 |
INTRODUCTION |
Due to the compartmentalization of eukaryotic cells, a
sophisticated protein trafficking system is an integral requirement for
homeostasis and growth. Proteins destined for compartments other than
the cytoplasm are synthesized with intrinsic signals that determine
their transport within the cell. Small peptide motifs often form the
necessary targeting determinants (1). For example, an N-terminal
hydrophobic sequence forms part of the typical secretory signal that
directs proteins across the endoplasmic reticulum
(ER)1 membrane (2-5).
Similarly, N-terminal amphipathic and bipartite sequences target
proteins to the chloroplast and mitochondria (6-8).
The malaria parasite, Plasmodium falciparum, spends part of
its life cycle inside mature human erythrocytes. The parasite invades
this quiescent host cell and develops within a parasitophorous vacuole
(PV). An unusual and highly specialized secretory system enables the
malaria parasite to survive within a cell that lacks its own machinery
for protein synthesis and trafficking. Indeed, the parasite targets
proteins, not only to compartments within its own confines, but to the
PV, in which it resides, as well as the PV membrane (PVM), the
erythrocyte cytoplasm, and host cell membrane (9-11).
Efforts have been made to understand the trafficking signals that
target parasite proteins to different compartments within and outside
the parasite. Proteins destined for the ER, the parasite plasma
membrane (PPM), the PV, or the PVM appear to have a "classical" hydrophobic N-terminal signal sequence (i.e. a stretch of
10-15 hydrophobic amino acids commencing 3-17 amino acids from the N terminus) (12, 13). For example, the secretory signal of the PVM-located integral membrane protein, exported protein-1 (Exp1, also
called antigen 5.1, QF119, or cross-reactive antigen) has a
characteristic N-terminal signal (14), which is cleaved by the malaria
parasite at a site adjacent to glutamate 23 (15). This signal sequence
is recognized by the translocation machinery of higher eukaryotes (16).
However, a number of proteins that are directed past the PVM to the
erythrocyte cytosol do not have classical N-terminal signals. Instead,
proteins such as the knob-associated histidine-rich protein (KAHRP)
have a hydrophobic stretch of amino acids starting 20-80 amino acids
from the N terminus (13, 17). This "internal" hydrophobic signal
does not appear to function in heterologous trafficking systems, since
KAHRP is not translocated across the ER membrane in a cell-free system
using mammalian microsomes (9, 16, 18).
Recently, the development of the technology for transfection of malaria
parasites has permitted an analysis of the signals that direct proteins
to different compartments. For example, Wickham et al. (19)
prepared a series of constructs in which gene fragments encoding the
N-terminal regions of KAHRP were appended to the reporter protein,
green fluorescent protein (GFP). These constructs were introduced into
P. falciparum using a stable transfection system (8, 19,
20), and the locations of the proteins were assessed by fluorescence
microscopy. The studies indicated that the atypical N-terminal signal
sequence of KAHRP contains information that is both necessary and
sufficient for entry into the ER and trafficking to the PV. These
studies suggested that secretion into the PV is the default pathway for
export of proteins in the malaria parasite. The studies of Wickham
et al. (19) also indicated that a separate sequence element
in the histidine-rich region of KAHRP is needed for the translocation
of this protein across the PVM. Trafficking of proteins to the
erythrocyte cytosol appears to be a two-step process, with the PV
acting as an intermediate compartment (19, 21). Following release into
the erythrocyte cytosol, soluble KAHRP appears to associate briefly
with the cytoplasmic surface of Maurer's clefts and eventually is
assembled into "knob" structures underneath the erythrocyte
membrane (19).
A separate transfection study employed constructs encoding the
classical N-terminal signal sequence of Exp1 appended to luciferase (22). Using a transient transfection system and a sensitive biochemical
assay for detection of the luciferase tag, these authors reported that
about 40% of the tagged Exp1 was directed to the PV and about 60% to
the erythrocyte cytosol. The authors concluded that export of parasite
proteins to the erythrocyte cytosol represents the default pathway for
protein transport and that additional signal sequence information is
required to trap the exported proteins in the PV.
