HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 44, 34353-34358, November 3, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34353    most recent M003323200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hayashi, M. Articles by Moriyama, Y. Search for Related Content PubMed PubMed Citation Articles by Hayashi, M. Articles by Moriyama, Y. Social Bookmarking                      What's this?

Vacuolar H⁺-ATPase Localized in Plasma Membranes of Malaria Parasite Cells, Plasmodium falciparum, Is Involved in Regional Acidification of Parasitized Erythrocytes^*

Mitsuko Hayashi^§¶, Hiroshi Yamada^¶, Toshihide Mitamura^**, Toshihiro Horii^**, Akitsugu Yamamoto, and Yoshinori Moriyama^§§

From the  Department of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, the ^** Institute of Microbial Disease, Osaka University, Suita, Osaka 565-0871, and the  Department of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan

Received for publication, April 18, 2000, and in revised form, July 20, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Recent biochemical studies involving 2',7'-bis-(2-carboxyethyl)-5,6-carboxylfluorescein (BCECF)-labeled saponin-permeabilized and parasitized erythrocytes indicated that malaria parasite cells maintain the resting cytoplasmic pH at about 7.3, and treatment with vacuolar proton-pump inhibitors reduces the resting pH to 6.7, suggesting proton extrusion from the parasite cells via vacuolar H⁺-ATPase (Saliba, K. J., and Kirk, K. (1999) J. Biol. Chem. 274, 33213-33219). In the present study, we investigated the localization of vacuolar H⁺-ATPase in Plasmodium falciparum cells infecting erythrocytes. Antibodies against vacuolar H⁺-ATPase subunit A and B specifically immunostained the infecting parasite cells and recognized a single 67- and 55-kDa polypeptide, respectively. Immunoelectron microscopy indicated that the immunological counterpart of V-ATPase subunits A and B is localized at the plasma membrane, small clear vesicles, and food vacuoles, a lower extent being detected at the parasitophorus vacuolar membrane of the parasite cells. We measured the cytoplasmic pH of both infected erythrocytes and invading malaria parasite cells by microfluorimetry using BCECF fluorescence. It was found that a restricted area of the erythrocyte cytoplasm near a parasite cell is slightly acidic, being about pH 6.9. The pH increased to pH 7.3 upon the addition of either concanamycin B or bafilomycin A₁, specific inhibitors of vacuolar H⁺-ATPase. Simultaneously, the cytoplasmic pH of the infecting parasite cell decreased from pH 7.3 to 7.1. Neither vanadate at 0.5 mM, an inhibitor of P-type H⁺-ATPase, nor ethylisopropylamiloride at 0.2 mM, an inhibitor of Na⁺/H⁺-exchanger, affected the cytoplasmic pH of erythrocytes or infecting parasite cells. These results constitute direct evidence that plasma membrane vacuolar H⁺-ATPase is responsible for active extrusion of protons from the parasite cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plasmodium falciparum is a parasitic unicellular protozoa that causes the most serious infectious disease for human beings. The malaria parasite has a complex life cycle. Upon invasion of a host, the parasite lies dormant for some hours and then starts rapid growth followed by cell division, resulting in the generation of new parasite cells. During this development, the malaria parasite develops complex membrane systems outside itself and takes up hemoglobin and digests it in food vacuoles, speculative counterparts of mammalian lysosomes, followed by energy acquisition for growth and cell division (reviewed in Refs. 1 and 2). Glucose is also rapidly taken up by the parasite plasma membrane through a facilitated hexose transporter and is metabolized through glycolysis (3), with an approximately 100-fold increase in glucose utilization ability as compared with uninfected erythrocytes (3-5, 7, 8). This higher glycolysis activity increases the formation and export of lactic acid by the infected cells. Consistently, malaria parasite infection gives rise to extensive extracellular acidosis (9). Thus, it is believed that the malaria parasite may somehow extrude protons outside cells, although little is known about the mechanism underlying the proton extrusion.

