From the School of Biochemistry and Molecular
Biology, Faculty of Science, Australian National University,
Canberra, Australian Capital Territory 0200, Australia and the
** Molecular and Biochemical Parasitology Group, Liverpool
School of Tropical Medicine, Pembroke Place,
Liverpool L5 3QA, United Kingdom
Received for publication, August 22, 2002, and in revised form, November 8, 2002
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ABSTRACT |
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As it grows within the human erythrocyte, the
malaria parasite, Plasmodium falciparum, ingests the
erythrocyte cytosol, depositing it via an endocytotic feeding mechanism
in the "digestive vacuole," a specialized acidic organelle. The
digestive vacuole is the site of hemoglobin degradation, the storage
site for hemozoin (an inert biocrystal of toxic heme), the site of
action of many antimalarial drugs, and the site of proteins known to be
involved in antimalarial drug resistance. The acidic pH of this
organelle is thought to play a critical role in its various functions;
however, the mechanisms by which the pH within the vacuole is
maintained are not well understood. In this study, we have used a
combination of techniques to demonstrate the presence on the
P. falciparum digestive vacuole membrane of two discrete
H+ pumping mechanisms, both capable of acidifying the
vacuole interior. One is a V-type H+-ATPase, sensitive to
concanamycin A and bafilomycin A1. The other is a
H+-pyrophosphatase, which was inhibited by NaF and showed a
partial dependence on K+. The operation of the
H+-pyrophosphatase was dependent on the presence of a
Mg2+-pyrophosphate complex, and kinetic experiments gave
results consistent with free pyrophosphate acting as an inhibitor of
the protein. The presence of the combination of a H+-ATPase
and a H+-pyrophosphatase on the P. falciparum
digestive vacuole is similar to the situation in the acidic tonoplasts
(vacuoles) of plant cells.
The malaria parasite is a unicellular eukaryote (genus
Plasmodium) that during the course of its life cycle invades
the erythrocytes of its vertebrate host. As it grows within its host
erythrocyte, the parasite ingests the host cell cytosol via an
endocytotic feeding mechanism. The host cell proteins (predominantly
hemoglobin) are deposited in an internal acidic compartment, the
digestive vacuole, where they are broken down by acid hydrolases to
their component amino acids (1), some of which are used for protein synthesis (2). As well as being the major site for hemoglobin digestion, the digestive vacuole serves as a site for the storage of
hemozoin (inert crystals of the potentially toxic heme that is
liberated in the course of hemoglobin degradation). It is believed to
be the primary site of action of many of the antimalarial drugs, and
the two proteins known to play a role in antimalarial drug resistance
in the most virulent of the human malaria parasites, Plasmodium
falciparum, Pgh1 (3) and PfCRT (4), have been localized to the
digestive vacuole membrane.
The acidic pH of the digestive vacuole is thought to play a key role in
the various physiological functions of this organelle and may also play
a role in the phenomenon of antimalarial drug resistance (5, 6).
Despite the likely significance of vacuolar pH, the mechanism(s)
underlying the acidification of the vacuole interior are not well
understood. There is immunological evidence for the presence on the
digestive vacuole membrane of a V-type H+-ATPase1
(7-9). Krogstad et al. (10) did show a
Mg2+ATP-stimulated acidification of the digestive vacuole,
but this was not characterized in any detail. In a later study (11), it
was shown that the ATPase activity of isolated digestive vacuoles can be inhibited by the nonspecific H+ pump inhibitors
N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, but the nature of the pump(s) involved was not investigated.
The aim of this study was to identify and characterize the mechanism(s)
involved in the acidification of the digestive vacuole of P. falciparum. Using a combination of techniques, we have obtained direct physiological evidence for the presence on the digestive vacuole
membrane of a V-type H+-ATPase, as well as a second
H+ pump, a H+-pyrophosphatase, which couples
the hydrolysis of the phosphoanhydride bond of pyrophosphate
(PPi) to the influx of H+ into the vacuole.
Parasite Culture--
P. falciparum parasites (strain
D10) were maintained in synchronous in vitro cultures as
described previously (12) with modifications (13). All experiments were
carried out on late stage trophozoites, 36-40 h postinvasion.
Loading Fluorescent pH Indicators into the Parasite's Digestive
Vacuole--
A membrane-impermeant, dextran-linked form of the
fluorescent pH indicator fluorescein (pKa ~6.4)
was loaded into the parasite's digestive vacuole as described
previously (10, 14), with only minor modifications. Packed erythrocytes
(1 ml) were lysed by the addition of 2.25 ml of lysis buffer (5 mM HEPES, 11 mM glucose, 2 mM
MgCl2, and 2 mM ATP, pH 7.4) containing 110 µM fluorescein-dextran (Mr 10,000;
Molecular Probes, Inc., Eugene, OR). The lysate was incubated for 10 min at 30 °C, and then the cells were resealed by the addition of
2.25 ml of a resealing buffer (280 mM NaCl, 40 mM KCl, 11 mM glucose, 1 mM HEPES,
pH 7.4). The dye-loaded erythrocytes were washed three times in RPMI (1,200 × g, 5 min) and then resuspended in culture
medium and inoculated with ~1 ml of a trophozoite-stage P. falciparum-infected erythrocyte suspension (3-4% hematocrit,
10-20% parasitemia). The parasites were maintained in 50 ml of medium
on an orbital shaker until the parasitemia was higher than 10-15%
(rarely more than two complete cycles), at which point the cells were
used for experiments.