The discrepancy between the data obtained from these two experimental
approaches prompted us to reexamine the Exp1 signal sequence in a
stable transfection system using GFP as the reporter. In this work, we
have prepared a construct encoding the N-terminal signal sequence of
Exp1 fused to GFP and used the plasmid to transfect P. falciparum-infected erythrocytes. Our results confirm that transit
through the ER to the PV is the default pathway for protein trafficking. Using dual labeling with the lipid probe,
BODIPY-TR-ceramide, we demonstrate that protrusions from the PV extend
into the erythrocyte cytoplasm and occasionally bud to form mobile
vesicular compartments containing PV components. The lipid probe was
also used to identify subcompartments of the PV that do not contain the
GFP chimera.
 |
MATERIALS AND METHODS |
PCR and Cloning--
The sequence of P. falciparum
exported antigen-1, Exp1, was obtained from the National Center for
Biotechnology Information data base (available on the World Wide Web at
www.ncbi.nlm.nih.gov/BLAST/; accession number P04926) (23, 24). The
sequence encoding amino acid residues 1-35 of Exp1 of P. falciparum 3D7 strain (EXP1-(1-105)) was
amplified by PCR from genomic DNA using primers: 5'-AGA TCT ATG AAA ATC TTC TTA TCA GTA TTT TTT C and 5'-CCT AGG GCT
GCT AAC ACC ACT TCC AGT TTC (restriction sites are underlined). The
resulting fragment, bearing the BglII and AvrII
restriction sites, was first cloned into the pCR2.1 TOPO vector
(Invitrogen) and then placed upstream of the mut 2 EGFP coding region
in the pHH2 vector (EXP1-(1-105)-GFP-pHH2) (8,
19). The insert was sequenced in both directions using fluorescent
dideoxynucleotide termination.
P. falciparum Culture and Transfection--
P.
falciparum-infected erythrocytes (ring stage, 3D7 strain) were
transfected with the EXP1-(1-105)-GFP-pHH2
plasmid and cultured in the presence of WR99210 (10 nM) as
described by Wickham et al. and Fidock et al.
(19, 25). Parasites expressing the GFP chimeric protein
(Exp1-(1-35)-GFP) were obtained 55 days after transfection and
thereafter maintained in the presence of 100 pM WR99210.
Harvesting and brefeldin A (BFA) treatment of parasites was carried
out as described by Adisa et al. (26).
Subcellular Fractionation of Transfectants--
Transfectants
were cultured to a parasitemia of 10% rings and then synchronized
using sorbitol treatment. To ensure tight synchronization, the
parasites were treated with sorbitol, allowed to develop for several
hours, and then treated with sorbitol again. These synchronous
parasites were cultured for a further 24 h (trophozoite stage) and
harvested on a Percoll-sorbitol cushion as described by Aley et
al. (27). To release the erythrocyte contents, ~5 × 107 parasites were treated with either 450 or 900 units of
streptolysin O (SLO; Sigma; corresponding to 4 or 8 hemolytic units, as
defined by Baumeister et al. (28)) and separated into
pellets and supernatants as described by Ansorge et al. (21,
29) and Burghaus and Lingelbach (22). To release the erythrocyte and PV
contents, 5 × 107 parasites were subjected to lysis
in the presence of 0.09% saponin (Sigma) and separated into soluble
and particulate fractions by centrifugation (21). Equivalent fractional
amounts of each of the samples were subjected to SDS-PAGE and Western analysis.