To explain how protons are extruded by the malaria parasite, three mechanisms have been postulated. At first, Mikkelsen et al. (10, 11) suggested that the proton-pump, possibly P-type ATPase, is involved in the extrusion of protons by the malaria parasite cells, since the cytoplasmic pH of rodent malaria cells, Plasmodium chabaudi, decreased upon treatment with N,N'-dicyclohexylcarbodiimide or vanadate. However, P-type H⁺-ATPase has not been identified yet in a malaria parasite. The second possibility is the involvement of Na⁺/H⁺ antiporter. Bosia et al. (12) found that the cytoplasmic pH of parasite-infected erythrocytes was affected by extracellular Na⁺, which was sensitive to amiloride and its analogue, EIPA,¹ inhibitors of the Na⁺/H⁺ antiporter (13). It is suggested that a gene product of PfATPase 1 (encoding MAL1) encoding a putative homologue of the  subunit of Na⁺/K⁺-ATPase is a primary Na⁺-pump for the functional operation of the Na⁺/H⁺ antiporter (14). However, the molecular nature of the P. falciparum Na⁺/H⁺ antiporter and putative primary Na⁺-pump remains obscure. Very recently, Saliba and Kirk (15) presented evidence for a third type of proton extrusion by a malaria parasite. They labeled P. falciparum cells with BCECF, a fluorescent pH indicator, and then measured the cytoplasmic pH in the presence or absence of various H⁺-pump inhibitors. They observed that bafilomycin A₁, a specific inhibitor of V-ATPase (16), caused acidification of the cytoplasm of the malaria parasite cells, whereas vanadate and amiloride analogues had little effect. From these observations, they proposed that active proton extrusion via V-ATPase may occur through the plasma membrane of the malaria parasite cells and that inhibition of V-ATPase may cause acidification of the cytoplasm due to the inevitable accumulation of protons.

V-ATPase is a multisubunit protein complex comprising distinct catalytic and membrane sectors, whose subunit structure is well conserved among animals, plants, fungi, and even some bacteria (17, 18). In P. falciparum, the cDNAs encoding V-ATPase subunits A and B have been cloned and sequenced, and immunological counterparts of these subunits have been identified (19, 20). The internal pH of food vacuoles is known to be acidic (21, 22). Mg²⁺-ATPase activity in the food vacuole fraction has also been identified and characterized (23). Although these results suggest the presence of functional V-ATPase in malaria parasite cells, the precise localization of V-ATPase at the subcellular level is less understood.

The present study was conducted to determine the localization of V-ATPase in P. falciparum cells infecting erythrocytes using immunoelectron microscopy with antibodies against V-ATPase subunits A and B. It was found that V-ATPase is localized at the plasma membrane as well as intracellular vesicular structures including food vacuoles. Moreover, it was found that a limited area of the erythrocyte cytoplasm outside the parasite cell is acidic. The acidic pH is sensitive to bafilomycin A₁ and concanamycin B, potent and specific V-ATPase inhibitors (16, 25, 26), but relatively insensitive to vanadate and EIPA. These results strongly suggest that V-ATPase is functionally expressed at the plasma membrane of malaria parasite cells and pumps protons out of the organisms.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

P. falciparum Culture-- P. falciparum strain FCR-3 cells were cultured with 10% heat-inactivated O⁺ human erythrocytes and then suspended at a hematocrit of 5% in RPMI 1640 medium (Life Technologies, Inc.) containing 50 mg/liter gentamicin (27) and maintained at 37 °C under an atmosphere comprising 5% CO₂, 5% O₂, and 90% N₂ gas. Erythrocytes exhibiting 0.3% parasitemia were added to each well of plates in 990 ml of culture medium to give a final hematocrit of 3%. Then the plates were incubated at 37 °C under 5% CO₂, 5% O₂, and 90% N₂ gas for 72 h. In some experiments, the cultures were synchronized by hemolysis of mature, trophozoite-stage parasitized erythrocytes by suspension in a 5% (w/v) sorbitol solution (27).

