From the Departments of Biochemistry and
¶ Medicinal Informatics, Faculty of Pharmaceutical Sciences,
Okayama University, Okayama 700-8530, Japan and the Department
of Physiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
Received for publication, December 26, 2000, and in revised form, January 24, 2001
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ABSTRACT |
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N-Ethylmaleimide-sensitive
factor (NSF) and its homologues play a central role in vesicular
trafficking in eukaryotic cells. We have identified a NSF homologue in
Plasmodium falciparum (PfNSF). The reported
PfNSF gene sequence (GenBankTM accession
number CAB10575) indicated that PfNSF comprises 783 amino acids with a
calculated molecular weight of 89,133. The overall identities of its
gene and amino acid sequences with those of rat NSF are 50.9 and
48.8%, respectively. Reverse transcription-polymerase chain
reaction analysis and Northern blotting with total P. falciparum RNA indicated expression of the PfNSF
gene. Polyclonal antibodies against a conserved region of NSF
specifically recognized an 89-kDa polypeptide in the parasite cells.
After homogenization of the parasite cells, ~90% of an 89-kDa
polypeptide is associated with particulate fraction, suggesting
membrane-bound nature of PfNSF. PfNSF was present within both the
parasite cells and the vesicular structure outside of the parasite
cells. The export of PfNSF outside of the parasite cells appears to
occur at the early trophozoite stage and to terminate at the merozoite
stage. The export of PfNSF is inhibited by brefeldin A, with 9 µM causing 50% inhibition. Immunoelectromicroscopy
indicated that intracellular PfNSF was associated with organelles such
as food vacuoles and that extracellular PfNSF was associated with
vesicular structures in the erythrocyte cytoplasm. These results
indicate that PfNSF expressed in the malaria parasite is exported to
the extracellular space and then localized in intraerythrocytic
vesicles in a brefeldin A-sensitive manner. It is suggested that a
vesicular transport mechanism is involved in protein export targeted to
erythrocyte membranes during intraerythrocytic development of the
malaria parasite.
Plasmodium falciparum, a human malaria parasite,
invades an erythrocyte during one stage of its life cycle. In an
infected erythrocyte, the P. falciparum organism develops a
membrane structure called the parasitophorus vacuolar membrane.
The parasitophorus vacuolar membrane extends into the host cell
cytoplasm and forms a complex membrane structure, thus called the
tubovesicular membrane network (reviewed in Refs. 1-3). These membrane
systems outside the P. falciparum cell are important for the
transport of various nutrients such as glucose, phospholipids, and
amino acids and for extrusion of antimalarial agents so as to maintain
suitable circumstances for them (3-6). In addition to the formation of such intraerythrocytic membrane systems, P. falciparum cells
also transport some proteins such as erythrocyte membrane protein-1 of
P. falciparum
(PfEMP1)1 and PfEMP3 to the
erythrocyte plasma membrane, which results in the formation of a
knob-like structure on the surfaces of the infected
erythrocytes. These proteins are responsible for protection against
immunological attack and attachment of infected erythrocyte to
endothelial cells, one of the crucial steps for cerebral malaria (1-3,
7-11). Importantly, the extraparasite protein transport process can
not rely upon the endogenous transport machinery in the host cells,
because mature erythrocytes are completely devoid of machinery for
protein trafficking. Thus, the malaria parasite must transport proteins
through the plasma membrane and the membrane structure in the cytoplasm
of the host cells by means of their own mechanism, although the
molecular pathway for the transport of proteins through the parasite
plasma membrane is less understood.
It has been shown that the transport of some proteins from malaria
parasites is sensitive to BFA (12-16), which is a well known macrolide
antibiotic produced by fungi that blocks eukaryotic protein trafficking
processes, especially transport from the endoplasmic reticulum to the
Golgi apparatus by inhibiting the activities of ADP-ribosylation
factors and guanine nucleotide exchange factors (17). These results
suggest that the transport pathway from the endoplasmic reticulum to
the Golgi apparatus is involved in the targeting of parasite proteins
to the plasma membranes of parasitized erythrocytes. Consistently, a
homologue of the ADP-ribosylation factor and a variety of small
GTP-binding proteins, including a Sar1 homologue, which are components
of the common machinery for membrane traffic, have been identified in
P. falciparum (18-22). However, in mature intraerythrocytic
malaria parasites, a morphologically distinguishable Golgi apparatus
has not yet been identified, although the cis-Golgi marker,
PfERD2, is present separately with the trans-Golgi marker,
PfRab6. Sphingomyelin synthase, a marker for the Golgi apparatus in
other eukaryotes, is present at least partially in tubovesicular
membrane networks (3, 23, 24). Upon treatment with BFA,
Plasmodium proteins exported and localized to erythrocyte membrane such as merozoite surface protein-1 are accumulated in a novel
compartment similar to but distinct from the endoplasmic reticulum
within the malaria parasite (25). Thus, the mechanism of protein
secretion through the malaria parasite plasma membrane seems to be
unusual, and it may be more complex than that in other eukaryotes.
