THE JOURNAL OF BIOLOGICAL CHEMISTRY © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 13, Issue of March 31, pp. 9709 –9715, 2000 Printed in U.S.A.
Oxidative Phosphorylation, Ca2ⴙ Transport, and Fatty Acid-induced Uncoupling in Malaria Parasites Mitochondria* (Received for publication, July 27, 1999, and in revised form, December 29, 1999)
Sergio A. Uyemura‡, Shuhong Luo, Silvia N. J. Moreno, and Roberto Docampo§ From the Laboratory of Molecular Parasitology, Department of Pathobiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61802
Malaria remains one of the leading causes of morbidity and mortality in the developing world. An estimated 300 –500 million cases of malaria each year result in about one million deaths, mainly children under 5 years of age in Africa (1). Malaria is caused by protozoan parasites of the genus Plasmodium; of which four species (P. falciparum, P. vivax, P. malariae, and P. ovale) infect humans. Other species such as P. knowlesi, P. yoelii, P. berghei, P. chabaudi, and P. gallinaceum, infect a number of wild and domestic animals and are fre-
* This work was supported in part by a Burroughs Wellcome New Initiatives in Malaria Research Award (to R. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Postdoctoral fellow of the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo. Permanent address: Departamento de Ana´lises Clı´nicas, Toxicolo´gicas e Bromatolo´gicas, Faculdade de Cieˆncias Farmace´uticas, Ribeira˜o Preto, Universidade de Sa˜o Paulo, Ribeira˜o Preto, 14049 Sa˜o Paulo, Brazil. § To whom correspondence should be addressed: Laboratory of Molecular Parasitology, Dept. of Pathobiology, College of Veterinary Medicine, University of Illinois at Urbana-Champaign, 2001 S. Lincoln Ave., Urbana, IL 61802. Tel.: 217-333-3845; Fax: 217-244-7421; E-mail:
[email protected]. This paper is available on line at http://www.jbc.org
quently used as models for the human parasites. It is usually claimed that mammalian malaria parasites do not have a functional citric acid cycle (2). They are also proposed to lack a mitochondrial ATP synthase and a rotenonesensitive Complex I (3). Because malaria parasites entirely depend on de novo pyrimidine synthesis, a major function of their mitochondrial electron transport chain was proposed as providing a means to dispose electrons generated by dihydroorotate (4). The generation of a proton-motive force necessary for other mitochondrial metabolic reactions was also suggested as an important role for their respiratory chain (3). There is conflicting evidence (5, 6) for the presence of a branched electron transport system involving an alternative oxidase in malaria parasites by which electrons are transferred from ubiquinol to oxygen without involving cytochrome c (3). Although mitochondria from P. yoelii and P. falciparum have been isolated, the process of oxidative phosphorylation or their ability to maintain a membrane potential (⌬⌿) could not be demonstrated (5, 7). A flow cytometry assay has recently been developed to monitor the mitochondrial membrane potential of P. yoelii inside erythrocytes and the changes induced by atovaquone (8). It was concluded that malarial mitochondria do not contribute much to the ATP pool and that these parasites lack the machinery for oxidative phosphorylation (8). This lack of information on mitochondrial physiology in Plasmodium sp. is in contrast to the relevance of mitochondrial activity for the chemotherapy of malaria (8 –11). The antimalarial drug atovaquone has been shown to inhibit electron transport at the bc1 complex (11) and to collapse the mitochondrial membrane potential (8) in malaria parasites, and this effect has been shown to be enhanced by another antimalarial agent: proguanil (12). In this work we report that the use of trophozoites permeabilized with digitonin, a procedure previously established to investigate in situ mitochondrial bioenergetics in trypanosomatids (13–16) and in the related apicomplexan parasite Toxoplasma gondii (17), allowed for the first time the functional characterization of the respiratory chain of a malaria parasite. Our results demonstrate that Complex I, oxidative phosphorylation, and Ca2⫹ transport occur in P. berghei mitochondria, and evidence is presented for the possible presence of an uncoupling protein in these mitochondria. EXPERIMENTAL PROCEDURES
Parasites—Plasmodium berghei berghei (strain NK65) was maintained in vivo in male Balb/c mice by weekly transfer infection. Blood was collected in phosphate-buffered saline (PBS), pH 7.4, containing 10 units of heparin/ml at approximately 60% parasitemia. Red blood cells were washed twice in PBS by centrifugation at 4 °C at 1,500 ⫻ g for 5 min. Washed erythrocytes were diluted 1:2 in PBS and passed over a powdered cellulose column (CF11; Whatman, Clifton, BJ) to remove leukocytes and platelets (18). Contamination of the resulting preparations with white blood cells was always less than 5% as determined by using a Neubauer chamber and Giemsa staining. Red blood cells de-
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Respiration, oxidative phosphorylation, calcium uptake, and the mitochondrial membrane potential of trophozoites of the malaria parasite Plasmodium berghei were assayed in situ after permeabilization with digitonin. ADP promoted an oligomycin-sensitive transition from resting to phosphorylating respiration. Respiration was sensitive to antimycin A and cyanide. The capacity of trophozoites to sustain oxidative phosphorylation was additionally supported by the detection of an oligomycin-sensitive decrease in mitochondrial membrane potential induced by ADP. Phosphorylation of ADP could be obtained in permeabilized trophozoites in the presence of succinate, citrate, ␣-ketoglutarate, glutamate, malate, dihydroorotate, ␣-glycerophosphate, and N,N,Nⴕ,Nⴕ-tetramethyl-p-phenylenediamine. Ca2ⴙ uptake caused membrane depolarization compatible with the existence of an electrogenically mediated Ca2ⴙ transport system in these mitochondria. An uncoupling effect of fatty acids was partly reversed by bovine serum albumin, ATP, or GTP and not affected by atractyloside, ADP, glutamate, or malonate. Evidence for the presence of a mitochondrial uncoupling protein in P. berghei was also obtained by using antibodies raised against plant uncoupling mitochondrial protein. Together these results provide the first direct biochemical evidence of mitochondrial function in ATP synthesis and Ca2ⴙ transport in a malaria parasite and suggest the presence of an Hⴙ conductance in trophozoites similar to that produced by a mitochondrial uncoupling protein.