Selective Permeabilization of the Erythrocyte and Parasite Plasma
Membranes--
Fig. 1 illustrates the different parasite/vacuole
preparations used throughout this study.
Parasitized erythrocytes (Fig. 1A) were permeabilized (as
described previously (15)) by brief exposure to saponin (0.005%, w/v
sapogenin), a plant-derived detergent that permeabilizes both the host
cell membrane and the parasitophorous vacuole membrane (16), thereby
allowing solutes present in the extracellular solution unimpeded access
to the parasite plasma membrane (Fig. 1B). Parasites within
saponin-permeabilized erythrocytes are referred to throughout this
paper as "isolated parasites." For all experiments other than those
giving rise to Figs. 2, 8, and 10C, the plasma membrane of
the parasite (within the saponin-permeabilized erythrocyte membrane)
was itself permeabilized using digitonin (10), thereby permitting
solutes added to the extracellular medium immediate access to the
digestive vacuole membrane (Fig. 1C). Isolated parasites were suspended in 2 ml of ice-cold intracellular saline A (110 mM KCl, 30 mM NaCl, 2 mM
MgCl2, 5 mM HEPES, pH 7.3). The cell suspension
(typically 5 × 107 cells/ml) was kept on ice for
5-10 min before adding digitonin (0.01%, w/v) from a 200 mg/ml stock
(in Me2SO). The cells were mixed gently and returned
to ice for a further 2 min, after which 1 ml of ice-cold intracellular
saline A containing 1 mg/ml bovine serum albumin was added. The
permeabilized parasites were centrifuged immediately (15,800 × g) for 1 min and then washed twice (1 min at 15,800 × g) with 1 ml of the same bovine serum albumin-containing solution and once in intracellular saline A without bovine serum albumin. The digitonin-permeabilized parasites were suspended in
intracellular saline A at an average cell density of ~8 × 106/ml and kept at 37 °C until being used in experiments
(usually within 2 h, but up to 5 h in the experiment giving
rise to Fig. 4). Within digitonin-permeabilized parasites, the
digestive vacuole remained intact, as indicated by the retention of the
dextran-linked fluorescent dye within the digestive vacuole of
parasites isolated from dye-loaded erythrocytes (see above).
Isolation of Digestive Vacuoles--
Digestive vacuoles (Fig.
1D) were isolated as described previously (17), with some
modifications. Approximately 2 × 109 isolated
parasites (see above) were lysed by suspension in ~1 ml of an acidic,
ice-cold, hypotonic lysis buffer (20 mM MES, pH 4.5). The
lysate was transferred to a watchglass on ice, triturated forcefully
twice through a 27-gauge needle, and then centrifuged (15,800 × g, 0.5 min). The supernatant solution was removed, and the
pellet was resuspended in ~1 ml of intracellular saline B (120 mM KCl, 10 mM NaCl, 25 mM HEPES, 2 mM MgCl2, 5 mM
Na2HPO4, pH 7.3), and triturated (as described
above) six more times. The suspension was centrifuged (15,800 × g, 0.5 min), and the supernatant solution was removed. The
pellet was again resuspended in ~1 ml of intracellular saline B, to
which was added DNase I (300-400 units/ml), and the suspension was
incubated at 37 °C for 5 min. The sample was centrifuged once more
(15,800 × g, 0.5 min) and then resuspended in 100 µl
of intracellular saline B and mixed with 1.3 ml of a Percoll solution
(80% v/v Percoll, 1.5 mM MgCl2, 200 mM sucrose, pH 7.3). The suspension was again triturated (2 ×) onto the watchglass, returned to microcentrifuge tubes, and centrifuged at 5,200 × g for 8 min at 4 °C.
Isolated vacuoles were retrieved from the bottom ~50 µl of the
Percoll solution, avoiding the hemozoin crystal pellet at the very
bottom of the tube, and resuspended in ice-cold intracellular saline B
until being used in experiments within 20 min of their isolation.
Microscopic examination of the preparation revealed a homogeneous
population of approximately spherical, hemozoin-filled organelles that
appeared to be free of contaminants.
Fluorescence Measurements--
Throughout this study, the pH
within the parasite's digestive vacuole (pHDV) was
monitored using the fluorescence arising from either the
membrane-impermeant pH indicator fluorescein-dextran, loaded into the
digestive vacuole as described above, or (in one series of experiments
with isolated digestive vacuoles) from the membrane-permeant compound
quinacrine. In preliminary experiments with a range of dextran-linked
pH indicators, we attempted to calibrate the measured fluorescence with
pHDV but found that the estimated pHDV varied
with the fluorescent indicator used. The origin of this variation is
presently under investigation. For the purpose of the present study,
the fluorescence was simply used as a semiquantitative indicator of
pHDV, and there was no attempt made to calibrate the
fluorescence measurements. For both fluorescein-dextran and quinacrine,
an upward deflection of the trace is indicative of an alkalinization,
and a downward deflection of the trace is indicative of an acidification.
Experiments with Cell Populations--
In experiments with
intact isolated parasites (Fig. 1B) in which the digestive
vacuole was preloaded with fluorescein-dextran, the cells were
suspended at a density of ~5 × 106 cells/ml in a
solution comprised of 125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 20 mM glucose, 25 mM HEPES, pH 7.1. With the exceptions of the experiments
giving rise to Figs. 5B and 6B,
digitonin-permeabilized parasites (Fig. 1C) were suspended
(at ~5 × 106 cells/ml) in intracellular saline A. For the experiment giving rise to Fig. 5B, permeabilized
parasites were suspended in intracellular saline A without
MgCl2. For the experiment giving rise to Fig. 6B, they were suspended in a K+-free solution
containing 140 mM NaCl, 2 mM MgCl2,
5 mM HEPES, pH 7.3.