Immunoblotting-- P. falciparum cells were isolated from the infected erythrocytes by saponin treatment (28). Then the cells (about 10⁸) were denatured with SDS sample buffer containing 1% SDS and 10% -mercaptoethanol and electrophoresed on a 10% polyacrylamide gel in the presence of SDS (29). Following electrotransfer at 0.3 A for 2 h, the nitrocellulose filter was blocked in a buffer consisting of 20 mM Tris-Cl (pH 7.6), 5 mM EDTA, 0.1 M NaCl, and 0.5% bovine serum albumin for 4 h and then probed with 1 µg/ml purified antibodies in the above buffer for 1 h. The filter was washed with 20 mM Tris-Cl buffer (pH 7.6) containing 5 mM EDTA, 0.1 M NaCl, and 0.1% Tween 20, treated with peroxidase-labeled anti-rabbit IgG at a dilution of 1:2000 for 30 min, washed further with the same buffer, and subjected to ECL amplification according to the manufacturer's manual (Amersham Pharmacia Biotech). Site-specific antibodies against V-ATPase subunit A were raised in rabbits using a synthetic peptide corresponding to residues 366-381 (AEMPADSGYPAYLGAR(C)) of subunit A of clathrin-coated vesicle ATPase in bovine brain (30). This region is conserved in all V-ATPases from plant and animal origin (17, 18). Polyclonal antibodies against subunit B of bovine chromaffin granule V-ATPase were raised in a rabbit by injecting subunit B isolated electrophoretically from the purified V-ATPase (31). These antibodies cross-reacted with the corresponding subunits of plants and animal origin. The specificity of the antibodies was extensively characterized previously (31-34).

Immunohistochemistry-- Erythrocytes infected with P. falciparum cells on glass coverslips were fixed in 100% methanol for 10 min at 20 °C, then incubated with PBS containing 2% goat serum and 0.5% bovine serum albumin, and finally reacted with antibodies at 10 µg/ml in PBS containing 0.5% bovine serum albumin for 1 h. The samples were washed three times with PBS and reacted with the second antibodies conjugated with fluorescein, and then the immunoreactivity (green color) was observed under an Olympus FLUOVIEW confocal laser microscope.

Immunoelectron Microscopy-- For immunoelectron microscopy, a pellet of either parasitized erythrocytes or isolated P. falciparum cells was fixed for 1 h at room temperature by immersion in a fixative comprising 4% paraformaldehyde dissolved in 0.1 M phosphate buffer (pH 7.4) and then rinsed several times with PBS. Then the pellet was dehydrated and embedded at 20 °C in LR-White, as described previously (35), using an ultraviolet polymerizer, TUV-200 (Dosaka EM; Kyoto, Japan). Then ultra-thin sections were cut and picked up on collodion carbon-coated nickel grids (150 mesh). The grids were floated for 10 min on drops of PBS containing 2% goat serum and 0.5% bovine serum albumin and then incubated for 30 min at room temperature on 20 µl of PBS containing 50 µg/ml antibodies and 0.5% bovine serum albumin. In the control experiment, PBS containing 50 µg/ml control IgG (IgG fraction from preimmune rabbit serum) and 0.5% bovine serum albumin was used. After washing with PBS containing 0.5% bovine serum albumin, the grids were further incubated for 15 min with goat anti-rabbit IgG conjugated with 10 nm gold particles (British BioCell International, Cardiff, UK), dissolved in PBS containing 0.5% bovine serum albumin, washed with 0.1 M cacodylate buffer (pH 7.4), and post-fixed with 5% glutaraldehyde in the same buffer. The sections were washed with distilled water, then successively stained with uranyl acetate for 10 min and lead citrate for 1 min, and finally observed under a Hitachi H7000 electron microscope. Quantitative analysis of the immunogold labeling was performed according to Refs. 34-38.