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1 The abbreviations used are: BSA, bovine serum albumin; BHAM, benzohydroxamate; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; PUMP, plant uncoupling mitochondrial protein; SHAM, salicylhydroxamate; TMPD, N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine.
g of protein) and an amount of white blood cells similar to the amount that could be present in P. berghei preparations were mixed with electrophoresis buffer and treated as described above. Electrophoresed proteins were transferred to nitrocellulose (27) using a Bio-Rad Transblot apparatus. Blots were blocked in 5% nonfat dry milk in PBS and kept overnight at 4 °C. Polyclonal antisera raised against PUMP protein from potato generously supplied by Drs. I. Maia and P. Arruda (Universidade Estadual de Campinas, Campinas, Brazil) were used. A 1:1,000 dilution of antiserum in blocking buffer was applied to blots at 25 °C for 60 min. The nitrocellulose was washed three times for 15 min each with T-PBS buffer (0.1% Tween 20 in PBS) before addition of a 1:20,000 dilution of goat anti-rabbit IgG in blocking buffer for 30 min. Immunoblots were visualized on radiographic film (Kodak) using the ECL chemiluminescence detection kit (Amersham Pharmacia Biotech). RESULTS
The use of digitonin to permeabilize P. berghei plasma membrane enabled us to study oxidative phosphorylation in mitochondria in situ. Fig. 1 (A and B) shows that addition of a low concentration of digitonin (16 M) to suspensions of P. berghei trophozoites (0.6 mg/ml) incubated in the standard buffer containing 5 mM succinate was followed by a small increase in the rate of oxygen uptake. This increase indicated that the plasma membrane became permeable to succinate. The subsequent addition of ADP induced the transition from resting (State 4) to phosphorylating (State 3) respiration. A respiratory control (State 3/State 4) of 1.9 (Table I) was estimated using the value of State 4 respiration after addition of oligomycin (for nomenclature of respiratory States see Ref. 28). FCCP addition, at the concentration used (1 M), partially released the State 4 respiration, which was then completely inhibited by antimycin A (Fig. 1A) or potassium cyanide (Fig. 1B). The inclusion of the respiratory substrate system TMPD/ascorbate, which reduces the respiratory Complex IV, reinitiated respiration that was fully inhibited by 2 mM cyanide (Fig. 1A), which also occurred in the absence of TMPD/ascorbate (Fig. 1B). Fig. 2 shows that addition of the alternative oxidase inhibitors salicylhydroxamate (SHAM, 2 mM) or benzohydroxamate (BHAM, 1 mM, not shown) after (Fig. 2A) or before 1.5 mM cyanide (Fig. 2B) did not affect respiration, which was completely blocked by KCN (Fig 2, A and B) or antimycin A (Fig. 2C). These results rule out the presence, as previously postulated in P. falciparum (6), of a cyanide-insensitive terminal oxidase in P. berghei trophozoites. The operation of a phosphorylating site III is suggested by the results shown in Fig. 1C. Addition of ADP to the antimycin A-poisoned preparation after TMPD/ascorbate (in the absence of succinate) stimulated respiration. The trace WBC in Fig. 1D shows results from an experiment performed to rule out any influence of white blood cell contamination in the results reported. This preparation of white blood cells (99% lymphocytes) was obtained from normal mice using exactly the same procedure used for the preparation of P. berghei trophozoites. Oxygen uptake was very low and addition of either ADP or oligomycin did not significantly affect this activity. The amount of white blood cells used (1 ⫻ 106 cells/ml) was the same as the amount that was present in the trophozoite preparation shown in Fig. 1D, trace Pb. Citrate, glutamate, ␣-ketoglutarate, and malate, substrates commonly used to demonstrate the presence of NADH-ubiquinone oxidoreductase (Complex I) activity in mitochondrial preparations, were also able to stimulate ADP phosphorylation in permeabilized trophozoites (Table I). Other potential NADHdependent substrates, such as pyruvate or NADH (Table I), were unable to stimulate respiration or ADP phosphorylation. In contrast, the potential flavoprotein-linked substrates ␣-glycerophosphate and dihydroorotate (4) (Table I) were able to stimulate phosphorylation. Mitochondrial membrane potential is probably the most sensitive indicator of the energy-coupling condition of the or-