In eukaryotic cells, protein transport along the secretory pathways is
mediated by vesicles that move between the organelles (26). Transport
vesicles are formed from the donor compartment and are targeted to
acceptor organelles, where they deliver cargo molecules through
membrane fusion. The docking and/or fusion of transport vesicles with
the target membranes is mediated by the supramolecular protein complex
consisting of NSF, soluble NSF-attachment protein (SNAP), and receptors
for soluble NSF attachment protein (SNARE) (27-29). During docking
and/or fusion of transport vesicles, NSF may form a 20 S complex
together with receptors for soluble NSF attachment protein at the
target membrane (tSNARE) and vesicular receptors for soluble
NSF-attachment protein (vSNARE) to trigger membrane fusion with the
plasma membrane (30, 31). It would be interesting to determine whether
proteins involved in the above mentioned docking/fusion of vesicles are
present in malaria parasites.
Very recently, Bowman et al. (32) reported the complete
nucleotide sequence of chromosome 3 of P. falciparum. In the
sequence, they found a gene homologous to the NSF gene and called the
protein MP03103 (GenBankTM accession number CAB10575). In
the present study, we found that this protein is actually expressed and
present in P. falciparum cells. To our surprise, part of
P. falciparum NSF (PfNSF) appears to be exported from the
parasite cells and localized in vesicular structures in the erythrocyte
cytoplasm in a BFA-sensitive manner. These results are consistent with
the idea that a supramolecular protein complex consisting of NSF, SNAP,
and receptors for soluble NSF attachment protein is involved in the
targeting of P. falciparum proteins to the plasma membranes
of host erythrocytes.
P. falciparum Culture and Drug Treatment--
P.
falciparum strain FCR-3 cells were cultured in RPMI 1640 medium
(Life Technologies, Inc.) containing 50 mg/liter gentamycin and 10%
A+ human serum at a hematocrit of 5%, according to the
method of Trager and Jensen (33). Erythrocytes exhibiting 0.3%
parasitemia were added to each well of plates in 990 µl 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 (34). Then BFA dissolved in ethanol was added to the cultures
at the concentrations shown in Fig. 7. After incubation for 2 h,
the parasitized erythrocytes were washed and used for further experiments.
Cell Fractionation--
P. falciparum cells were
obtained by saponin treatment as follows (34). Parasitized erythrocytes
exhibiting 3.5-7% parasitemia (about 1 × 108 cells)
were washed three times with PBS containing 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride and suspended in 40 ml of the same buffer. Then saponin was added to a
final concentration of 0.075%. The solution was incubated for 5 min at
4 °C and centrifuged at 10,000 × g for 10 min. The pellet (malaria parasites) was washed three times with the same buffer,
suspended in 100 µl of 20 mM MOPS-Tris buffer (pH. 7.0) containing 5 mM EDTA, 0.25 M sucrose, 10 µg/ml pepstatin A, 10 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride, and kept in an ice bath until use. The
supernatant, which contained the particulate fraction in the
extraparasitic space, was centrifuged at 105,000 × g
for 1 h, and the pellet and supernatant were obtained. The pellet
was suspended in the same buffer and designated as the extracellular
particulate fraction.
Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
Analysis--
Total RNA extracted from parasitized erythrocytes (1 µg each) was transcribed into cDNA in a final volume of 20 µl
of reaction buffer containing 0.5 mM each dNTP, 10 mM dithiothreitol, 100 pmol of random octamers, and 200 units of Molony murine leukemia virus reverse transcriptase (Amersham
Pharmacia Biotech). After a 1-h incubation at 42 °C, the reaction
was terminated by heating at 90 °C for 5 min. For PCR amplification,
the 100-fold diluted synthesized cDNA solution was added to the
reaction buffer containing 0.12 mM dNTPs (30 µM each dNTP), 25 pmol of primers, and 1.5 units of
AmpliTaq-Gold polymerase (PerkinElmer Life Sciences). 35 temperature cycles were conducted as follows: denaturation at 94 °C
for 30 s, annealing at the temperature specific for each set of
primers for 30 s, and extension at 72 °C for 30 s. The
amplification products were finally analyzed by polyacrylamide gel
electrophoresis. The sequences of the oligonucleotides used as primers
were based on published sequences (32). For amplification of the
PfNSF gene, the specific sense primer was
5'-GGGAATATAGGGAGAGAAGAA-3' (bases 163-183), and the antisense primer
was 5'-AGCCACTAAATCCCGAAGAAT-3' (bases 539-559). The antisense primer
(5'-CTCATCAAAATCGGCTGTCTT-3' (bases 475-495) was also used for 2 second amplification. For amplification of the parasite actin-1
gene (GenBankTM accession number M22719), the specific
sense primer was 5'-GCAGCAGGAATCCACACAAC-3' (bases 1119-1138), and the
antisense primer was 5'-GTGGACAATACTTGGTCCTG-3' (bases 1402-1421).
Northern Blotting--
Total RNA (25 µg) isolated from
parasitized eryhtrocytes were separated on a formaldehyde-agarose gel
(1%) and transferred to a nylon membrane (Amersham Pharmacia Biotech).
The immobilized RNA was probes with a cDNA fragment of
PfNSF, amplified as described above, labeled with
[32P]dATP by random priming. After extensive washing, the
membrane was subjected to autoradiography using BAS 1000 film
(Fuji Film Co.).
Antibodies--
Site-specific antibodies against PfNSF were
raised in rabbits by injecting the following peptide conjugated to
thyroglobulin with glutaraldehyde:
412DLIDEALLRPGR423 (which is conserved among
mammalian NSFs; see Fig. 1). The antibodies recognized NSFs of
mammalian origin and plant sources (data not shown). Site-specific
polyclonal antibodies against vacuolar H+-ATPase subunit A
were prepared as described previously (35). Polyclonal antibodies
against the serine repeat antigen protein and H+-pumping
pyrophosphatase from mung bean were kindly provided by Dr. Mitamura
(Institute of Microbial Diseases, Osaka University, Japan) and Dr.
Maeshima (Nagoya University, Japan), respectively.
Immunoblotting--
Samples were denatured with SDS-sample
buffer containing 1% SDS and 10% Indirect Immunofluorescence Microscopy--
Parasitized
erythrocytes on polylysine-coated glass coverslips were fixed in PBS
containing 4% paraformaldehyde for 30 min, washed three times with
PBS, then incubated with PBS containing 0.1% Triton X-100 for 20 min
and further with 2% goat serum and 0.5% bovine serum albumin in PBS,
and finally reacted with antibodies at 10 µg/ml 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 or Olympus BX60 fluorescence microscope.
Immunoelectromicroscopy--
The pre-embedding silver
enhancement immunogold method described by Burry et al. (36)
was used with a slight modification. The parasitized erythrocytes on
polylysine-coated plastic coverslips were fixed in 4% paraformaldehyde
plus 0.1% glutaraldehyde dissolved in 0.1 M sodium
phosphate buffer (pH 7.4) for 30 min and then washed three times with
sodium phosphate buffer. Then the cells were incubated in a blocking
buffer containing 0.25% saponin and 5% bovine serum albumin for 30 min and reacted with anti-NSF antiserum (200× dilution) in
blocking buffer containing 0.005% saponin, 10% bovine serum albumin,
10% goat serum, and 0.1% cold water fish gelatin at 4 °C
overnight. Then the cells were washed in sodium phosphate buffer
containing 0.005% saponin and incubated with goat anti-rabbit IgG
conjugated with colloidal gold (1.4-nm diameter, Nanogold, Nanoprobes)
in blocking buffer for 2 h at room temperature. Cells were washed
five times with sodium phosphate buffer containing 0.005% saponin for
10 min, washed with sodium phosphate buffer for 5 min, and fixed with
1% glutaraldehyde for 10 min. After washing, the gold particles were
intensified using a silver enhancement kit (HQ silver, Nanoprobes) for
5 min at 20 °C in the dark. After washing in distilled water, the
cells were post-fixed with 0.5% OsO4 for 90 min at
4 °C, washed with distilled water, dehydrated with a graded series
of ethanol, and embedded in epoxy resin. Ultrathin sections were doubly
stained with uranyl acetate and lead citrate and observed under a
Hitachi H7000 electron microscope.