Changes in pHDV were monitored at 37 °C on a
Perkin-Elmer Life Sciences LS-50B spectrofluorometer using a dual
excitation Fast Filter accessory. The sample was excited at 490 and 450 nm successively, and the fluorescence was measured at 520 nm. The ratio
of the fluorescence intensity measured using the two excitation wavelengths (490 nm/450 nm) provides a measure of pHDV.
Experiments with Single Cells--
Digitonin-permeabilized
parasites (Fig. 1C) preloaded with fluorescein-dextran were
immobilized on poly-L-lysine-coated coverslips and then
mounted in a Bioptechs FCS2 perfusion chamber and immersed in
intracellular saline A at 37 °C. The cells were imaged using a Zeiss
LSM510 confocal microscope through a Plan-Apochromat 63 × 1.4 NA
oil objective. The fluorescein-dextran was excited using the 488-nm
wavelength line of an argon ion laser through a 488-nm HFT filter.
Emitted fluorescence was captured off a 545-nm dichroic mirror through
a 505-550-nm bandpass filter.
Experiments with Suspensions of Isolated
Vacuoles--
pHDV in isolated P. falciparum
digestive vacuole suspensions was monitored using a method based on one
used previously by Hirata et al. (18) to measure the pH in
vacuoles isolated from yeast. Vacuoles were isolated from unloaded
parasites and suspended (at 4-8 × 107 vacuoles/ml)
in intracellular saline B to which had been added 25 nM
quinacrine. Fluorescence intensity was measured using excitation and
emission wavelengths of 425 and 495 nm, respectively. The experiments
with this preparation were carried out at 20 °C, as in Ref. 18.
31P NMR Measurements--
Hydrolysis of
PPi by suspensions of digitonin-permeabilized parasites
(Fig. 1C) and isolated P. falciparum digestive
vacuoles (Fig. 1D) was
monitored using 31P NMR spectroscopy.
In experiments with digitonin-permeabilized parasites, the cells were
suspended at a cell density of ~6 × 107 cells/ml in
intracellular saline A containing 4 mM PPi and
incubated in the presence or absence of 10 mM NaF at
37 °C. At predetermined time intervals, 250-µl aliquots were
removed and centrifuged (0.5 min at 15,800 × g). A
200-µl aliquot of the supernatant solution was transferred to 100 µl of ice-cold 30% perchloric acid in order to halt any enzyme
activity. The pH was subsequently adjusted to 7.4 using NaOH.
In experiments with isolated vacuoles, the organelles were suspended in
intracellular saline A at a density of ~1 × 107
vacuoles/ml and incubated in the presence of 0.5 mM
PPi for 2 h at 37 °C. Samples were then processed
as described above.
The volume of each neutralized extract sample was made up to 400 µl,
and the solution was then transferred to a 5-mm (outside diameter) NMR
tube. Methylene diphosphonic acid was added to a final concentration of
either 5 mM (in the permeabilized parasite experiments) or
0.5 mM (in the isolated vacuole experiments) as an internal
31P NMR reference standard. EDTA was added to a
concentration of 24 mM to preclude signal broadening by
trace concentrations of multivalent metal ions, and
2H2O (10% v/v) was added to provide a field
frequency lock. 31P NMR spectra were acquired at 25 °C
and at 121.4 MHz, using a Varian VXR-300 spectrometer with a 45°
pulse and a 1-s relaxation delay between successive pulses.
Calculation of the Concentrations of Free PPi and
Mg·PPi Complexes--
The concentrations of free
PPi (the sum of PP Regulation of pHDV in Isolated Parasites--
When
isolated parasites in which the digestive vacuole was preloaded with
fluorescein-dextran were suspended in a glucose-containing medium, they
gave rise to a steady fluorescent signal. The addition of the V-type
H+-ATPase inhibitors concanamycin A (Fig.
2A) or bafilomycin
A1 (not shown) resulted in a progressive increase in the
(490 nm/450 nm) fluorescence ratio, indicative of the dye-loaded
digestive vacuole undergoing an alkalinization. This is consistent with a V-type H+-ATPase playing a role in maintaining the acidic
pH of this organelle.
The intraerythrocytic parasite derives its ATP from glycolysis (20). As
shown in Fig. 2B, removal of glucose from the extracellular medium (a maneuver shown previously to result in a rapid depletion of
parasite ATP (21)) resulted in an alkalinization of the vacuole. Upon
restoration of glucose (20 mM) to the medium, the vacuoles reacidified. A similar reacidification was seen upon the addition to
glucose-depleted parasites of 2 mM ATP (Fig.
2C), which is known to cross the parasite plasma membrane
via an ATP/ADP translocase (22, 23). Atractyloside (100 µM), an inhibitor of the ATP/ADP translocase, prevented
the ATP-induced reacidification while having no effect on the
reacidification caused by the subsequent addition of glucose (Fig.
2D). Unlike ATP, PPi (added to the extracellular solution at a concentration of 2 mM) failed to induce an
acidification of the vacuole in glucose-depleted parasites, although it
did cause a modest decrease in the rate of alkalinization (Fig.
2E).