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pleted of leukocytes and platelets were washed once as described above and resuspended in PBS. Infected erythrocytes were enriched using the Percoll method (19). Infected erythrocytes within the 70% Percoll interphase were collected and contained predominantly trophozoites (85– 90% of the red blood cells, the rest containing ring stage and schizont forms), which were found by examination of Giemsa-stained thin blood smears. To isolate the parasites, infected erythrocytes were lysed with 0.1 mg/ml saponin in PBS at room temperature for 5 min. After centrifugation at 1,500 ⫻ g for 5 min at 4 °C to remove red blood cell membranes, the parasites were washed five times in buffer A (116 mM NaCl, 5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, and 50 mM Hepes, pH 7.2). The parasites were resuspended at 1 ⫻ 108 cells/ml in the same buffer. Contamination of the preparation with red blood cells was negligible. Contamination with white blood cells (mostly lymphocytes) was always less than 1% as determined using a Neubauer chamber and Giemsa staining (typically we obtained about 1.2–1.3 ⫻ 109 trophozoites contaminated with 1.0 –1.15 ⫻ 107 white blood cells per mouse). Control experiments were also done with preparations of white blood cells from control mice in amounts similar to those that could contaminate trophozoite preparations. Those numbers of white blood cells were insufficient to detect the changes in respiration, Ca2⫹ transport, or mitochondrial membrane potential observed in the experiments with trophozoites (see “Results”). The protein concentration was determined by the biuret assay (20) in the presence of 0.2% deoxycholate. P. falciparum was obtained as described previously (21). Chemicals—Arsenazo III, ADP, antimycin A, ascorbate, ATP, bovine serum albumin (BSA),1 calcium ionophore A23187, digitonin, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), GTP, oligomycin, potassium cyanide, rotenone, N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine (TMPD), and valinomycin, were purchased from Sigma. Dulbecco’s PBS was from Life Technologies, Inc. All other reagents were of analytical grade. Measurements of Oxygen Uptake—Measurements of oxygen consumption were made with a Clark-type electrode fitted to an oxygraph (Gilson Medical Electronics, Inc., Middleton, WI) in medium containing 0.5% BSA (unless indicated otherwise) and 125 mM sucrose, 65 mM KCl, 10 mM Hepes-KOH (pH 7.2), 5 mM MgCl2, 2 mM potassium phosphate, 0.5 mM EGTA, and (where indicated) 5 mM succinate and 5 M rotenone. Other additions are indicated in the figure legends and in Table I. Representative traces from experiments conducted on at least three separate cell preparations are shown in the figures and Table. Estimation of Mitochondrial Membrane Potential (⌬⌿)—The mitochondrial membrane potential was measured by the safranine method according to Vercesi et al. (13). The calibrations were made in potassium-free medium containing 0.5% BSA, 200 mM sucrose, 10 mM NaHepes buffer (pH 7.2), 5 mM MgCl2, 2.0 mM sodium phosphate, and 0.5 mM EGTA. Absorption spectra and time-dependent absorption changes of safranine O were recorded with an SLM Aminco DW2000 spectrophotometer. Representative traces from experiments conducted on at least three separate cell preparations are shown in the figures. Determination of Ca2⫹ Movements—Variations in free Ca2⫹ concentrations were followed by measuring the changes in the absorbance spectrum of Arsenazo III (22), using the SLM Aminco DW2000 spectrophotometer at the wavelength pair 675– 685 at 28 °C. Each experiment was repeated at least three times, and the figures shown are from representative experiments. Concentrations of the ionic species and complexes at equilibrium were calculated by employing an iterative computer program (23) modified from that described by Fabiato and Fabiato (24) and taking into account the dissociation constants reported by Schwarzenbach et al. (25). SDS Electrophoresis and Preparation of Western Blots— P. berghei and P. falciparum trophozoites (1 ⫻ 109) were resuspended in 100 l of Dulbecco’s PBS. Aliquots (10 l, about 30 g of protein) were mixed with 10 l of electrophoresis buffer (125 mM Tris-HCl, pH 7, 10% (w/v) -mercaptoethanol, 20% (v/v) glycerol, 4.0% (w/v) SDS, 4.0% (w/v) bromphenol blue) and boiled for 5 min prior to application to 10% SDS-polyacrylamide gels. White potato tubers (Solanum tuberosum) were obtained from a local supermarket, and crude mitochondria were prepared as described before (26). White blood cells from normal mice were obtained using the same procedure used for the preparation of P. berghei trophozoites. Aliquots of crude potato mitochondria (about 30