Other Procedures, Preparations, and Chemicals--
ATPase
activity was measured as described previously (37). Synaptic vesicles
were prepared from rat brains as described previously (38). A Golgi
membrane-rich fraction was prepared from CHO cells as described
previously (38). BFA and C5-ceramide were purchased from Molecular
Probes. Other chemicals used in the study were the highest grade
available commercially.
Expression of PfNSF--
The complete nucleotide sequence of
chromosome 3 of P. falciparum enabled us to identify the
gene encoding NSF or its homologue. A gene that is homologous to the
mammalian NSF gene consisting of 2352 base pairs without any intron
structures (GenBankTM accession number CAB10575) encodes
783 amino acids with a calculated molecular weight of 89,133 (Fig.
1). The overall identities of its gene
sequence and amino acid sequence with those of rat NSF are 50.9 and
48.8%, respectively. This gene product is abbreviated as PfNSF in this
study. Like other NSFs, PfNSF possesses two conserved ATP-binding
motifs (Walker motifs), which are GXXXXGKT at
positions 294-301 and 575-582 (Fig. 1, boxed).
We examined whether or not the PfNSF gene is actually
expressed in P. falciparum cells. We prepared a total RNA
fraction from parasitized erythrocytes, and then RT-PCR reaction was
performed using specific primers for a PfNSF gene as
described under "Experimental Procedures." When the reverse
transcripts were used, an amplified product with the expected size was
obtained (Fig. 2A, lane
3), whereas no amplified product was obtained when samples without reverse transcriptase reaction were used (Fig. 2A,
lane 4). The nucleotide and deduced amino acid sequences of
the amplified DNA product (333 base pairs corresponding to 55-165
amino acid sequence positions) exactly matched those of the
PfNSF gene (Fig. 1). Northern blotting with the amplified
product as a probe demonstrated the presence of a single species of
mRNA of PfNSF (~3.6 kilobases) (Fig. 2B). These
results indicate that the PfNSF gene is actually expressed
in P. falciparum cells.
To identify PfNSF at the protein level, we raised polyclonal antibodies
specific to the conserved region of NSF (see Fig. 1, dashed
line). The anti-NSF antibodies recognized single 83- and 89-kDa
polypeptides in rat brain synaptic vesicles and parasitized erythrocytes, respectively (Fig.
3A, lanes 1 and
2). The apparent molecular masses of these polypeptides are
those expected from their cDNA sequences (Fig. 1). No immunological
cross-reactivity was detected when noninfected control erythrocytes
were used (data not shown). The presence of the antigenic peptide,
which was used for preparation of anti-NSF antibodies, during
immunodecoration, prevented the immunological cross-reactivities (Fig.
3A, lanes 3 and 4). These results
strongly suggest that PfNSF was present in the parasitized
erythrocytes.
Isolated parasites (about 108 cells) were homogenized
vigorously and then centrifuged again at 105,000 × g
for 1 h. More than 90% of the anti-NSF immunoreactivity was
recovered in the pellet, suggesting that most PfNSF is present as a
membrane-bound form.
Properties of PfNSF--
Mammalian NSFs have been shown to be
N-ethylmaleimide-sensitive ATPases; upon the addition of
MgATP, the enzymes may hydrolyze ATP, thereby forming ADP and inorganic
phosphate, although the rate of hydrolysis is quite slow (39). We
isolated PfNSF by solubilization with polyoxyethylene lauryether
followed by immunoprecipitation with anti-NSF antibodies (Fig.
3B). The isolated PfNSF showed MgATP hydrolytic activity
(0.11 µmol of Pi liberated/h/mg of protein), which was
inhibited completely by N-ethylmaleimide at 1 mM. The N-ethylmaleimide-sensitive ATPase
activity was weak but comparable with those in mammalian NSFs
(39). These results suggested that PfNSF is a
N-ethylmaleimide-sensitive ATPase as in the case of mammalian NSFs.
NSF in the CHO Golgi fraction is known to be released from the membrane
upon treatment with MgATP (26, 27), although NSF in neuronal synaptic
vesicles or endocrine synaptic-like microvesicles does not have such an
effect (38, 40). We examined whether or not PfNSF is released from the
membrane upon the addition of ATP. As shown in Fig.
4, neither PfNSF nor NSF in synaptic
vesicles was released from parasite membranes upon treatment with
MgATP, whereas the same treatment released NSF from CHO Golgi
membranes. These results indicated that PfNSF shares properties with
NSFs of synaptic vesicles and synaptic-like microvesicles.