Measurement of Intracellular pH-- An Attofluor ratio imaging system (Zeiss) was used to measure the intracellular pH of infecting parasite cells (13, 14). Erythrocytes infected with P. falciparum cells (10⁷) were incubated on a coverslip precoated with 100 µg/ml poly-L-lysine in RPMI 1640 medium and then washed twice with the same medium. Then the cells were incubated in the above medium containing 10 µM BCECF-AM for 50 min at 37 °C and then washed twice with the same medium without BCECF-AM. The cells were perfused with a warmed Ringer's solution comprising 128 mM NaCl, 1.9 mM KCl, 1.2 mM KH₂PO₄, 2.4 mM CaCl₂, 1.3 mM MgSO₄, 26 mM NaHCO₃, 10 mM glucose, and 10 mM Hepes (pH 7.4), and then fluorescence images were continuously taken at 37 °C with a CCD camera with a photomultiplier (Photometer Option MPM200; Zeiss). The fluorescence emission at 520 nm was monitored, and the velocity of data acquisition for F488 by F460 images was at 2-s intervals with a resolution of 512 × 512 pixels per image. A personal computer with appropriate software (Attofluor Ratio Vision; Atto Instruments, Rockville, MD) was used to control the optical equipment and recording and data analysis. The software enabled subtraction of background fluorescence and pixel-to-pixel division of F488 by F460 images in the arbitrary area in a cell where the pH was measured (see Fig. 3B). Intracellular pH was calibrated by the nigericin/high potassium method (14). In brief, cells were perfused in a high K⁺ solution comprising 130 mM KCl, 10 mM NaCl, 1 mM MgSO₄, and 10 mM Hepes (pH. 6.80, 7.10, or 7.80) containing 1 µM nigericin. Then the ratio of the BCECF fluorescence intensity observed was plotted against pH, which was almost linear within the pH range examined, and used to make a calibration curve.

Chemicals-- BCECF-AM was obtained from Dojin Co., Japan. Bafilomycin A₁ was obtained from Wako Chemical Co., Japan. Concanamycin B was prepared as described previously (26). Other chemicals used in this study were of the highest grade available commercially.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Immunological Detection of V-ATPase Subunit A and B in P. falciparum Cells-- We have used immunological techniques to investigate the localization of V-ATPase in P. falciparum cells infecting human erythrocytes. Western blotting with antibodies against the V-ATPase subunit A and B recognized a single polypeptide with apparent molecular mass of 67 and 55 kDa in the total proteins of P. falciparum, respectively (Fig. 1A). The apparent molecular mass of the polypeptide coincides well with that of the corresponding subunit of the malaria parasite, as reported previously (19, 20), and is slightly smaller than those of subunit A and B of mammalian V-ATPases (Fig. 1A) but similar to those of higher plant ones (31). These antibodies specifically immunostained the malaria cells, whereas no immunologically positive signals were observed for non-infected erythrocytes (Fig. 1A, lanes 3 and 6, B, and C). These results indicated that these antibodies are useful for investigating the localization of V-ATPase in P. falciparum cells infecting erythrocytes. The fact that the antibodies immunostained the whole body of the infecting parasite cells (Fig. 1, B and C) suggested that V-ATPase is present in various organelles other than food vacuoles, a known acidic organelle in the malaria parasite cells (21, 22). Essentially the same immunostaining pattern was observed for P. falciparum cells at all developmental stages (data not shown), suggesting that V-ATPase is expressed throughout the cell cycle.

View larger version (44K): [in this window] [in a new window]   Fig. 1.   Immunological detection of V-ATPase subunit A and B in parasitized erythrocytes. A, rat brain synaptic vesicles were prepared as described (31), and an aliquot (50 µg of protein) was dissolved in SDS-containing sample buffer and used as a positive control (lanes 1 and 4). Total proteins of malaria parasites (108 cells) (lanes 2 and 5) and non-infected erythrocytes (100 µg of protein) (lanes 3 and 6) dissolved with SDS sample buffer were also subjected to electrophoresis. Then immunoblotting with either anti-V-ATPase subunit A antibodies (lanes 1-3) or anti-V-ATPase subunit B antibodies (lanes 4-6) was performed as described under "Experimental Procedures." The positions of subunit A and B are indicated by arrows. B and C, parasitized erythrocytes were immunostained with either anti-V-ATPase subunit A antibodies (B) or anti-V-ATPase subunit B antibodies (C) and then observed under a confocal laser microscope and Nomarski microscope. A superimposing picture was shown. An immunostained malaria parasite is indicated by an arrow. Bar, 1 µm.