Antibodies and Lipid Probes--
Western blot analyses were
performed as previously described (26) using antibodies recognizing GFP
(murine monoclonal; Roche Molecular Biochemicals), P. falciparum S-antigen (rabbit antiserum against 3D7 strain obtained
from Prof. Robin Anders, La Trobe University) and Exp1 (rabbit
antiserum kindly provided by Prof. Klaus Lingelbach,
Philipps-University, Marburg, Germany). For immunoblot analyses,
asynchronous P. falciparum cultures were harvested,
subjected to SDS-PAGE (12% acrylamide), transferred to polyvinylidene
difluoride membrane, probed with antiserum followed by horseradish
peroxidase-conjugated secondary antibodies (Sigma), and developed with
enhanced chemiluminescence (ECL) reagent. BODIPY-TR-ceramide was
obtained from Molecular Probes and used to label parasitized erythrocytes as described by Behari and Haldar (30). Briefly, parasitized erythrocytes were resuspended in complete medium (5% parasitemia, 10% hematocrit) and incubated in the presence of 1 µM BODIPY-TR-ceramide in complete medium at 37 °C for
60 min, washed three times in complete medium and examined by
fluorescence microscopy. The extent of conversion of the exogenously
added ceramide to sphingomyelin was assessed by extracting the lipids and subjecting them to thin-layer chromatography as described by Haldar
et al. (31).
Confocal Fluorescence Microscopy--
Parasitized erythrocytes
expressing Exp1-(1-35)-GFP were mounted wet on a glass slide, covered
by a glass coverslip, and imaged within 20 min at ambient temperature
(maintained at 20 °C) using a Leica TCS confocal microscope
(Confocal Imaging Facility, Monash University, Clayton, Australia)
equipped with a ×100 planapochromatic oil immersion objective (1.4 numerical aperture). The 488- and 568-nm lines of a 60-milliwatt
krypton-argon laser were used to excite GFP and BODIPY-TR,
respectively. The fluorescence from each probe was detected through a
530/30-nm band pass filter and a 590-nm long pass filter, respectively.
Differential interference contrast (DIC) images were generated using
the transmitted light.
Photobleaching experiments were typically conducted as follows. Two
prebleach images were obtained, and the laser was focused on a single
spot (representing a diffraction-limited point) defined on the
prebleach image. The point was illuminated for a specified time (1 s)
using a bleach pulse at maximum laser power at the excitation wavelength characteristic of the fluorophore to be bleached.
In the case of fluorescence recovery after photobleaching (FRAP)
measurements, postbleach images were obtained immediately after the
high intensity illumination (delay ~1 s) and at time intervals after
bleaching. For fluorescence loss in photobleaching (FLIP) measurements,
four high intensity bleach pulses were applied at 30-s intervals prior
to the acquisition of each image. The gain on the photomultiplier was
kept high, and the laser power was adjusted using an acousto-optical
tunable filter to a level sufficient to minimize photobleaching of the
sample during image acquisition. Images were typically obtained at
0.049-nm pixel resolution and were the average of four acquisitions
(~2-s total image acquisition time).
Image Analysis--
Confocal images were processed and analyzed
using ImageJ software (available on the World Wide Web at
rsb.info.nih.gov/ij). For photobleaching measurements, images were
processed using a median filter followed by smoothing (both employing a
1-pixel radius) to improve the signal/noise ratio and were corrected
for background. The images were also corrected for the small degree of
bleaching occurring during image acquisition using a correction factor
obtained from the intensity of the two prebleach images and applying it
exponentially to the remaining images. The bleaching between successive
images was typically 2-5%. Two criteria were rigorously employed when
quantitating the intensity of regions of interest between successive
images: (i) the regions of interest should not contain saturated pixel
values (i.e. all pixel values should be less than 255), and
(ii) there should be no significant movement of the regions analyzed.
Note that the confocal images presented in this paper have been
adjusted for presentation purposes to highlight and accentuate features
of interest and do not represent the actual images analyzed.
The spatial distribution of bleaching was visualized by constructing a
bleach (B) image from a prebleach (PRE) and a
postbleach (POST) image as follows,
|
(Eq. 1)
|
where B, PRE, and POST
represent the pixel intensities at the x and y
pixel coordinates of the respective images. The spatial distribution of
the fluorescence recovery following bleaching was visualized using a
Quench/Recovery (QR) image. This was generated from a
prebleach (PRE) image and postbleach images obtained
immediately after bleaching (POST0) and at time
t following bleaching (POSTt).