Presence of Extracellular PfNSF--
During isolation of malaria
parasites from parasitized erythrocytes with saponin (see
"Experimental Procedures"), we noticed that an appreciable level of
anti-NSF immunoreactivity remained in the supernatant after isolation
of the parasite cells (Fig. 5A). This fraction contained
the erythrocyte cytoplasm, erythrocyte plasma membranes, and
extraparasitized membrane structures including tubovesicular membrane
networks. This fraction was then centrifuged at 105,000 × g for 1 h, a pellet (the extracellular particulate fraction) and a supernatant being obtained. Western blotting
experiments indicated that PfNSF was present in the extracellular
particulate fraction as well as in the parasites but not in the
supernatant (Fig. 5A). Vacuolar H+-ATPase and
H+-pumping pyrophosphatase, which are known to be present
in the parasite (35, 41), were detected in parasite cells but not detected in either the extracellular particulate fraction or the supernatant (Fig. 5A). These results strongly suggest the
presence of extraparasitized membrane-bound PfNSF.
Indirect immunofluorescence microscopy with anti-NSF antibodies further
demonstrated the presence of extraparasitized PfNSF in erythrocytes.
The anti-NSF antibodies immunostained the parasite cells in parasitized
erythrocytes (Fig. 5B), whereas no immunoreactivity was
found in noninfected erythrocytes (Fig. 5C). Significantly, the PfNSF immunoreactivity was present within the vesicular structures outside the parasite cells (Fig. 5B). Consistent with the
distribution observed on the immunoblotting shown in Fig.
5A, no such extraparasitized vesicular structures were
observed in the immunoreactivities against antibodies for vacuolar
H+-ATPase (Fig. 5D), H+-pumping
pyrophosphatase (Fig. 5E), serine repeat antigen protein, markers for the peripheral space between the parasitophorus
vacuolar membranes, and the plasma membrane of the malaria parasite
(Fig. 5F). Vital staining with C5-ceramide revealed
tubovesicular membrane networks (6) (Fig. 5G). From these
results, we concluded that PfNSF is present in both parasite cells and
vesicular structures outside of parasite cells.
During development, a similar degree of anti-PfNSF immunoreactivity was
observed in all stages of intraerythrocyte parasites, indicating that
PfNSF is expressed in all cell stages. At the early trophozoite stage,
PfNSF appeared in the extraparasite space and seemed to be associated
with several apparent vesicular structures outside the parasites in the
erythrocyte cytoplasm. At the trophozoite stage, the PfNSF-positive
vesicular structure seems to be more discrete and distributed
throughout erythrocyte cytoplasm, which becomes weak at the schizont
stage and disappears at the merozoite stage (Fig.
6). These results indicate that export of
PfNSF occurs at the early trophozoite stage.
Export of PfNSF Is BFA-sensitive--
To obtain information on the
mechanism by which PfNSF is transported outside the parasites, we next
examined the effect of BFA (Fig. 7). It
was found that BFA effectively blocked the export of PfNSF from
malarial parasites; the concentration required for 50% inhibition was
9 µM. Almost all immunoreactivity against anti-NSF antibodies outside of parasite cells disappeared upon treatment with 50 µM BFA for 2 h. The effect of BFA was reversible,
because the immunoreactivity outside the parasite cells appeared again when the erythrocytes were washed several times and resuspended in
culture medium. Under similar assay conditions, BFA did not affect the
distribution of either serine repeat antigen protein or C5-ceramide
(data not shown). These results indicated that the export of PfNSF is
sensitive to BFA.
Prolonged exposure to 50 µM BFA for 24 h inhibited
maturation of the parasite; BFA-treated cells did not progress to the
trophozoite stage, whereas control cells matured normally. This arrest
was also reversible. These results were consistent with previous
observations (12, 22).
Subcellular Localization of PfNSF--
Finally, the localization
of PfNSF in parasitized erythrocytes at the subcellular level was
investigated by immunoelectromiscroscopy. Consistent with the
immunohistochemistry described in Fig. 5A, immunogold
particles for PfNSF were selectively and intensely labeled in
infected P. falciparum cells, whereas few immunogold particles were observed in erythrocyte cytoplasm (Fig.
8A). The immunogold particles
seem to be associated with intracellular organelles such as food
vacuoles (Fig. 8A) and plastid-like organelle (Fig.
8B), supporting the membrane-bound nature of PfNSF (Fig. 4).
As shown in Fig. 8, C and D, immunogold particles
were also associated with vesicular structures with a diameter of
30-70 nm and electron translucent contents in erythrocytes. The
immunogold particles associated with vesicles outside the
parasite cells disappeared when the cells were treated with BFA,
although the BFA treatment did not change the number or morphology of
the vesicles (data not shown). Immunogold particles were also
associated with larger membrane structure in erythrocyte cytoplasm,
which may correspond to part of the tubovesicular membrane network
(Fig. 8A, arrowhead).