ATP- and PPi-induced Acidification of the Parasite's
Digestive Vacuole in Suspensions of Digitonin-permeabilized
Parasites--
In isolated parasites (as used in the experiments
giving rise to Fig. 2), the parasite plasma membrane remains intact,
and compounds added to the extracellular medium have to traverse this membrane in order to gain access to the digestive vacuole (Fig. 1B). The parasite plasma membrane can be permeabilized,
thereby exposing the surface of the digestive vacuole directly to the extracellular solution (as well as releasing the contents of the cytosol, including those compounds required to energize the
acidification of the vacuole), by treating the isolated parasites with
digitonin (Fig. 1C).
Fig. 3 illustrates the effects of
PPi and ATP on pHDV in digitonin-permeabilized
parasites. As shown in Fig. 3A, the addition of 0.2 mM PPi to digitonin-permeabilized parasites
suspended in a high K+ solution resulted in a rapid
decrease in the fluorescence ratio, indicative of the vacuole
undergoing an acidification. The subsequent addition of the
H+-ATPase inhibitor concanamycin A (75 nM) had
no significant effect. The data are consistent with the presence in the
vacuole membrane of a H+ pumping PPase, distinct from the
V-type H+-ATPase.
The addition of 1 mM ATP to digitonin-permeabilized
parasites also caused a marked acidification of the digestive vacuole (Fig. 3B). The acidification was reversed upon the addition
of concanamycin A, consistent with the ATP-induced acidification being
mediated by a V-type H+-ATPase. The subsequent addition of
0.2 mM PPi resulted in the complete
reacidification of the vacuole (Fig. 3B).
The rate (and magnitude) of the vacuolar acidification induced by 2 mM ATP decreased with the time after permeabilization of
the parasite plasma membrane by digitonin (Fig.
4, closed symbols). By ~1 h after the addition of digitonin, the ATP-induced
acidification was reduced to an almost negligible level. By contrast,
the rate of acidification measured in response to the addition of 0.2 mM PPi was maintained at an approximately
constant level for >4 h following exposure of the parasites to
digitonin (Fig. 4, open symbols).
The time-dependent decrease of the ATP-induced
acidification made an analysis of the ATP concentration dependence of
the process difficult, and this was not attempted. It was, however,
possible to analyze the concentration dependence of the
PPi-induced acidification of the vacuole in
digitonin-permeabilized parasites. The process showed biphasic
kinetics, illustrated in Fig.
5A. The rate of acidification
increased with [PPi] up to a concentration of ~0.5 mM and then decreased as [PPi] was increased
further. When PPi was added at a concentration of 5 mM, the rate of acidification was less than 20% of its
maximum value, whereas the addition of 10 mM
PPi had a negligible effect on pHDV.
A likely explanation for the biphasic kinetics is that the true
substrate for the H+-PPase is a Mg·PPi
complex (MgPP
Further evidence in support of the view that Mg·PPi is
the substrate for the H+-PPase is provided in Fig.
5B, which shows that in the absence of MgCl2 the
addition of 0.2 mM PPi had no effect on
pHDV, whereas the subsequent addition of 2 mM
MgCl2 resulted in the immediate acidification of the
digestive vacuole. The addition of MgCl2 in the absence of
PPi had no effect on pHDV (not shown).
The rate and magnitude of the PPi-induced acidification of
the digestive vacuole in digitonin-permeabilized parasites were reduced
by replacement of K+ with Na+ in the medium
(Fig. 6). The PPi-induced
acidification was also susceptible to competitive inhibition by the
H+-PPase inhibitor NaF (Fig.
7). When the digestive vacuole was acidified by the addition of 2 mM PPi to the
digitonin-permeabilized parasite suspension, the subsequent addition of
10 mM NaF resulted in a modest alkalinization (Fig.
7A). Reduction of the PPi concentration increased the sensitivity to inhibition by NaF, indicative of competitive behavior. When the digestive vacuole was acidified by the
addition to the permeabilized parasite suspension of 0.2 mM
PPi, NaF at a concentration as low as 2.5 mM
induced a progressive alkalinization of the vacuole (Fig.
7B).
ATP- and PPi-induced Acidification of the Parasite's
Digestive Vacuole in Isolated Digestive Vacuoles and in Single
Permeabilized Parasites--
In addition to the measurements carried
out on suspensions of intact and digitonin-permeabilized parasites, two
alternative approaches were taken to confirm the presence of discrete
ATP- and PPi-dependent acidification mechanisms
on the parasite's digestive vacuole. The first utilized preparations
of isolated digestive vacuoles. The second entailed monitoring the
fluorescence arising from within the (dye-loaded) digestive vacuoles of
single digitonin-permeabilized parasites using confocal microscopy.
Fig. 8 shows representative fluorescence
traces obtained from suspensions of isolated digestive vacuoles to
which had been added quinacrine, a membrane-permeant fluorescent
indicator that partitions into acidic compartments where it undergoes a
decrease in fluorescence. The isolated vacuoles underwent a marked
acidification in response to the addition of ATP to the medium,
consistent with the presence of a functional H+-ATPase. The
subsequent addition of the H+-ATPase inhibitor concanamycin
A caused the vacuoles to realkalinize (Fig. 8A). The
addition of PPi to isolated vacuoles resulted in an
acidification, consistent with the operation of an H+-PPase
(Fig. 8B). Similar results were obtained from suspensions of
digestive vacuoles isolated from parasites grown in erythrocytes preloaded with fluorescein-dextran (not shown).