|
(Eq. 2)
|
The differences between the postbleach images are normalized
relative to the prebleach image. Regions that increase in value (i.e. exhibit recovery) appear blue, whereas regions that
decrease in value (i.e. are quenched) appear red using the
lookup table shown in Fig. 9. In the case of B and
QR images, only regions of interest within the image that
did not appear to move during the course of the measurements were
visualized in the image.
 |
RESULTS |
Amino Acids 1-35 of Exp1, When Fused to GFP, Allow Secretion of
the Chimera in Transfected P. falciparum--
The arrangement of the
gene and the protein sequence encoded by EXP1 (3D7 strain)
are shown in Fig. 1. Exon 1 of the
EXP1 gene encodes the first 40 amino acids of the protein
(Fig. 1, A and B). The first 22 amino acids of
this protein have been shown to comprise a classical signal sequence
that functions both in vivo and in vitro to
direct proteins into the ER (14-16).

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Organization of the EXP1
gene and gene product. A, schematic diagram of
the EXP1 gene. Exons 1-3 are indicated by
rectangles, and introns and gene flanking sequences are
shown by solid lines. B, amino acid
sequence of 3D7 Exp1. The N-terminal signal sequence (which terminates
at the proteolytic cleavage site) is underlined, and the
hydrophobic core of the signal is double
underlined. The putative transmembrane region is in
italics. C, the N-terminal 35-amino acid fragment
of Exp1 appended to GFP (Exp1-(1-35)-GFP). It is likely that the
signal peptidase in the ER processes the chimera to a mature
form.
|
|
Recently, it has been suggested that the N-terminal region of Exp1 also
contains sequence elements that direct the protein beyond the PV (22).
To further examine this suggestion, we generated a chimera comprising
an N-terminal fragment of Exp1 linked to the reporter GFP. A portion of
the EXP1 gene that encodes the first 35 amino acids of the
protein was joined upstream of the mut 2 enhanced GFP coding sequence
in the transfection vector pHH2 (pHH2-EXP1-(1-35)-GFP) (Fig.
1C). This region of Exp1 includes the signal sequence
comprising 13 hydrophobic amino acids flanked by charged residues and
the cleavage site between residues 22 and 23. The resultant GFP fusion
protein was expressed from a stably maintained episome (8, 19) within
the transformed 3D7 P. falciparum blood stage parasites.
Parasites expressing the GFP chimeric protein (Exp1-(1-35)-GFP) were
obtained 55 days after transfection and were maintained in culture in
the presence of a 10 nM concentration of the antifolate drug, WR99210. They showed similar growth rates to untransfected parasites and maintained the plasmid even under lower (100 pM) drug concentrations. This suggests that the expression
of the GFP chimera did not confer a growth disadvantage on the parasites.
To confirm expression of the Exp1-(1-35)-GFP fusion protein in the
P. falciparum transfectants, we performed Western blots on
parasite-infected erythrocytes (Fig. 2).
When probed with antibodies recognizing GFP, no reactivity was observed
in uninfected erythrocytes or in the untransfected parental line, 3D7.
By contrast, the transfected parasites expressed proteins that were
resolved as a doublet of ~28 and 26 kDa. The predicted size of the
full-length Exp1-(1-35)-GFP chimera is 30 kDa. It is likely that the
28-kDa band represents a processed form of Exp1-(1-35)-GFP in which
the 22-amino acid signal sequence has been removed. The 26-kDa band
presumably represents a further degradation product. Lower molecular
weight degradation products of GFP chimeras have been reported
previously (8, 19).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 2.
Western blot analysis of parasites
transfected with the Exp1-(1-35)-GFP construct. Total cell
lysates from ~106 cells (10% parasitemia) were lysed by
freezing and thawing and subjected to electrophoresis (10-15%
acrylamide), transferred to polyvinylidene difluoride membrane, and
probed with anti-GFP antibody (Roche Molecular Biochemicals; 1:1000
dilution). UI, UT, and TR represent
uninfected erythrocytes and untransfected and transfected parasitized
erythrocytes, respectively.
|
|
The expression of the Exp1-(1-35)-GFP reporter in different stages of
the intraerythrocytic cycle of transfected P. falciparum was
examined using confocal fluorescence microscopy (Fig.