NSF and its homologue are key proteins that comprise a
supramolecular complex with SNAP and its receptors (which
contain synaptotagmin, vesicular-associated membrane protein (VAMP),
and syntaxin), catalyze the docking and fusion of vesicles, and
facilitate protein transport during the biogenesis of organelles in
eukaryotes (26-29). Because the gene encoding NSF or its homologue was
identified in P. falciparum cells, one can expect that NSF
or its homologues is expressed and functions in P. falciparum cells. The identification and characterization of the
PfNSF protein may provide a clue as to the vesicular transport systems
in the malaria parasites and the mechanism underlying protein transport
to the erythrocyte membrane. The present study was, therefore,
undertaken to obtain the direct evidence of PfNSF in P. falciparum cells.
We detected the mRNA of PfNSF by RT-PCR and Northern blotting (Fig.
2), and identified PfNSF by Western blotting with site-directed polyclonal antibodies against a conserved region of NSF (Fig. 3). The antibodies immunostained the whole body of malaria parasites infecting erythrocytes (Fig. 5). Furthermore, the immunoprecipitated polypeptide showed a weak N-ethylmaleimide-sensitive ATPase
activity. Taken together, these results constitute evidence for the
functional occurrence of the PfNSF protein in the malaria parasite.
The presence of PfNSF suggests that a vesicular transport mechanism is
operating in the malaria parasite. PfNSF is associated with organelles
such as food vacuoles, suggesting that PfNSF plays a role in the
biogenesis of organelles through vesicular transport. It is noteworthy
that PfNSF is also associated with plastid-like organelles (Fig.
8B). The plastid of P. falciparum is an
evolutionary homologue of the plant chloroplast. Very recently, signal
and plant-like transit peptides were found to be involved in the
protein trafficking to plastids in P. falciparum (42). The
association of PfNSF with plastid-like organelles suggests that PfNSF
plays some role in protein trafficking to plastids. Consistently, a NSF
homologue was shown to be important for vesicle fusion and/or membrane
protein translocation in plastids of the higher plant Capsicum
annuum (43).
Another important finding of the present study is that PfNSF is
exported from parasite cells and localized in vesicular structures in
the erythrocyte cytoplasm. This suggests that PfNSF plays some role in
protein transport from the parasite to the erythrocyte plasma membrane.
Immunoelectromicroscopy clearly revealed that extraparasitized PfNSF is
associated with vesicles with diameters of 30-70 nm. In this respect,
it is noteworthy that PfEMP1 and PfEMP3, which are targeted to the
plasma membrane of infected erythrocytes, are also known to be
associated with vesicles (11). PfNSF containing vesicles and PfEMP1-
and PfEMP3-containing vesicles have electron translucent contents and
are morphologically similar to each other. It is possible that PfNSF is
involved in the targeting of PfEMP1 and pfEMP3 into erythrocyte plasma membrane.
Moreover, very recently, it was reported that P. falciparum
cells have the ability to transport erythrocyte membrane proteins to
internal organelles of the parasite cells and that cholesterol and
sphingomyelin are important for this process (44). Because the vacuolar
uptake of host components seems to correspond to the endocytotic
process in other eukaryotes, it appears that a vesicular transport
mechanism is involved in the endocytotic process as well as the
targeting of the parasite membrane to the erythrocyte membrane.
PfNSF is the first example of the presence of the SNAP receptor complex
in the malaria parasite. Since other constituents of the SNAP receptor
complex have not yet been detected in the malaria parasite, the
identification and characterization of such proteins will be important
in revealing all of the features of the putative vesicular transport
mechanism in the malaria parasite. Phylogenetically, the vesicular
machinery participating in membrane biogenesis, such as docking and
fusion of vesicles, is broadly conserved across the species barrier in
higher eukaryotes. Consistent with this idea, Toxoplasma
uses trafficking mechanisms, that is the NSF/SNAPs/SNAP
receptor/Rabs, suggesting a role in exocytotic and endocytotic pathways
(45, 46). It is possible that a vesicular transport mechanism operates
in pathogenic protozoa in general.
