the expressed rat and hydrogenosomal AACs (Fig.2B,C) Notably, the addition of the specific inhibitors of mitoch-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 [35S]methionine.Details of induction, purification and
autoradiography are given in Experimental procedures.Lane 1, E coli
control cells; lane 2, E coli cells expressing the hydrogenosomal AAC
from Neocallimastix spec.L2; lane 3, E coli cells expressing the AAC1
from Rattus norwegicus; lane 4, E coli cells expressing the AAC2 from
Rattus norwegicus; lane 5, E coli cells expressing the AAC1
from Arabidopsis thaliana; lane 6, E coli cells expressing the
AAC2 from Arabidopsis thaliana; lane 7, E coli cells expressing
the AAC3 from Arabidopsis thaliana; lane 8, E coli cells expressing the
AAC1 from Solanum tuberosum.
Trang 5pet16b control plasmid (without any insert) as well as E coli
wildtype cells are not able to transport ADP or ATP [13]
The import of [a-32P]ADP and [a-32P]ATP by the
recombinant AACs into E coli displays typical
Michaelis–Menten kinetics.The results were plotted as
Lineweaver–Burk and Eadie–Hofstee diagrams, as sum-marized in Table 2.The Kmvalues were determined with or without addition of the protonophore CCCP because nucleotide uptake into well-coupled mitochondria (high energy state) from mammals was shown to be controlled by the membrane potential (proton motive force; PMF) across the inner mitochondrial membrane [36,37].The addition of CCCP also causes a decrease of the PMF across bacterial membranes [15].The apparent Km values for AAC1 and AAC2 from rat mitochondria are about 40 lM for ADP and about 105 and 140 lM for ATP, respectively.The presence of the protonophore CCCP increases the ATP affinity for both AACs up to 10-fold, whereas the ADP affinity is not affected.In contrast, the four plant AACs show significantly higher affinities for both, ADP and ATP The corresponding Kmvalues range between 10 and 22 lM They are only slightly influenced by CCCP.On the other hand, the kinetic data of the hydrogenosomal AAC from Neocallimastixreveals 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 (Vmax) 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 induced E coli cells, then the export of adenylates by the recombinant AACs must depend on the presence of the appropriate externally applied substrate.Therefore, we preloaded IPTG-induced E coli cells (expressing different AACs) with radioactively labeled ADP (or ATP) and performed back exchange experiments in the presence of the various unlabeled external substrates.The radioactively labeled nucleotides, which were released after preloading, were analyzed by thin-layer (poly(ethylene amine) chro-matography.To investigate to what degree incorporated nucleotides are metabolized by the E coli cells, we disrupted the cells after preloading with ADP (or ATP)
Fig 2 Time dependency of [a 32 P]ADP (j), [a 32 P]ATP (s) uptake into intact E coli cells IPTG-induced E coli cells harbouring plasmid encoding several AACs were incubated with (A) 7.5 l M ADP or ATP (AAC2, Arabidopsis thaliana) (B) 12 5 l M ADP or ATP (AAC2, Rattus norwegicus) and (C) 500 l M ADP or ATP (hdgAAC Neocal-limastix spec.L2) for the indicated time periods.Data is the mean of three independent experiments.SE <7% of the mean values.