As a second means of confirming that the activities detected are both
present on the digestive vacuole, we used confocal imaging to collect
the fluorescence emanating from the digestive vacuole of individual
digitonin-permeabilized parasites. The addition of ATP to the
permeabilized parasite resulted in an acidification of the digestive
vacuole, and the addition of the H+-ATPase inhibitor
concanamycin A caused it to realkalinize (Fig. 9A). The addition of
PPi also caused a vacuolar acidification (Fig.
9B).
31P NMR Measurements of PPi
Hydrolysis--
The fluorescence data presented so far are consistent
with the presence in the digestive vacuole membrane of two discrete H+ pumping mechanisms: a concanamycin A-sensitive
H+-ATPase and a NaF-sensitive H+-PPase. The
presence of PPase activity (i.e. the hydrolysis of PPi to Pi) was confirmed in both
digitonin-permeabilized parasites and in isolated digestive vacuoles
using 31P NMR spectroscopy.
PPi added at a concentration of 4 mM to a
suspension of digitonin-permeabilized parasites underwent progressive
hydrolysis to Pi, with complete conversion by ~3 h (Fig.
10A). As is clear from the
time courses of Fig. 10B, the rate of hydrolysis (indicated by the slope of the lines) increased as time progressed, and the concentration of PPi in the sample decreased. The
dotted line in Fig. 10B indicates the
changing ratio of the concentration of Mg·PPi complex to
the concentration of free PPi as the experiment progressed.
During the course of the experiment, the relative concentration of the
(inhibitory) free PPi species decreased, and it is probably
this that underlay the observed time-dependent increase in
the rate of PPi hydrolysis. The inclusion of NaF (10 mM) in the medium had the effect of slowing the rate of
hydrolysis substantially (Fig. 10B).
31P NMR spectroscopy was also used to monitor the
conversion of PPi to Pi by isolated digestive
vacuoles. The difficulty of preparing the large quantities of isolated
digestive vacuoles required for the 31P NMR analysis
limited the experiments that we were able to do using this approach. As
illustrated in Fig. 10C, however, the isolated vacuoles
mediated the complete conversion of 0.5 mM PPi
to Pi within 2 h.
The digestive vacuole of the malaria parasite is an acidic
organelle in which host erythrocyte proteins (predominantly hemoglobin) are degraded to small peptides and amino acids. In this paper, we
present evidence for the presence on the P. falciparum
digestive vacuole membrane of two discrete H+ pumps. One is
a V-type H+-ATPase, inhibited by concanamycin A and
bafilomycin A1. The other is a H+-PPase,
inhibited by NaF and showing a partial dependence on K+.
The presence of the two types of H+ pump resembles the
situation in the acidic intracellular vacuole of plant cells (26) and
perhaps in the acidocalcisome (an acidic organelle that serves as a
calcium store) of a number of protozoan parasites (27, 28), in which a
H+-ATPase and a H+-PPase act in concert to
maintain a low internal pH.
The Characteristics and Physiological Role of the Digestive Vacuole
H+-ATPase and H+-PPase--
The presence of a
V-type H+-ATPase on the digestive vacuole of P. falciparum has been inferred previously from immunological data
(7-9), from the previous demonstration of
Mg2+-ATP-stimulated acidification of the digestive vacuole
in permeabilized parasites (10), and from the demonstration that ATP
hydrolysis by isolated digestive vacuoles can be inhibited by
(nonspecific) H+ pump inhibitors such as
N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (11). In this study, we provide direct evidence (Fig. 2) that a V-type
H+-ATPase plays a major role in maintaining the acidic pH
of the digestive vacuole in intact parasites (within
saponin-permeabilized erythrocytes). Under normal conditions, the pump
was reliant on glucose in the medium to ensure that it was adequately
supplied with ATP (Fig. 2B). However, the pump was able to
function in the absence of glucose (for at least 10 min) under
conditions in which the parasite was able to import ATP directly from
the extracellular medium via the atractyloside-sensitive ATP/ADP
exchanger (Fig. 2C).
The first reports of a H+-PPase in the intraerythrocytic
malaria parasite were from Docampo and co-workers (29, 30), who presented evidence for PPi-dependent
acidification of a subcellular compartment in total cell homogenates of
mature Plasmodium parasites. The results of the present
study, showing both PPi-induced acidification of the
digestive vacuole and hydrolysis of PPi to Pi
by isolated vacuoles, provide direct evidence for the presence of a
functional H+-PPase on the digestive vacuole membrane.
As with other H+-PPases described to date (e.g.
in mung bean (Vigna radiata) hypocotyls (31), Avena
sativa L. roots (32), and Kalanchoë
daigremontiana leaves (33)), the H+-PPase described in
this study exhibited biphasic kinetics, with the
PPi-induced acidification rate increasing as
[PPi] was increased up to ~0.5 mM and then
decreasing as [PPi] was increased further (Fig.
5A). These observations may be explained by inhibition of the H+-PPase by free PPi (24, 25), the
concentration of which increased to high levels as the total
concentration of PPi was increased under conditions of
constant [Mg2+] (Fig. 5A). This explanation is
also consistent with the results of the 31P NMR experiment
(Fig. 10A) in which the rate of hydrolysis of PPi to Pi by digitonin-permeabilized parasites
(indicated by the slope of the solid lines in
Fig. 10B) increased with time. As the PPi was
hydrolyzed (under conditions of constant [Mg2+]), the
ratio of Mg·PPi to free PPi increased (Fig.
10B, dotted line), thereby alleviating
inhibition of the H+-PPase and increasing the rate of
hydrolysis of PPi.