In conclusion, we obtained evidence of NSF or its homologue in P. falciparum cells. Like the Sar1 protein, parts of PfNSF are
associated with vesicles in the erythrocyte cytoplasm. It is possible
that these vesicles are involved in protein targeting to the
erythrocyte plasma membrane and that the SNAP receptor complex is
involved in this transport process. P. falciparum cells may
constitute a unique experimental system for studies on vesicular trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol and then
electrophoresed on a 12% polyacrylamide gel in the presence of SDS
(36). Following electrotransfer at 0.3 amperes for 2 h, the
nitrocellulose filters were blocked in a buffer consisting of 20 mM Tris-HCl (pH 7.6), 5 mM EDTA, 0.1 M NaCl, and 0.5% bovine serum albumin for 4 h and
then probed with 1000-fold diluted antiserum for 2 h. The
filters were washed with 20 mM Tris-HCl buffer (pH 7.6)
containing 5 mM EDTA, 0.1 M NaCl, and 0.05%
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 then
subjected to ECL amplification according to the manufacturer's manual
(Amersham Pharmacia Biotech).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the amino acid sequence of PfNSF
with those of NSFs of other origins. The amino acid sequence of
PfNSF (PF-NSF, top line) is aligned with those of
NSFs of other origins as indicated. Asterisks indicate
conserved amino acid residues. The consensus amino acid sequences for
ATP binding in ATPase (Walker motifs 1 and 2) are boxed. The
conserved region, which was used for the preparation of anti-NSF
antibodies, is also boxed with a dashed line. The
overall identities of gene sequences and amino acid sequences with
those of human and yeast NSF are 51.7, 47.2, 55.1, and 47.2%,
respectively.
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Fig. 2.
Identification of the transcript of the
PfNSF gene. A, the presence of
mRNA for actin 1 (lanes 1 and 2) and PfNSF
(lanes 3 and 4) was examined by RT-PCR, and the
amplified products were visualized on an acrylamide gel stained with
ethidium bromide. The amplified products were not detected when samples
without reverse transcriptase reaction were used in lanes
2 and 4, respectively. The position of the PfNSF
transcript is indicated by an arrow. The positions of
molecular markers are also indicated (from top to
bottom, 1412, 517, 396, and 221 base pairs (bp)).
B, Northern blotting. Total RNA (25 µg) obtained from
asynchoronous parasites was hybridized to the amplified product for
PfNSF shown above, washed, and visualized using BAS 1000 film after
overnight exposure.
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Fig. 3.
Immunological detection of PfNSF.
A, rat brain synaptic vesicles (50 µg protein)
(lanes 1 and 3) and malarial cells isolated by
saponin treatment (10 µl) (lanes 2 and 4) were
dissolved in SDS-sample buffer. After electrophoresis, the proteins
were transferred to a nitrocellulose sheet followed by
decoration with anti-NSF antibodies (1000 diluted antiserum),
and the immunoreactivity was visualized with ECL. For lanes
3 and 4, the nitrocellulose sheet was incubated with 2 mg of antigenic peptide during the antibody treatment. B,
isolated parasites (about 108 cells) were solubilized with
1% polyoxyethylene lauryether and then centrifuged at 105,000 × g for 1 h. The supernatant was carefully removed,
anti-NSF antiserum (10 µl) or control serum (10 µl) was added, and
then successively 30 µl of protein A-labeled Sephadex (Bio-Rad) was
added. The solution was then gently shaken at 4 °C overnight. Then
protein A-labeled particles were washed several times with PBS,
dissolved in the sample buffer as described above, and finally
electrophoresed. Immunoblotting with anti-NSF antibodies was performed
as described above. Lane 1, supernatant (30 µg of
protein); lane 2, anti-NSF antibody; lane 3,
control serum.
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Fig. 4.
The effect of MgATP on the membrane-bound
PfNSF. Isolated parasites (about 108 cells) were
vigorously homogenized and then centrifuged again at 105,000 × g for 1 h, and a particulate fraction was obtained. CHO
Golgi membranes, rat brain synaptic vesicles, and a particulate
fraction of P. falciparum cells were then incubated in 25 mM MOPS-Tris (pH 7.2) containing 1 mM
dithiothreitol, 0.1 M KCl, 0.3 M sucrose, and
2% polyethylene glycol 4000 plus 5 mM MgCl2
(lane 1), 5 mM MgCl2, and 0.5 mM ATP (lane 2), or 2 mM EDTA and
0.5 mM ATP (lane 3), according to Ref. 37. After
incubation on ice for 10 min, the mixtures were centrifuged at
400,000 × g for 30 min in a Beckman
micro-ultracentrifuge. The proteins in the supernatant were dissociated
with SDS-sample buffer, subjected to polyacrylamide gel electrophoresis
in the presence of SDS, and then analyzed by immunoblotting with
anti-NSF antibodies.