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TABLE I Substrate oxidation by trophozoite mitochondria in situ Experimental conditions are as described in the legend to Fig. 1 and under “Experimental Procedure.” Rate of respiration Substrate State 3
RCRa
State 4
nmol of O2/min ⫻ mg of protein
Succinate Citrate Glutamate ␣-Ketoglutarate Malate ␣-Glycerophosphate Dihydroorotate Pyruvate NADH
18.1 ⫾ 0.4b 4.9 ⫾ 0.7 2.6 ⫾ 0.4 6.1 ⫾ 0.8 4.3 ⫾ 0.3 6.6 ⫾ 0.5 2.5 ⫾ 0.1 NDc ND
9.6 ⫾ 0.3 1.9 ⫾ 0.2 1.6 ⫾ 0.1 2.1 ⫾ 0.4 2.4 ⫾ 0.2 2.7 ⫾ 0.4 1.3 ⫾ 0.3 ND ND
1.9 2.7 1.6 2.9 1.8 2.4 1.9
a
RCR, respiratory control ratio. Mean ⫾ S.E. values of four determinations. c ND, not detected. b
ganelle. The experiment depicted in Fig. 3A shows that the mitochondrial membrane potential of digitonin-treated trophozoites could also be accurately estimated using safranine O. In this experiment, the cells were treated with digitonin and the reaction was initiated by the addition of succinate. A fast upward deflection of the trace was observed. Addition of ADP, after the absorbance had stabilized, was followed by a small downward deflection compatible with the utilization of the electrochemical proton potential to drive ADP phosphorylation by the FoF1-ATP synthase (29). As expected, this absorbance decrease was totally reversed by oligomycin, a known inhibitor of the FoF1-ATP synthase. The inclusion of either FCCP (Fig. 3A) or antimycin A (see below) promoted a fast downward deflection of the trace compatible with depolarization of the inner mitochondrial membrane and return of safranine to the water phase. The magnitude of the membrane potential of
mitochondria respiring on succinate was about 140 mV on the basis of the extent of the safranine shift and comparison to calibrations such as that of Fig. 3 (B and C). In this experiment the permeabilized cells were suspended in a potassium-free medium and the addition of valinomycin did not cause any change in absorbance, but the subsequent titration with potassium was followed by the concomitant decrease in membrane potential due to the electrogenic influx of the cation (30). Further addition of FCCP completely collapsed ⌬⌿. The membrane potential after each K⫹ addition was calculated according to the Nernst equation (29). A mitochondrial membrane potential of similar value to that obtained with succinate (Figs. 3A and 4B, trace Pb) could also be generated by the addition of malate/glutamate (Fig. 4A, trace Pb) or TMPD/ascorbate (Fig. 4C, trace Pb). Addition of cyanide totally collapsed ⌬⌿ (Fig. 4C, trace Pb). Rotenone (1–2 M) was also effective in collapsing ⌬⌿ when malate/glutamate was used as the substrate (Fig. 4A, trace Pb). Traces WBC in Fig. 4 show the membrane potential of digitonin-permeabilized white blood cells under similar conditions. The amount of white blood cells used (1 ⫻ 106 cells/ml) was the same as the amount that was present in the trophozoite preparation shown in Fig. 4, traces Pb. Addition of Ca2⫹ to digitonin-permeabilized trophozoites evoked a cycle of respiratory stimulation (Fig. 5A). The higher rate of State 4 respiration in Fig. 5A, as compared with Fig. 1 (A and B) is due to the absence of EGTA in the buffer used. Fig. 5A shows that successive Ca2⫹ additions resulted in Ca2⫹
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FIG. 1. Oxygen consumption by digitonin-permeabilized trophozoites and white blood cells. The test system (final volume, 1.6 ml; 28 °C) contained 125 mM sucrose, 65 mM KCl, 10 mM Hepes-KOH, pH 7.2, 5 mM MgCl2, 2.0 mM potassium phosphate, 0.5% BSA, and 0.5 mM EGTA, in the presence (A, B, D) or absence (C) of 5 mM succinate and 5 M rotenone. Additions were: trophozoites (Pb, 0.6 mg of protein, digitonin (DIG, 16 M), ADP (300 nmol), oligomycin (OLIGO, 2 g), FCCP (1 M), antimycin A (AA, 0.5 g), ascorbate (0.5 mM) plus TMPD (300 M in A and 120 M in C) (TMPD/ASC), and potassium cyanide (KCN, 2 mM). Note that the scale is different and the amount of TMDP added is lower in C to allow a better visualization of ADP-stimulated respiration. D, oxygen uptake by digitonin-permeabilized trophozoites (trace Pb) or digitonin-permeabilized white blood cells (trace WBC). The arrow indicates the addition of either trophozoites (0.6 mg of protein/ ml) or white blood cells (1.0 ⫻ 106 cells/ml). The numbers in parenthesis indicate the rate of oxygen uptake in nanomoles/min ⫻ mg of protein.