The extent to which the H+-PPase contributes significantly
to the maintenance of the acidic pH of the digestive vacuole under physiological conditions is not clear. The addition of the PPase inhibitor NaF to intact parasites did cause an alkalinization of the
digestive vacuole (results not shown); however, the fact that NaF is
also a potent inhibitor of glycolysis (and, thereby, of ATP production)
makes the interpretation of this observation difficult. The observation
that the specific H+-ATPase inhibitor concanamycin A caused
a pronounced alkalinization shows that under the conditions of the
experiment, the H+-PPase alone was not able to maintain
pHDV.
The finding that PPi is present in the blood stage of at
least one species of Plasmodium (P. berghei) (34)
does suggest that the digestive vacuole H+-PPase may be
active in situ, at least under some conditions. However, the
fact that earlier 31P NMR studies in P. berghei
failed to detect PPi (35), together with the observation
that the PPi content of different stages of
Trypanosoma cruzi has been shown to fluctuate with the
growth stage and with the level of "stress" to which the parasites
are subjected (36), raises the possibility that the concentration of
PPi in the intraerythrocytic forms of P. falciparum may, at least under certain conditions, be too low (or
perhaps even too high) (Fig. 5A) to allow the
H+-PPase to contribute significantly to the maintenance of
pHDV.
Following permeabilization of the parasite plasma membrane, the ability
of ATP to induce a vacuolar acidification decreased in a
time-dependent manner (Fig. 4). The fact that the
PPi-induced acidification did not show a similar decrease
over a longer period indicates that the decrease in H+
pumping by the ATPase is not simply a reflection of a general loss of
digestive vacuole function or integrity. A loss of
H+-ATPase activity has been reported to occur following
fractionation of organelles in T. brucei (37). In yeast
vacuoles, V-type H+-ATPase activity decreases in the
prolonged absence of glucose, reportedly through disassembly of
subunits from the multisubunit complex (38-42). Enzyme oxidation after
isolation has also been implicated in loss of H+-ATPase
activity (43).
Whatever the underlying mechanism(s), the observation that the
H+-ATPase activity is lost under conditions in which the
H+-PPase activity remains active suggests a possible role
for the PPase as a "back-up system," which might operate to acidify
the digestive vacuole under conditions in which the vacuolar
H+-ATPase is impaired. However, the pronounced vacuolar
alkalinization observed to occur upon inhibition of the
H+-ATPase, either by concanamycin A (Fig. 2A) or
by glucose deprivation (Fig. 2, B-E), suggests that the
H+-PPase was unable to fulfill such a back-up role under
the conditions of our experiments. The fact that in some systems V-type
H+-ATPases have the ability to "run backward" and
synthesize ATP when subjected to a sufficiently large H+
electrochemical gradient (e.g. see Refs. 18 and 44) raises the possibility that under conditions in which the parasite has a
significant cytosolic [PPi] and low [ATP], the presence
of a H+-PPase-induced H+ electrochemical
gradient across the digestive vacuole membrane might cause the vacuolar
H+-ATPase to run backward, thus enabling the digestive
vacuole to act as an ATP-generating organelle. Again, however, this is
unlikely to have occurred under the conditions of our experiment, since in the absence of parasite ATP the vacuolar pH increased to such an
extent as to make it unlikely that there was a significant H+ gradient across the digestive vacuole membrane. Just as
a H+-PPase-induced H+ gradient could, in
principal, cause the H+-ATPase to run backward and
synthesize ATP, so too could a H+-ATPase-induced
H+ gradient cause the H+-PPase to run backward
and synthesize PPi. Whether this occurred under the
conditions of our experiments is unclear.
Whereas ATP added to intact parasites induced an acidification of the
intracellular vacuole, the addition of PPi did not
(cf. Fig. 2, C and E). This may be due
to PPi not crossing the parasite plasma membrane at a
sufficiently high rate to fuel the vacuolar H+-PPase and/or
to the hydrolysis by cytosolic PPases (present in intact parasites but
lost on permeabilization of the parasite plasma membrane) of the
PPi entering the cell.
The Identity of the Digestive Vacuole
H+-PPase--
The H+-PPases that have been
characterized to date are composed of single polypeptides and fall into
two distinct classes, distinguished on the basis of their
K+ dependence; type I H+-PPases have an
absolute requirement for K+ in order to function, whereas
type II H+-PPases operate in the absence of K+
(45). Analysis of the P. falciparum genome has revealed the presence of two H+-PPase genes, PfVP1 and
PfVP2, encoding homologues of the type I
(K+-dependent) H+-PPase and type II
(K+-independent) H+-PPase, respectively, of
Arabidopsis (46). The mRNAs from both were shown to be
expressed throughout the intraerythrocytic phase of the parasite life
cycle, with the level of PfVP1 mRNA much higher than the
level of PfVP2 mRNA (46). PfVP1 and PfVP2 have predicted
molecular masses of 76.4 and 115.8 kDa, respectively. In two
previous studies, Western blots of P. falciparum trophozoite lysates or membranes using polyclonal antibodies generated against a
peptide sequence (TKAADVGADLVGKIE) from the A. thaliana
H+-PPase showed a single band (albeit at two different
estimated molecular masses, 77 kDa (29) and 67 kDa (46)). On the basis of immunofluorescence experiments, it was concluded that the protein is
distributed over the parasite surface as well as intracellularly (29,
46). However, there was no fluorescence detected from the parasite's
digestive vacuole (46). A similar localization pattern was seen in
experiments in which parasites were transfected with green fluorescent
protein-PfVP1 fusion protein (46).