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[in a new window]
Fig. 5.
Evidence of the extraparasitized
PfNSF A, after saponin treatment, P. falciparum
cells (lane 1), an extracellular particulate fraction
(lane 2), and a soluble fraction (lane 3) were
isolated as described under "Experimental Procedures." After
dissociation with SDS-sample buffer, each sample was analyzed by
immunoblotting with the antibodies as indicated. B-G,
immunohistochemical detection of PfNSF (B), PfNSF in
noninfected erythrocyte (C), vacuolar H+-ATPase
subunit A (V-ATPase) (D),
H+-pumping pyrophosphatase (V-PPase)
(E), and the serine repeat antigen protein (F) in
parasitized erythrocytes. The parasitized erythrocytes were
immunostained with the indicated antibodies and then observed by
fluorescence microscopy. Vital staining with C5-ceramide was also
performed to reveal localization of the tubovesicular membrane networks
(TVM) (G). P, P. falciparum
cell; EMP, erythrocyte plasma membrane. Bar, 5 µm. (Note: during preparation, H+-pumping
pyrophosphatase was digested by protease, resulting in a low molecular
weight fragment (A)).
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Fig. 6.
Change in the distribution of PfNSF during
development. Parasitized erythrocytes at various stages as
indicated were doubly labeled with antibodies against NSF and
4',6-diamidino-2-phenylindole HCl and observed under a Nomarski
microscope (upper panel) and a fluorescence microscope
(lower panel). After superpositioning the two images, the
localization of PfNSF (green) and nuclei (blue)
is shown. R, ring; ET, early trophozoite;
T, trophozoite; S, schizont; M,
merozoite. Bar, 5 µm.
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Fig. 7.
The effect of BFA on the export of
PfNSF. Parasitized erythrocytes were treated in the absence
(A) or presence (B) of BFA at 50 µM
as described under "Experimental Procedures," and then indirect
immunofluorescence microscopy for PfNSF was performed. Bar,
10 µm. C, dose dependence of the effect of BFA. 100 parasitized erythrocytes grown under synchronized conditions
(trophozoite stage) were treated with the listed concentrations of BFA,
and the resultant numbers of PfNSF-positive vesicles were determined.
The results are expressed as relative percent, taking the number of
vesicles in the absence of BFA as 100%, which corresponds to
220.
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Fig. 8.
Morphological evidence of the association of
PfNSF with vesicular structures in parasitized erythocytes.
Immunoelectromicroscopy for PfNSF was performed as described under
"Experimental Procedures," and the localization of PfNSF was
visualized with immunogold particles after silver enhancement. The
whole body of a parasitized erythrocyte (A), plastid-like
organelle (B), or cytoplasm of erythrocyte (C and
D) is shown. Vesicles containing PfNSF in erythrocyte
cytoplasm are indicated by an arrowhead in A and
by arrows in C and D. FV,
parts of food vacuoles; E, erythrocyte cytoplasm;
EPM, erythrocyte plasma membrane; P, parasite
cytoplasm; TVM, tubovesicular membrane network.
Bar, 1 µm in A and 0.2 µm in
B-D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Yoshimori (Institute of Basic Biology, Okazaki, Japan) for critically reading the manuscript and Drs. T. Mitamura and T. Horii (Osaka University) for providing the antibodies against the serine repeat antigen protein.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid 08281105 for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports and Culture 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) CAB10575.
The first two authors contributed equally to the present work.
§ Supported by the Hayashi Memorial Foundation for Female Natural Scientists and by a Research Fellowship from the Japan Society for Promotion of Science for Young Scientists.
** To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan. Tel. and Fax: 81-86-251-7933; E-mail: moriyama@pheasant.pharm.okayama-u.ac.jp.
Published, JBC Papers in Press, February 1, 2001, DOI 10.1074/jbc.M011709200
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ABBREVIATIONS |
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The abbreviations used are: PfEMP, erythrocyte membrane protein of Plasmodium falciparum; BFA, brefeldin A; C5-ceramide, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)sphingosine; NSF, N-ethylmaleimide-sensitive factor; PfNSF, N-ethylmaleimide-sensitive factor from P. falciparum; PBS, phosphate-buffered saline; SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; CHO, Chinese hamster ovary; MOPS, 4-morpholinepropanesulfonic acid; RT-PCR, reverse transcription-polymerase chain reaction.
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REFERENCES |
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