Immunoelectron microscopy was performed to investigate the localization of V-ATPase in P. falciparum cells at the subcellular level. As shown in Fig. 2, immunogold for V-ATPase subunit A selectively labeled food vacuoles (Fig. 2, A and C), small clear vesicles (Fig. 2, A and D), and the plasma membrane (Fig. 2, A and E). Immunogold particles were also occasionally observed in the parasitophorus vacuolar membrane (Fig. 2F), whereas essentially no labeling was seen in the nuclear envelope, nucleus (Fig. 2G), and mitochondrion (Fig. 2H). Control antibodies gave the essentially no labeling (Fig. 2B). A comparable immunogold labeling was also obtained with antibodies against V-ATPase subunit B (Fig. 2, I-L).

View larger version (150K): [in this window] [in a new window]   Fig. 2.   Immunoelectron microscopy of V-ATPase subunits A and B in a P. falciparum cell. Localization of V-ATPase subunit A (A-G) or subunit B (I-L) was visualized with immunogold particles (diameter, 10 nm), as shown by arrows. A and B show the whole body of the parasite immunostained with anti-V-ATPase subunit A antibodies (A) or control IgG (B); C, parts of food vacuoles (FV); D, small clear vesicles (SV); E, plasma membrane (PM); F, parasitophorus vacuolar membrane (PVM); G, nucleus (N) and nuclear envelope (NE); H, mitochondrion (M); I and J, immunogold particles for subunit B at plasma membrane (PM), parasitophorus vacuolar membrane (PVM), food vacuoles (FV), and small clear vesicles (SV). K, nucleus (N) and nuclear envelope (NE); L, mitochondrion (M). Bar, 1 µm in A and B; 0.1 µm in C-F; 0.2 µm in G-L.

Quantification of this labeling data is summarized in Table I. After subtracting the density of immunogold particles by control antibodies, 0.65 and 0.88 of the immunogold particles per 1 µm were found to be associated with plasma membrane and food vacuole of the parasite cells, respectively (Table I). By using specific binding, the absolute number of V-ATPase at plasma membrane and food vacuole was roughly estimated to be 16,000 and 2200 particles based on the assumption that parasite cell contains a single ball-shaped plasma membrane and a ball-shaped food vacuole with a diameter of 2.5 and 0.8 µm, respectively, and that the labeling efficiency is 20% (35-38) (Table I). These results strongly suggest that V-ATPase is predominantly localized in the plasma membrane in P. falciparum cells. It is noteworthy that the cytoplasmic side but not the luminal side of these organelles was selectively labeled by immunogold, suggesting that the catalytic parts of V-ATPase face the cytoplasm. This orientation of the V-ATPase is the same as that in known various organelles of plants and animals (17, 18) and implies the orientation of the proton transport across these membranes.

Identification of a Weakly Acidic Compartment of Parasitized Erythrocytes-- It is tempting to speculate that the V-ATPase identified at the plasma membrane extrudes protons from a parasite cell in cytoplasm of an infected erythrocyte, causing acidification of a restricted region outside the plasma membrane. To monitor such presumptive proton extrusion from the parasite cell, we measured the cytoplasmic pH of BCECF-loaded parasitized erythrocytes fluorometrically.