In the course of this work, we used antibodies raised against the
corresponding peptide from the mung bean H+-PPase
(DVGADLVGKVE) (47) in an attempt to detect one or more proteins in
Western blots of the isolated digestive vacuole preparation. The
corresponding sequence of PfVP1 has the sequence DVGADLSGKNE, whereas
that of PfVP2 has the sequence DIGADLVGKVE (46). Western blots of whole
saponin-isolated parasites showed a single band at an estimated
molecular mass of 65 kDa, close to the molecular mass predicted for
PfVP1 and very similar to that observed by McIntosh et al.
(46). However, the protein was not present at detectable levels in
isolated digestive vacuole preparations (data not shown). This,
together with the previous immunolocalization data, argues against
PfVP1 being the digestive vacuole H+-PPase. It is possible
that the vacuolar H+-PPase is the substantially larger
PfVP2 and that this protein is not recognized by the antibodies used
both by us and by others (despite the similarity of the
target sequence) and/or that the level of expression of PfVP1 (or
PfVP2) in the digestive vacuole is too low to have been detected on our
Western blots. PfVP2, like its K+-independent
Arabidopsis homologue, AVP2, possesses a conserved lysine
(at position 817) (46) that has recently been shown to confer
K+ independence to H+-PPases (48). PfVP2 might
therefore also be expected to be K+-independent. The
finding that the parasite digestive vacuole H+-PPase shows
a pronounced stimulation by K+ (Fig. 6) might therefore
argue against it being PfVP2, raising the possibility that the
H+-PPase activity on the digestive vacuole is due to
another, as yet unidentified, P. falciparum protein. The
resolution of this issue awaits further research.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Schematic representation of the different
parasite preparations used in this study. Mature P. falciparum trophozoites (A) were "isolated" from
their host (human) erythrocytes by permeabilization of the erythrocyte
and parasitophorous vacuole membranes by brief exposure to saponin
(B). The plasma membrane of isolated parasites was
permeabilized (releasing the contents of the parasite cytosol and
giving solutes in the external solution direct access to the digestive
vacuole) by brief exposure of isolated parasites to digitonin
(C). Digestive vacuoles were isolated (D) by
hypotonic lysis of isolated trophozoite-stage parasites and purified
using Percoll density gradient centrifugation (see "Experimental
Procedures").
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
Alkalinization and reacidification of the
digestive vacuole in intact isolated parasites. The digestive
vacuole was loaded with fluorescein-dextran, and the host erythrocyte
membrane was permeabilized with saponin as described under
"Experimental Procedures." Fluorescence was measured at 520 nm and
is expressed as a ratio of that measured on excitation at 490/450 nm.
An increase in the ratio (i.e. an upward deflection)
indicates an alkalinization. The addition of the H+-ATPase
inhibitor concanamycin A (75 nM) to parasites suspended in
a glucose-containing medium (A, black
arrowhead) caused an alkalinization of the digestive
vacuole, as did the removal of glucose from the medium
(B-E, black arrowheads). The
readdition of 20 mM glucose to glucose-depleted cells
(B, white arrowhead) caused a
reacidification of the digestive vacuole, as did the addition to the
extracellular medium of 2 mM ATP (C,
gray arrowhead). The ATP/ADP translocase
inhibitor, atractyloside (100 µM, white
arrowhead) prevented the ATP-induced reacidification
(gray arrowhead) but did not prevent the
glucose-induced reacidification (D, white
arrowhead). The addition of 2 mM PPi
to glucose-depleted cells (E, white
arrowhead) slowed the rate of alkalinization slightly but
failed to induce a reacidification. The traces are from a single
experiment and are representative of those obtained in at least three
separate experiments.
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Fig. 3.
Effect of ATP and PPi on the pH
of the digestive vacuole in isolated P. falciparum trophozoites in which the plasma membrane
was permeabilized by digitonin. The digestive vacuole was loaded
with fluorescein-dextran, and the erythrocyte membrane, parasitophorous
vacuole membrane, and parasite plasma membrane were permeabilized as
described under "Experimental Procedures." The addition of either
PPi (A, 0.2 mM, white
arrowhead) or ATP (B, 1 mM,
gray arrowhead) caused an acidification.
Concanamycin A (75 nM), added at the points indicated by
the black arrowheads, had no effect on the pH of
PPi-acidified vacuoles (A) but reversed the
ATP-induced acidification (B). The addition of 0.2 mM PPi to ATP- and concanamycin A-treated cells
(B, white arrowhead) resulted in the
complete reacidification of the digestive vacuoles (B). The
traces are from a single experiment and are representative of those
obtained in at least three separate experiments.
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Fig. 4.
Initial rate of acidification of the
digestive vacuole of digitonin-permeabilized parasites following the
addition of 2 mM ATP (closed
symbols) or 0.2 mM PPi
(open symbols), plotted as a function of
the time postpermeabilization. Initial rates of acidification were
estimated by fitting traces such as those illustrated in Fig. 3 to the
first order equation fluorescence ratio =
fluorescence
ratiomax × (1
e
kt), where:
fluorescence ratio is
the difference between the resting fluorescence ratio prior to the
addition of ATP or PPi (averaged over the 20 s
immediately prior to the addition) and the fluorescence ratio at a
given time (t) following the addition of ATP or
PPi;
fluorescence ratiomax is the maximum
decrease in fluorescence ratio attained following the addition of ATP
or PPi; and k is the first order rate constant
for the acidification. The time dependence of the ATP-induced
acidification rate was fitted to a first-order exponential decay
function. The PPi data were fitted to a straight line. The
data are from a single experiment and are representative of those
obtained in five separate experiments.