Trang 6alone.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
experiments
Plant mitochondria were isolated according to the method
of Neuburger et al.[24] with some improvements, as
described in the Experimental procedures.The percentage
of intactness ranged from 85 to 96%.Contaminations with
intact peroxisomes and plastids were not detectable.To
obtain energized mitochondria, we added glycine, malate,
pyruvate and NAD+to the media used for isolation and
storage [39,40]
To analyse uptake of radioactively labeled nucleotides
into intact plant mitochondria a rapid filtration technique
was used [28].At the end of the incubation period,
mitochondria were filtered through membrane filters
previ-ously set under vacuum.An advantage of this accurate and
reproducible method in comparison to the silicon oil
centrifugation is the complete removal of the pool of nucleotides which are presumably present in the space between the inner and outer membranes and thus no corrections for external pool variations are required [28] The time course of [a-32P]ADP or [a-32P]ATP uptake into potato tuber mitochondria and Arabidopsis leaf mitochon-dria at 0C is shown in Fig.4.Uptake is linear for about
10 s and reaches saturation after 20–30 s.Nucleotide uptake was significantly faster for ADP than for ATP into 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 Kmvalues, 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 lM (Table 3) for both, ADP and ATP, and thus significantly higher than those described for rat mitochondria [28,36]
D I S C U S S I O N
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 measure the uptake of radioactively labeled ADP and ATP into intact E coli cells
Two AACs from rat, which were studied in a similar way, could be expressed, and the nucleotide exchange data could
be compared with those published by different research groups for isolated rat mitochondria.The Kmvalues are described to be around 10–30 lMfor ADP and about 100–
150 lMfor ATP in energy-rich mitochondria [28,36].These values closely resemble those obtained with the energized
E coli expressing the two rat AACs which show an
Table 2 K m and V max values for ATP and ADP of several heterologously expressed AACs determined on intact E coli cells under various energy conditions (coupled and uncoupled) K m is given in [l M ], V max is given in (nmolÆmg protein)1Æh)1), E coli cells were preincubated with 100 l M CCCP for 2 min for uncoupling.
Trang 7apparent Kmvalue of 38 lM (AAC1) and 40 lM (AAC2)
for ADP and an apparent Kmvalue of 105 lM(AAC1) and
140 lM(AAC2) for ATP (Table 2).In energy-depleted rat
mitochondria, the affinity for ADP is nearly unchanged in
contrast to the highly increased affinity for ATP [36]
Similar findings were made by uncoupling E coli cells
expressing the rat AACs with CCCP.The affinity for ADP
of both rat AACs was slightly changed in contrast to the
significantly affected ATP affinity which increased up to
12–30 l (Table 2)
Importantly, E coli cells must be kept energized after induction in order to obtain biochemical characteristics for recombinant AACs, comparable with the data observed for energized mitochondria.We conclude that a direct corre-lation exists between the nonretarded growth of E coli and the energization of the cells, which is necessary for building
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 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-32P]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
E coli cells after preloading; lane 2 (A–C), separation of radioactive
compounds exported by E coli in counter exchange without any
exogenous substrate (control); lane 3 (A–C), separation of radioactive
compounds exported by E coli in counter exchange to exogenous
ATP [(A): 75 l M ; (B): 125 l M ; (C): 500 l M )]; lane 4 (A–C), separation
of radioactive compounds exported by E coli in counter exchange to
exogenous ADP [(A): 75 l M ; (B): 125 l M ; (C): 500 l M )]; lane 5 (A–C),
separation of radioactive compounds exported by E coli in counter
exchange to exogenous ATP (concentrations see lane 3) in the presence
of CCCP (100 l M ); lane 6 (A–C), separation of radioactive
com-pounds exported by E coli in counter exchange to exogenous ADP
(concentrations see lane 4) in the presence of CCCP (100 l M ).
Fig 4 Time dependency of [a- 32 P]ADP, [a- 32 P]ATP uptake into iso-lated mitochondria Isoiso-lated potato tuber mitochondria (A) and Ara-bidopsis leaf mitochondria (B) were incubated at 0 C with 1 l M
radioactively labeled ADP (j) or 1 l M radioactively labeled ATP (d) for the indicated time periods.For inhibition of ADP (h) or ATP (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.