Upon incubation with BCECF-AM, erythrocytes and infected malaria parasites were labeled (Fig. 3). Much greater labeling of the malaria parasite cell than the erythrocyte cytoplasm was always observed. The ratio of the fluorescence intensities at the two different excitation wavelengths enabled us to determine the internal pH in a restricted area of a parasitized erythrocyte and uninfected erythrocyte (Fig. 4A). The cytoplasmic pH values of parasitized erythrocytes and infecting parasite cells as well as uninfected erythrocytes were found to be essentially the same (7.30 ± 0.02, 30 determinations for 4 independent preparations), which is consistent with previous observations (10, 12, 13, 15).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Identification of weakly acidic regions in the cytoplasm of parasitized erythrocytes. Parasitized erythrocytes were labeled with BCECF-AM, and then the internal pH was measured as described under "Experimental Procedures." A, labeling with BCECF was observed by fluorescence microscopy. A fluorescence micrograph, with excitation at 460 nm, is presented to show BCECF-loaded erythrocytes. Heavily labeled areas represents the infecting parasites. B, the same view as in A to indicate intracellular pH transients, as calculated by the BCECF fluorescence intensity ratio. Crescent-shaped weakly acidic areas observed are indicated by arrows. The parasitized erythrocyte without an acidic area is also indicated by *, which contained schizontal parasites by hematoxylin staining. Bar, 10 µm.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   The time course for the intracellular pH change. A, the labeled erythrocytes shown in Fig. 3B were boxed and numbered. Boxes 1-3 corresponded to the cytoplasm of an uninfected erythrocyte, the cytoplasm of parasitized erythrocyte, and the cytoplasm of parasite, respectively. Then the time course of the pH change in the box 1 (B), box 2 (C), and box 3 (D) were simultaneously monitored. Concanamycin B at 0.1 µM was added at the time indicated by an arrow.

We found a crescent-shaped weakly acidic region, pH 6.9 ± 0.03, near the parasite cells in erythrocytes (Figs. 3 and 4). This region corresponded to the extracellular domain of an infecting parasite, as judged from the fluorescence image of parasitized erythrocytes (Fig. 3). In synchronized cultures, the acidic area was observed in about 88% of parasitized erythrocytes at the trophozoite stage (more than 186 parasitized erythrocytes were examined), whereas no acidic area was observed in either ring or schizont stage parasite cells, indicating that the acidic area is a characteristic of trophozoite stage parasite cells.

Concanamycin B increased the acidic pH to 7.3 (Fig. 4C). Simultaneously, the cytoplasmic pH of BCECF-loaded malaria parasite cells in the same erythrocytes decreased with a similar time course to that of concanamycin B-evoked alkalinization of the cytoplasm of erythrocytes (Fig. 4D). On the other hand, concanamycin B did not affect cytoplasmic pH of uninfected erythrocyte at all (Fig. 4B). Essentially the same results were obtained when bafilomycin A₁ was used (Table II). Vanadate, an inhibitor of P-type H⁺-ATPase, or EIPA, an inhibitor of the Na⁺/H⁺ antiporter, on the other hand, had no effect on the cytoplasmic pH of parasitized erythrocytes or infecting malaria cells (Table II). These results suggest that the acidic pH in the crescent-shaped area outside a malaria parasite cell is maintained by V-ATPase. Taking the results of the immunological localization of V-ATPase shown in Fig. 2 together, it is concluded that V-ATPase at the plasma membrane of P. falciparum cells is functional and pumps protons into the extracellular space.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

V-ATPase is present in various endomembrane organelles and plasma membranes and pumps protons at the expense of ATP hydrolysis (17, 18). The resultant acidic pH in organelles and extracellular space is important for various biological phenomena including the accumulation of ions and nutrients, extrusion of ions and harmful drugs, and so on (17, 18). Previous reports have been shown that in the malaria parasite V-ATPase is present in food vacuoles and acidifies the internal space for hydrolysis of hemoglobin for energy acquisition (19, 20). In the present study, we found that V-ATPase is mainly associated with small clear vesicles and the plasma membrane in addition to food vacuoles.

Saliba and Kirk (15) reported that trophozoite stage malaria parasites within saponin-permeabilized human erythrocytes extrude protons via a Na⁺-independent mechanism. The proton extrusion was sensitive to bafilomycin A₁, indicating the possible involvement of V-ATPase in the extrusion process. The immunohistochemical distribution of the V-ATPase in trophozoite stage parasites that we observed is fully consistent with their observation. The immunogold particles for V-ATPase labeling the plasma membrane face the cytoplasmic side, suggesting that the catalytic sector of V-ATPase faces the cytoplasm. This orientation of the V-ATPase makes it possible for it to pump protons from the cytoplasm into the extracellular space across the plasma membrane. Consistently, a restricted area outside a malaria parasite in a parasitized erythrocyte is acidic (Figs. 3 and 4). Treatment with either bafilomycin A₁ or concanamycin B caused disappearance of the acidic region and simultaneously acidification of the cytoplasm of the malaria parasite. The bafilomycin A₁-evoked acidification of the cytoplasm of the parasite cell is supposed to be due to blockade of proton extrusion through V-ATPase (15). These observations constitute strong evidence for the functional occurrence of proton extrusion via V-ATPase at the plasma membrane.