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Fig. 5.
[PPi] and Mg2+
dependence of the PPi-induced acidification of the
digestive vacuole in digitonin-permeabilized parasites.
A, initial rates of acidification were estimated as
described in the legend to Fig. 4 and are expressed as a fraction of
the maximum rate of acidification attained (0.08 ± 0.01 fluorescence ratio units/s (mean ± S.E.) at [PPi] = 0.3 mM). The data are averaged from three independent
experiments and are fitted to the Weibull Distribution equation
(SigmaPlot; Jandel). Error bars represent S.E.
values. The dashed line represents the calculated
concentration of free PPi; the dotted
line represents the calculated concentration of
PPi complexed with Mg2+ (i.e. the
sum of the concentrations of MgPP
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Fig. 6.
K+ dependence of the
PPi-induced acidification of the digestive vacuole in
digitonin-permeabilized parasites. The traces show the effect of
0.2 mM PPi, added as either the K+
salt (A) or the Na+ salt (B), at the
point indicated by the white arrowhead, to
digitonin-permeabilized parasites suspended in a solution containing
either 110 mM K+ plus 30 mM
Na+ (A) or 0 mM K+ plus
140 mM Na+ (B). Traces are from a
single experiment, representative of those obtained in three separate
experiments.
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Fig. 7.
NaF-induced reversal of the
PPi-induced acidification of the digestive vacuole in
digitonin-permeabilized parasites. The addition of 10 mM NaF (black arrowhead) to parasites
treated with 2 mM PPi caused a modest
alkalinization of the digestive vacuole (A). The addition of
2.5-10 mM NaF (black arrowhead) to
parasites treated with 0.2 mM PPi caused a much
more pronounced alkalinization (B). The data are from a
single experiment, representative of those obtained in at least three
separate experiments.
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Fig. 8.
ATP- and PPi-induced
acidification of digestive vacuoles isolated from P. falciparum trophozoites. The traces show the
effect of the addition of ATP (1 mM; gray
arrowhead), followed by the addition of the
H+-ATPase inhibitor concanamycin A (75 nM;
black arrowhead) (A) and the addition
of PPi (1 mM, white
arrowhead) (B) on the fluorescence from a
suspension of isolated digestive vacuoles containing 25 nM
quinacrine. A downward deflection of the trace is indicative of
vacuolar acidification. The traces are representative of those obtained
in two or more similar experiments.
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Fig. 9.
Single cell measurements of the ATP- and
PPi-induced acidification of the digestive vacuole of
digitonin-permeabilized parasites. Digitonin-permeabilized
parasites (in which the digestive vacuole was preloaded with
fluorescein-dextran) were attached to coverslips with polylysine and
visualized using a confocal microscope. The fluorescence from the
digestive vacuoles of individual parasites was recorded. The addition
of 2 mM ATP (A, gray
arrowhead) caused an acidification that was reversed by the
addition of 70 nM concanamycin A (black
arrowhead). The addition of 2 mM PPi
(B, white arrowhead) induced a somewhat slower
acidification. The data are averaged from measurements on 27 (A) and 40 (B) individual parasites and are
expressed as a percentage of the initial resting fluorescence.
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Fig. 10.
Hydrolysis of PPi by
digitonin-permeabilized parasites and by isolated digestive vacuoles.
A, stacked 31P NMR spectra showing the
time-dependent hydrolysis of PPi (added at a
concentration of 4 mM) and the concomitant production of
Pi by digitonin-permeabilized parasites. The spectra are
derived from an average of 750 pulses, processed with a line-broadening
factor of 3. B, time courses showing the hydrolysis of
PPi (4 mM) in the absence (closed
circles) or presence (open symbols) of
digitonin-permeabilized parasites suspended in the presence
(squares) or absence (triangles) of 10 mM NaF. The data were averaged from two separate
experiments and are shown as mean ± range/2. There was no
hydrolysis in the absence of parasites. The dotted
line shows the calculated time-dependent change
in the ratio of Mg·PPi to free PPi in the
sample in which parasites were suspended in the absence of NaF
(i.e. in the time course indicated by the open
triangles). C, 31P NMR spectra
showing the complete conversion of PPi (0.5 mM)
to Pi after a 2-h incubation with isolated digestive
vacuoles. The spectra are derived from an average of 4,000 pulses,
processed with a line-broadening factor of 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We are grateful to the staff of the ACT Red Cross Blood Transfusion Service for the provision of blood, to Prof. M. Maeshima for the provision of H+-PPase antibody, and to Dr. P. Ryan for assistance with the use of GEOCHEM-PC.
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FOOTNOTES |
---|
* This work was supported by Australian National Health and Medical Research Council Grant 179804 and grants from the Australian National University Faculties Research Grants Scheme and the Ramaciotti Foundations.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
¶ To whom correspondence should be addressed. Tel.: 61-2-6125-0640; Fax: 61-2-6125-0313; E-mail: kevin.saliba@anu.edu.au.
Supported by an Australian postgraduate student award.
Supported by the Wellcome Trust.
Published, JBC Papers in Press, November 8, 2002, DOI 10.1074/jbc.M208648200
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ABBREVIATIONS |
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The abbreviations used are: H+-ATPase, proton-translocating ATPase; H+-PPase, proton translocating PPase; pHDV, digestive vacuole pH; MES, 4-morpholineethanesulfonic acid.
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