Trang 8and 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
Nevertheless, these results indicate different characteristics
for plant AACs compared to mammalian AACs.In
mammals, the uptake into well-coupled mitochondria (high
energy state) of cytosolic ADP in exchange for organellar
ATP is controlled via the membrane potential [36,37].This
suggests an asymmetrical exchange of external ADP against
internal ATP although the ATP/ADP ratio is greater
outside the rat mitochondria than inside [43–46].Keeping in
mind that plants have also a high cytosolic ATP/ADP ratio
of about 10 [47,48], it remains unclear how plant
mitochon-dria control their nucleotide exchange between cytosolic
ADP and intramitochondrial ATP if the external affinities
for both nucleotides are similar under energized conditions
However, back-exchange experiments after preloading
E colicells expressing different AACs demonstrate clearly
that under energized conditions the preferred export
nuc-leotide is ATP rather than ADP, especially for the plant
AACs (Fig.3)
Investigations of plant nucleotide transport using
reconstituted mitochondrial membranes from pea leaves
exhibit apparent affinities (94 lM for ADP; 53 lM for
ATP), which do not appear to be in accordance with our
data [49].This difference may be attributed to several
reasons: (a) the lack of cardiolipin might create a quite
artificial environment for mitochondrial membrane
pro-teins; (b) in addition, the presence of a PMF in this
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 Kmvalues
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 values for ADP (26 lM) and ATP (17 lM) 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 specific or occurs at certain stages of plant development or under special environmental conditions.Future studies with promotor–glucuronidase fusions might give an insight into these open questions
The anaerobic chytrid fungus Neocallimastix spec.L2 is the first anaerobic eukaryote in which a mitochondrial-type AAC has been identified in its hydrogenosomes by protein sequencing and phylogenetic analysis of the corresponding (nuclear) gene [3].Here, we have shown that the nucleotide affinities of this fungal AAC are significantly lower than those of plant and mammalian AACs (Table 2).This fact might be due to the substantially different metabolism of hydrogenosomes (anaerobic) and mitochondria (aerobic), and the composition of the organelle membranes.Notably, the hydrogenosomal AAC possesses similar transport characteristics as the mitochondrial AACs from mammals concerning the PMF dependency (Table 2).Back exchange experiments demonstrate clearly that the nucleotide trans-port mediated by the hydrogenosomal AAC takes place in a counter exchange mode (Fig.2).It is noteworthy that the affinity for ATP import is strongly dependent on the PMF over the bacterial membrane.Upon addition of the membrane uncoupler CCCP, the affinity for ATP increased about 10-fold (Table 2).The indicated PMF-dependence of energy transport mediated by the hydrogenosomal AAC postulates also the presence of a PMF across the inner membrane of hydrogenosomes.Indeed, there is ample evidence that a PMF is present in hydrogenosomes of Neocallimastixspec.L2 [52,53]
The functional heterologous expression in E coli of 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
A C K N O W L E D G E M E N T S
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 is given in [l M ], V max is given in (nmolÆmg protein)1Æh)1).Mitochondria were preincubated with 100 l M CCCP for 2 min for uncoupling.
Trang 9R E F E R E N C E S
1.Klingenberg, M.(1976) The state of ADP or ATP fixed to the
mitochondria by bongkrekate Eur J Biochem 65, 601–605.
2 Winkler, H.H & Neuhaus, H.E (1999) Non-mitochondrial
ade-nylate transport TIBS 24, 64–68.
3.Voncken, F.(2001) Hydrogenosomes: eukaryotic adaptations to
anaerobic environments.PhD Thesis, University of Nijmegen, the
Netherlands.
4 Powell, S.J., Medd, S.M., Runswick, M.J & Walker, J.E.
(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.,
Nash, D.& Ashburner, M.(1999) stress sensitive B encodes an
adenine nucleotide translocase in Drosophila melanogaster.
Genetics 153, 891–903.
13 Tjaden, J., Schwo¨ppe, C., Mo¨hlmann, T.& Neuhaus, H.E.(1998)
Expression of the plastidic ATP/ADP transporter gene in
Escherichia coli lead to the presence of a functional adenine
nucleotide transport system in the bacterial cytoplasmic
mem-brane J Biol Chem 273, 9630–9636.
14.Mo¨hlmann, T., Tjaden, J., Schwo¨ppe, C., Winkler, H.H.,
Kampfenkel, K.& Neuhaus, H E.(1998) Occurence of two
plastidic ATP/ADP transporters in Arabidopsis thaliana:
Molecular characterisation and comparative structural analysis of
homologous ATP/ADP translocators from plastids and Rickettsia
prowazekii Eur J Biochem 252, 353–359.