FIG. 2. Effect of inhibitors on the oxygen consumption by permeabilized trophozoites. The conditions were the same as described in A, with the addition of 16 M digitonin and 1 M FCCP. Additions were: trophozoites (Pb, 0.6 mg of protein), potassium cyanide (KCN, 1.5 mM), salicylhydroxamate (SHAM, 2 mM), and antimycin A (AA, 0.5 g). The number in parentheses indicates the rate of oxygen uptake in nanomoles/min ⫻ mg of protein.
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FIG. 4. Generation of a mitochondrial membrane potential in permeabilized trophozoites and white blood cells in the presence of different substrates. The reaction medium (2.5 ml; 30 °C) contained 125 mM sucrose, 65 mM KCl, 10 mM Hepes buffer, pH 7.2, 5 mM MgCl2, 2 mM potassium phosphate, 0.5% BSA, 0.5 mM EGTA, 16 M digitonin, 10 M safranine O, and 0.4 mg of protein/ml of the trophozoite suspension (Pb) or 1 ⫻ 106 white blood cells (WBC). Malate/glutamate (MAL/GLU, 5 mM of each), succinate (SUC, 5 mM), 300 M TMPD/0.5 mM ascorbate (TMPD/ASC), rotenone (ROT, 1 M), antimycin A (AA, 0.5 g), and KCN (1 mM) were added where indicated by the arrows.
uptake. Addition of EGTA decreased State 4 respiration. Fig. 5B shows that the addition of Ca2⫹ caused a decrease in the membrane potential of these mitochondria in situ that was compatible with the electrophoretic influx of Ca2⫹ into the mitochondria and in agreement with previous results (Fig. 5A). This decrease in ⌬⌿ was completely reversed when the medium-free Ca2⫹ was lowered to ⬍10⫺8 M by the addition of excess EGTA. When succinate was added to a reaction medium containing trophozoites, 3.5 M free Ca2⫹, and digitonin, a rapid decrease in Ca2⫹ concentration started and continued until a steady state was attained at a free Ca2⫹ concentration of about 0.6 M
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FIG. 3. Changes in safranine absorbance (511–533 nm) following digitonin permeabilization of trophozoites (A) and determination of mitochondrial membrane potential of trophozoites in situ by the safranine method (B, C). A, the reaction medium (2.5 ml; 30 °C) contained 125 mM sucrose, 65 mM KCl, 10 mM Hepes buffer, pH 7.2, 5 mM MgCl2, 2 mM potassium phosphate, 0.5% BSA, 5 mM succinate, 5 M rotenone, 0.5 mM EGTA, 16 M digitonin, 10 M safranine, and 0.4 mg of protein/ml of the cell suspension. ADP (300 nmol), oligomycin (OLIGO, 2 g), and FCCP (1 M) were added where indicated by the arrows. B, the cells (0.4 mg of protein/ml) were added to a potassium-free medium (2.5 ml; 30 °C) containing 200 mM sucrose, 10 mM Na-Hepes buffer (pH 7.2), 0.5 mM EGTA, 5 mM MgCl2, 2 mM sodium phosphate, 0.5% BSA, 5 mM succinate, 5 M rotenone, 16 M digitonin, and 10 M safranine. Valinomycin (V, 1 M) and FCCP (1 M) were added where indicated. A titration of ⌬⌿ was obtained by the sequential additions of KCl to give final concentrations of 0.6, 1.2, 3.2, 5.2, and 10.4 mM. The ⌬⌿ values after each KCl addition were determined by the Nernst equation. The calibration curve is plotted in C.