Of course our results do not completely rule out the participation of a Na⁺/H⁺ antiporter in the proton extrusion through the plasma membrane, although vanadate and EIPA apparently did not affect the cytoplasmic pH of infecting malarial cells (Table II). Studies for identification of the Na⁺/H⁺ antiporter and primary Na⁺-pump, and their characterization at the molecular level, allow re-evaluation of the possible contribution of the Na⁺/H⁺ antiporter, if any, to proton extrusion from the parasite.

The finding that V-ATPase is localized in various organelles other than food vacuoles is important for understanding the physiology of the parasite. A V-ATPase-evoked acidic environment has been realized to be important for various biological phenomena including the transport of nutrients, ions, toxins, and organellar fusion events. Small clear vesicles are morphologically distinct from dense granules containing subtilisin-like protein or rhoptry (39). It is possible that small clear vesicles are involved in endocytosis/exocytosis of the parasites (40, 41). Furthermore, as in the case of the plasma membranes of mammals and insects (6, 17, 18), proton pumping by plasma membrane V-ATPase and resultant acidification of the extracellular space might be energetically coupled with transporters for the accumulation of nutrients and regulation of ions in and extrusion of antimalarial agents from the parasites. It is also possible that the acidic pH is responsible for the bafilomycin-evoked increase in sensitivity to chloroquine (24). Further studies on the function of the plasma membrane V-ATPase and the resultant acidification will facilitate elucidation of novel physiological features of the parasite.

In conclusion, we presented evidence of the presence of V-ATPase in food vacuoles, small clear vesicles, and the plasma membrane of P. falciparum cells. The V-ATPase at the plasma membrane is responsible for extrusion of protons through the membrane and thereby acidifies the area outside a parasite. We suppose that the malaria parasite uses this acidic pH to transport ions and nutrients for its survival.

	ACKNOWLEDGEMENTS

We are grateful to Profs. Y. Wataya (Okayama University) and K. Kita (University of Tokyo) for their encouragement during this study.

	FOOTNOTES

^* This study was supported in part by Grant-in-Aid 08281105 for Scientific Research on Priority Areas from the Ministry of Education, Science, Culture and Sports of Japan.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.

^§ Supported by the Hayashi Memorial Foundation for Female Natural Scientists and a research fellowship from the Japan Society for the Promotion of Science for Young Scientists.

^¶ Contributed equally to the present work.

Supported by the Venture Business Laboratory of Okayama University. Present address: Dept. of Biochemistry, Faculty of Medicine, Okayama University, Okayama 700-8558, Japan.

^§§ To whom correspondence should be addressed. Tel./Fax: 81-86-251-7933; E-mail: moriyama@pheasant.pharm.okayama-u.ac.jp.

Published, JBC Papers in Press, July 27, 2000, DOI 10.1074/jbc.M003323200

	ABBREVIATIONS

The abbreviations used are: EIPA, ethylisopropylamiloride; BCECF, 2',7'-bis-(2-carboxyethyl)-5,6-carboxylfluorescein; BCECF-AM, 2',7'-bis-(2-carboxyethyl)-5,6-carboxylfluorescein acetoxymethyl ester; PBS, phosphate-buffered saline; V-ATPase, vacuolar H⁺-ATPase.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				N. Konno, S. Hyodo, Y. Yamaguchi, H. Kaiya, M. Miyazato, K. Matsuda, and M. Uchiyama African lungfish, Protopterus annectens, possess an arginine vasotocin receptor homologous to the tetrapod V2-type receptor J. Exp. Biol., July 15, 2009; 212(14): 2183 - 2193. [Abstract] [Full Text] [PDF]