15 Tjaden, J., van der Laan, M., Schwo¨ppe, C., Mo¨hlmann, T.,
Winkler, H.H & Neuhaus, H.E (1999) Two nucleotide transport
proteins in Chlamydia trachomatis.One for net nucleoside
tri-phosphate uptake and the other for the transport of energy.
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 (Stahl, E., ed.), pp 749–769 Springer, Heidelberg, Germany.
24 Neuburger, M , Journet, E -P , Bligny, R , Carde, J P & Douce, R.(1982) Purification of plant mitochondria by isopycnic cen-trifugation in density gradients of Percoll Arch Biochem Biophys.
217, 312–323.
25 Batz, O , Scheibe, R & Neuhaus, H E (1992) Transport processes and corresponding changes in metabolite levels in relation to starch synthesis in barley (Hordeum vulgare L.) etioplasts Plant Physiol 100, 184–190.
26.Herna´ndez, J.A., Corpas, F.J., Go´mez, M., del Rı´o, L.A & Sevilla, F.(1993) Salt-induced oxidative stress mediated by acti-vated oxygen species in pea leaf mitochondria Physiol Plant 89, 103–110.
27.Pastori, G M.& del Rı´o, L.A (1994) An activated-oxygen-mediated role for peroxisomes in the mechanism of senescence of Pisum sativum L.leaves.Planta 193, 385–391.
28 Winkler, H.H., Bygrave, F.L & Lehninger, A.L (1968) Char-acterization of the atractyloside-sensitive adenine nucleotide transport system in rat liver mitochondria J Biol Chem 243, 20–28.
29.Dowhan, W.(1997) Molecular basis for membrane phospholipid diversity: why are there so many lipids? Ann Rev Biochem 66, 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 van der Does, C , Swaving, J , van Klompenburg, 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 Biophys Acta 305, 185–198.
37 Vignais, P.V., Vignais, P.M & Doussiere, J (1975) Functional relationship between the ADP/ATP-carrier and the F 1 -ATPase in mitochondria Biochim Biophys Acta 376, 219–230.
Trang 1038.Klingenberg, M.(1993) Dialectics in carrier research: The ADP/
ATP carrier and the uncoupling protein J Bioenerg Biomem 25,
447–457.
39 Ebbighausen, H., Jia, C & Heldt, H.W (1985) Oxaloacetate
translocator in plant mitochondria Biochim Biophys Acta 810,
184–199.
40.Douce, R.& Neuburger, M.(1989) The uniqueness of plant
mitochondria Annu Rev Plant Physiol Plant Mol Biol 40, 371–
414.
41.Brierley, G.& O’Brien, R L.(1965) Compartmentation of heart
mitochondria.II.Mitochondrial adenine nucleotides and the
action of atractyloside J Biol Chem 240, 4532–4539.
42 Walker, G.H., Sarojini, G & Oliver, D.J (1982) Identification of a
glycine transporter from pea leaf mitochondria Biochem Biophys.
Res Commun 107, 856–861.
43 Heldt, H.W., Klingenberg, M & Milovancev, M (1972)
Differences between the ATP-ADP ratios in the mitochondrial
matrix and in the extramitochondrial space Eur J Biochem 30,
434–440.
44 Vignais, P.V., Vignais, P.M., Lauquin, G & Morel, F (1973)
Binding of adenosine diphosphate and of antagonist ligandes to
the mitochondrial ADP carrier Biochimie 55, 763–778.
45 Zuurendonk, P.F & Tager, J.M (1974) Rapid separation of
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 , De Santis, 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 relationship with mitochondria Mycol Res 98, 205–212.
53 Biagini, G.A., van der Giezen, M., Hill, B., Winters, C & Lloyd, D.(1997) Ca2+accumulation in the hydrogenosomes of Neo-callimastix frontalis L2: a mitochondrial-like physiological role FEMS Microbiol Lett 149, 227–232.