(Fig. 5C). The subsequent addition of FCCP was followed by a large increase in medium Ca2⫹, indicating the existence of an important mitochondrial Ca2⫹-transporting activity. Subsequent addition of the Ca2⫹ ionophore A23187 resulted in the additional release of Ca2⫹ from an extramitochondrial Ca2⫹ pool. When the order of additions was reversed, FCCP did not cause further Ca2⫹ release. We noticed that addition of 0.5% BSA was necessary to obtain better coupled mitochondria of trophozoites in situ. Uncoupling of mitochondria is usually considered an unwanted pathological effect or an isolation artifact. Because parasitized erythrocytes, as well as malaria parasites, are known to contain large amounts of free fatty acids (31–34), the effect of BSA, which removes fatty acids, can be interpreted as prevention of free fatty acid-induced uncoupling. However, regulated uncoupling mediated by uncoupling proteins or other carriers (35) could also play an important role in several mitochondria where a sudden or transient cutoff of efficient ATP production is required (35). Fig. 6 shows that linoleic acid exerted an uncoupling effect on trophozoite mitochondria in situ as manifested by the increase in the resting state respiration with succinate as substrate. Addition of BSA (Fig. 6C), which removes free fatty acids, reversed this effect, which was further weakly reversed by addition of GTP, a known inhibitor of the uncoupling protein (thermogenin) of brown adipose tissue (35). Subsequent FCCP addition stimulated oxygen consumption. BSA and GTP were ineffective when added after FCCP instead of linoleic acid (data not shown). Fig. 6 also shows that these mitochondria were more tightly coupled, as indicated by the lower resting state respiration, when suspended in media containing BSA and GTP (Fig. 6A, State 4 respiration: 6.4 nmol of O2/min/mg of protein; respiratory control ratio: 2.4) than in medium without these compounds (Fig. 6B, State 4 respiration: 9.8 nmol of O2/min/mg of protein, respiratory control ratio: 1.6). Addition of linoleic acid also decreased the membrane potential of these mitochondria (Fig. 7A), and this effect was partially reversed by BSA (Fig. 7A), GTP (Fig. 7B), or ATP (Fig. 7A). The lack of complete reversal by BSA, especially when incubations were done in the absence of EGTA (Fig. 7, A and B) or for prolonged time (Fig. 8), could also indicate some additional nonspecific effects of the fatty acids. Fig. 7C shows that addition of GTP produced an increase in the value of the membrane potential, which was further increased by bovine serum albumin. Similar results were obtained when the order of additions was reversed (Fig. 7, D and E). Fig. 7E shows that 2,4-dinitrophenol was also able to partially collapse ⌬⌿, which was then completely collapsed by antimycin A. Uncoupling by free fatty acids, at least in animal mitochondria, is mediated not only by uncoupling proteins but also by other carriers such as the ATP/ADP antiporter (36 –38), the aspartate/glutamate antiporter (38, 39) or the dicarboxylate carrier (40). Neither, the ATP/ADP-antiporter inhibitor atractyloside (Fig. 8A), nor the substrate ADP (Fig. 8B) suppressed the effect of linoleic acid on the membrane potential of trophozoites mitochondria in situ. Fig. 8A shows that palmitic acid was also effective in decreasing the membrane potential of these mitochondria in situ. In addition, neither glutamate (Fig. 8C) nor malonate (Fig. 8D), substrates of the aspartate/glutamate antiporter and the dicarboxylate carrier, respectively, was able to restore the mitochondrial membrane potential dissipated by addition of linoleic acid. These results suggest that neither the ADP/ATP antiporter, the glutamate antiporter, nor the dicarboxylate carrier participate in the protonophoric action of fatty acids in P. berghei mitochondria. Immunoblotting of the total P. berghei, P. falciparum, and potato protein (Fig. 9) allowed immunological detection of a
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FIG. 6. Fatty acid-induced uncoupling of digitonin-permeabilized trophozoites. Trophozoites (Pb, 0.6 mg of protein) were added to a reaction medium (final volume: 1.6 ml) containing 125 mM sucrose, 65 mM KCl, 10 mM Hepes, pH 7.2, 0.5 mM EGTA, 5 mM MgCl2, 2.0 mM potassium phosphate, and 5 mM succinate. A, plus 0.5% BSA and 1 mM GTP; B, no additions; C, plus 2 g of oligomycin. Additions were 16 M digitonin (DIG), 300 nmol of ADP, 2 g of oligomycin (OLIGO), 10 M linoleic acid (LA), 0.5% BSA, 1 mM GTP, and 1 M FCCP.
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FIG. 5. Ca2⫹ transport by digitonin-permeabilized trophozoites. A, respiratory stimulation by Ca2⫹. The experimental conditions were the same as described in Fig. 1A in the absence of EGTA and with 0.3 mg of protein/ml. Additions were 16 M CaCl2 and 1.0 mM EGTA. B, effect of CaCl2 on the mitochondrial membrane potential of digitoninpermeabilized trophozoites. The experimental conditions were the same as described in Fig. 3A in the absence of EGTA and with 0.3 mg of protein/ml. Additions were 16 M CaCl2, 1 mM EGTA, and 1 M FCCP. C, calcium uptake by digitonin-permeabilized trophozoites. The reaction medium (final volume: 2.5 ml) contained 3.5 M Ca2⫹, 200 mM sucrose, 10 mM Hepes buffer, pH 7.2, 40 M Arsenazo III, 5 mM succinate, 5 M rotenone, 16 M digitonin, and 0.45 mg of protein/ml. Additions were 5 mM succinate (SUC), 1 M FCCP, and 0.1 M calcium ionophore A23187. The calibration was performed by the sequential addition of known concentrations of EGTA.