				D. A. van Schalkwyk, W. Priebe, and K. J. Saliba The Inhibitory Effect of 2-Halo Derivatives of D-Glucose on Glycolysis and on the Proliferation of the Human Malaria Parasite Plasmodium falciparum J. Pharmacol. Exp. Ther., November 1, 2008; 327(2): 511 - 517. [Abstract] [Full Text] [PDF]

				P. Ellekvist, J. Maciel, G. Mlambo, C. H. Ricke, H. Colding, D. A. Klaerke, and N. Kumar Critical role of a K+ channel in Plasmodium berghei transmission revealed by targeted gene disruption PNAS, April 29, 2008; 105(17): 6398 - 6402. [Abstract] [Full Text] [PDF]

				N. Marchesini, M. Vieira, S. Luo, S. N. J. Moreno, and R. Docampo A Malaria Parasite-encoded Vacuolar H+-ATPase Is Targeted to the Host Erythrocyte J. Biol. Chem., November 4, 2005; 280(44): 36841 - 36847. [Abstract] [Full Text] [PDF]

				T. Wassmer, M. Froissard, H. Plattner, R. Kissmehl, and J. Cohen The vacuolar proton-ATPase plays a major role in several membrane-bounded organelles in Paramecium J. Cell Sci., July 1, 2005; 118(13): 2813 - 2825. [Abstract] [Full Text] [PDF]

				R. J. W. Allen and K. Kirk The Membrane Potential of the Intraerythrocytic Malaria Parasite Plasmodium falciparum J. Biol. Chem., March 19, 2004; 279(12): 11264 - 11272. [Abstract] [Full Text] [PDF]

				J. P. Shelby, J. White, K. Ganesan, P. K. Rathod, and D. T. Chiu A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes PNAS, December 9, 2003; 100(25): 14618 - 14622. [Abstract] [Full Text] [PDF]

				K. J. Saliba, R. J. W. Allen, S. Zissis, P. G. Bray, S. A. Ward, and K. Kirk Acidification of the Malaria Parasite's Digestive Vacuole by a H+-ATPase and a H+-pyrophosphatase J. Biol. Chem., February 14, 2003; 278(8): 5605 - 5612. [Abstract] [Full Text] [PDF]

				F. Wissing, C. P. Sanchez, P. Rohrbach, S. Ricken, and M. Lanzer Illumination of the Malaria Parasite Plasmodium falciparum Alters Intracellular pH. IMPLICATIONS FOR LIVE CELL IMAGING J. Biol. Chem., September 27, 2002; 277(40): 37747 - 37755. [Abstract] [Full Text] [PDF]

				H. M. Staines, J. C. Ellory, and K. Kirk Perturbation of the pump-leak balance for Na+ and K+ in malaria-infected erythrocytes Am J Physiol Cell Physiol, June 1, 2001; 280(6): C1576 - C1587. [Abstract] [Full Text] [PDF]

				K. Kirk Membrane Transport in the Malaria-Infected Erythrocyte Physiol Rev, April 1, 2001; 81(2): 495 - 537. [Abstract] [Full Text] [PDF]

				K. J. Saliba and K. Kirk H+-coupled Pantothenate Transport in the Intracellular Malaria Parasite J. Biol. Chem., May 18, 2001; 276(21): 18115 - 18121. [Abstract] [Full Text] [PDF]

				M. Hayashi, S. Taniguchi, Y. Ishizuka, H.-S. Kim, Y. Wataya, A. Yamamoto, and Y. Moriyama A Homologue of N-Ethylmaleimide-sensitive Factor in the Malaria Parasite Plasmodium falciparum Is Exported and Localized in Vesicular Structures in the Cytoplasm of Infected Erythrocytes in the Brefeldin A-sensitive Pathway J. Biol. Chem., April 27, 2001; 276(18): 15249 - 15255. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/44/34353    most recent M003323200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hayashi, M. Articles by Moriyama, Y. Search for Related Content PubMed PubMed Citation Articles by Hayashi, M. Articles by Moriyama, Y. Social Bookmarking                      What's this?