FIG. 7. Effect of GTP, ATP, and BSA on the membrane potential of digitonin-permeabilized trophozoites. The test system (final volume, 2.5 ml; 28 °C) contained 125 mM sucrose, 65 mM KCl, 10 mM Hepes-KOH, pH 7.2, 2 mM MgCl2, 2.5 mM potassium phosphate, 10 M safranine, 16 M digitonin, and 1.0 g/ml oligomycin. C, D, and E also contained 0.5 mM EGTA. Additions were 5 mM succinate (SUC), 10 M linoleic acid (LA), 0.5% BSA, 1 mM ATP, 1 mM GTP, 1 M FCCP, 1 mM 2,4-dinitrophenol (DNP), and 2 g of antimycin A (AA). Note that the ⌬⌿ achieved in A and B is lower than in C, D, and E, because these experiments were done in the absence of EGTA.
protein with antibodies developed against potato PUMP (41). These same antibodies have recently been used to demonstrate the presence of an uncoupling protein in the free living protozoa Acanthamoeba castellani (42). A single protein band with a molecular mass of approximately 32 kDa was revealed in potato mitochondria (lane 1), P. falciparum (lane 2), and P. berghei (lane 3) trophozoite lysates, indicating cross-reaction of the plant antibodies with parasite proteins. No reaction was
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FIG. 9. Immunological identification of a mitochondrial uncoupling protein in P. berghei and P. falciparum trophozoites. Primary antibodies were raised against potato PUMP. Potato crude mitochondria (lane 1), P. falciparum (lane 2), and P. berghei (lane 3) trophozite lysates (30 g), and white blood cells (amount similar to that contaminating P. berghei preparations) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose. The PUMP antibody recognized a polypeptide with an apparent molecular mass of 32 kDa in potato mitochondria, P. falciparum, and P. berghei lysates.
observed when white blood cells in the amount that could contaminate P. berghei preparations were used (lane 4). DISCUSSION
This work demonstrates that, as in trypanosomatids (13–16) and T. gondii (17), digitonin can be used to selectively permeabilize the plasma membrane of P. berghei trophozoites to ions, nucleotides, respiratory substrates, and safranine O without affecting the functional integrity of mitochondria. As shown in the experiments found in Figs. 1 and 3A, addition of ADP to digitonin-permeabilized cells was followed by the transition of respiration or ⌬⌿ from the resting to the phosphorylating state. This is one of the most sensitive tests to demonstrate mitochondrial integrity (28). Using permeabilized cells, the effects of various mitochondrial inhibitors on mitochondrial respiration and on the mitochondrial membrane potential were studied. The presence of a NADH-ubiquinone oxidoreductase (Complex I) was suggested by the ability of potential NADH-linked substrates such as
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FIG. 8. Lack of effect of atractyloside, glutamate, and malonate on membrane potential changes during fatty acid-induced uncoupling of digitonin-permeabilized trophozoites. The experimental conditions were as in Fig. 7A except that 5 mM each of malate plus glutamate (MAL/GLU) and 10 M palmitic acid (PA) were used in A and 300 M TMPD/0.5 mM ascorbate (TMPD/ASC) was used in D. Additions were 7 M atractyloside (ATR), 1 M FCCP, 300 nmol of ADP, 10 mM glutamate (GLU), and 5 mM malonate (MALO).
citrate, ␣-ketoglutarate, glutamate, and malate to stimulate ADP phosphorylation in these mitochondria in situ (Table I) and the ability of rotenone to collapse ⌬⌿ (Fig. 4A). This is in contrast to previous reports regarding the possible lack of NADH-ubiquinone oxidoreductase in P. yoelii (5, 8). We cannot rule out, however, that a rotenone-sensitive NADH dehydrogenase could be absent in other developmental stages of P. berghei or in other Plasmodium sp. Potential flavoprotein-linked substrates such as succinate, ␣-glycerophosphate, and dihydroorotate (4) were also able to stimulate ADP phosphorylation by these mitochondria in situ (Table I). The inhibition of respiration (Figs. 1 and 2) and collapse of ⌬⌿ by antimycin A (Figs. 4B, 7B, and 7E) and cyanide (Fig. 4C) supports the presence of ubiquinol-cytochrome c oxidoreductase (Complex III) and cytochrome c oxidase (Complex IV) in the respiratory chain of these cells and the absence of a postulated (6) alternative oxidase. SHAM and BHAM, two inhibitors of the plant alternative oxidase, were also unable to affect respiration by these preparations (Fig. 2). These results agree well with those of Deslauriers et al. (43) who used oxyhemoglobin in the erythrocytes as an indicator of oxygen consumption by P. berghei-infected cells and found only a weak inhibitory effect of SHAM but a complete block of oxyhemoglobin deoxygenation with the cytochrome c oxidase inhibitor NaN3. We cannot rule out, however, the presence of an alternative oxidase in other developmental or physiological states of the parasite or in other Plasmodium sp. In this regard, an inverse correlation has been found between the presence of alternative oxidase and uncoupling protein activities in plant mitochondria (44). The sensitivity of respiration and ⌬⌿ to ADP and oligomycin suggests that the machinery for oxidative phosphorylation is similar to that observed in most vertebrate cells. Likewise, the sensitivity of ⌬⌿ to the standard mitochondrial inhibitors and ionophores such as FCCP and valinomycin supports the notion that these mitochondria are also similar to vertebrate mitochondria in regard to the generation and utilization of an electrochemical proton gradient. The present study also shows that P. berghei mitochondria in situ take up Ca2⫹ from the incubation medium by a mechanism associated with depolarization of the membrane potential as shown by the safranine O experiments. The present results suggest that mitochondria of digitonin-treated trophozoites possess a Ca2⫹ uniport similar to that of mammalian mitochondria. These mitochondria were also able to buffer external free Ca2⫹ at a concentration of about 0.6 M, which occurs with mammalian mitochondria (45). Uncoupling of mitochondrial oxidation and phosphorylation by free fatty acids was described in 1956 by Pressman and Lardy (46). Later it was reported that the uncoupling protein (thermogenin), the protein responsible for the thermoregulatory uncoupling in brown fat mitochondria, was activated by fatty acids (47). More recently, it was found that uncoupling proteins (reviewed in Ref. 35) or other anion carriers such as the ATP/ADP antiporter (36 –38), the aspartate/glutamate antiporter (38, 39), and the dicarboxylate carrier (40) could mediate free fatty acid-induced uncoupling. It was postulated (48) that the free fatty acid binds to these hydrophobic proteins, and the fatty acid carboxylate group moves to the cytosolic side of the membrane by a flip-flop mechanism. Once in the cytosolic side of the membrane, the fatty acid diffuses laterally away from the conductance pathway and is protonated. The protonated fatty acid rapidly flip-flops again, delivering protons electroneutrally to the mitochondrial matrix and completing the cycle. This mechanism bypasses the ATP synthase, and as a consequence dissipates the H⫹ transmembrane electrochem-
Functional Mitochondria in Malaria Parasites
Acknowledgments—We thank Anibal E. Vercesi, Ivan Maia, and Paulo Arruda for the gift of polyclonal antibodies against PUMP. REFERENCES 1. Anonymous (1997) in Tropical Disease Research, Thirteenth Programme Report, UNDP/World Bank/World Health Organization Special Programme for Research and Training in Tropical Diseases, pp. 40 –73, World Health Organization, Geneva 2. Sherman, I. W. (1998) in Malaria, Parasite Biology, Pathogenesis and Protec-
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ical gradient. Addition of free fatty acids, such as linoleic acid, to P. berghei mitochondria resulted in mitochondrial uncoupling, whereas the presence of purine nucleotides such as ATP or GTP, which inhibit uncoupling proteins (35), and bovine serum albumin, which removes free fatty acids, recoupled respiration (Fig. 6) and increased the membrane potential (Fig. 7) of these mitochondria in situ. Our inhibitor studies suggest that the uncoupling effect of free fatty acids on P. berghei mitochondria in situ is mediated by an uncoupling protein and not by the ATP/ADP antiporter, the aspartate/glutamate antiporter, or the dicarboxylate carrier. In agreement with these results, a protein band of 32 kDa present in P. berghei and P. falciparum lysates cross-reacts with antibodies against PUMP (41). Uncoupling proteins are expressed in mammalian cells (35) and also in plants (49), and they were thought to have emerged recently in evolution (35). When our manuscript was under revision, Jarmuszkiewicz et al. (42) reported the immunological identification in A. castellani of a protein (AcUCP) that crossreacts with the same anti-PUMP antibodies that we used in this work and investigated the functional properties of this putative uncoupling protein in isolated mitochondria. Our results, together with those of Jarmuszkiewicz et al. (42), suggest that uncoupling proteins could be more widespread than previously believed and could also occur in lower eukaryotes. Its presence in malaria parasites would explain the difficulties found by other authors in detecting oxidative phosphorylation in these parasites. Since a Plasmodium-induced erythrocyte oxidant sensitivity has been postulated (reviewed in Ref. 50) and malaria parasites contain large amounts of free fatty acids (31–34), it is possible that an uncoupling protein could be a cellular defense system preventing formation of superoxide anion by the malaria mitochondria as it has been postulated to occur in animal cells (35). Attempts to test the digitonin-permeabilization method with P. falciparum trophozoites isolated as we described before (21) were unsuccessful (data not shown). No oxidative phosphorylation could be detected. Oxygen uptake by digitonin-treated trophozoites did not respond to the inhibitors added, possibly indicating complete lysis of the cells by digitonin and autoxidation processes due to the presence of free radical-generating systems associated to the hemozoin present in these preparations. Differences in the intracellular membrane composition of P. falciparum as compared with P. berghei trophozoites could be involved in this different response to digitonin. However, cross-reaction was detected with anti-PUMP antibodies in P. falciparum lysates (Fig. 9, lane 2). In conclusion, the respiratory chain and oxidative phosphorylation are functional in P. berghei, although the presence of large amounts of fatty acids and the possible presence of an uncoupling protein results in partially uncoupled mitochondria.
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Oxidative Phosphorylation, Ca2+ Transport, and Fatty Acid-induced Uncoupling in Malaria Parasites Mitochondria Sergio A. Uyemura, Shuhong Luo, Silvia N. J. Moreno and Roberto Docampo J. Biol. Chem. 2000, 275:9709-9715. doi: 10.1074/jbc.275.13.9709
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