3). The Exp1-(1-35)-GFP chimera is
expressed under the control of the hsp86 promoter and is thus produced
at all stages of intraerythrocytic growth (32). By contrast, endogenous
Exp1 is expressed most strongly in mature stage parasites (33) (data
not shown). The protein appears to be largely located in the PV that
surrounds the parasite (Fig. 3). Some parasites showed a "necklace of
beads" pattern around the periphery of the parasite (Fig. 3,
B and D) as has been reported previously for a
chimera of an N-terminal fragment of KAHRP with GFP (19).
In some doubly infected erythrocytes (Fig. 3B),
only one of the parasites appeared to be expressing the transfection
construct. The second parasite has presumably expelled the plasmid but
is protected from the deleterious effects of WR99210 by its sister
parasite. In more mature stages of the intraerythrocytic development of
the parasite, protrusions from the PV were commonly observed (Fig. 3,
B, C, and E). The protrusions largely
appeared to remain connected to the PV. As suggested previously (19),
the short protrusions of the PV may reflect evaginations of the PV into
blind appendices that form part of the tubulovesicular network (TVN).
In some cells, fluorescence was also observed in a compartment within
the cytoplasm of the parasite, which appears to be the food vacuole
(FV) (Fig. 3, C and E). In the schizont stage, a
segmented pattern around a highly fluorescent central remnant body was
obtained (Fig. 3F). This indicates that the fusion protein
surrounds the individual merozoites, which again is consistent with
trafficking of the protein to the PV. These results suggest that the
first 35 amino acids of the Exp1 protein are sufficient for entry into
the secretory system and secretion from the parasite into the PV but do
not contain information that would direct the protein into the
erythrocyte cytosol.

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 3.
Expression of Exp1-(1-35)-GFP chimeric
proteins at different stages of the intraerythrocytic life cycle of
P. falciparum. The first
image in each set represents the DIC image, and the
second is the fluorescence signal from the GFP chimeric
protein, with an overlay of these images in the third
panel. A, ring stage parasite showing PV
labeling. B, erythrocyte infected with two late ring stage
parasites, only one of which is expressing the Exp1-(1-35)-GFP
transgene. The fluorescence signal resembles a necklace of "beads,"
some of which are marked with arrows. C-E,
trophozoite stage parasites showing PV expression. Some cells retain
the necklace of beads pattern (D). In some cases,
distortions and evaginations of the PV were observed (C and
E, yellow arrowheads). In some cells,
GFP was observed in the FV (C and E,
blue arrowheads). F, schizonts show a
segmented pattern with a highly fluorescent central remnant body.
Fluorescence from GFP was captured in live cells using a Leica TCS NT
confocal microscope. Bar, 5 µm.
|
|
Exp1-(1-35) Is Present in the PV and FV of Transfected P. falciparum but Is Not Released into the Erythrocyte Cytosol--
The
volume of the erythrocyte cytosol is much larger than that of the PV
(22). Therefore, it remained formally possible that a significant
proportion of the Exp1-(1-35) chimera was exported as a soluble
protein to the erythrocyte cytosol but was below the level of detection
using fluorescence microscopy. In order to confirm the location of the
reporter protein, transfected parasites were subjected to selective
permeabilization protocols employing SLO and saponin. Treatment of the
transfected parasitized erythrocytes with 4 or 8 hemolytic units of SLO
released more than 97% of the hemoglobin from the parasitized
erythrocytes (data not shown), but did not release PfERC (Fig.
4C), which has previously been shown to be located within the ER lumen (34). S-antigen (a PV marker
(35)) was also largely associated with the pellet fraction (0.5 and
4% in the supernatant, respectively, for 4 and 8 units of SLO; Fig.
4B). The trace amount of S-antigen that is released may be
due to a small percentage of lysed cells. Similarly, the SLO treatment
released only a trace amount of the GFP reporter (2 and 3%,
respectively for 4 and 8 units of SLO) (Fig. 4A). This suggests that very little GFP is trafficked to the erythrocyte cytosol
in these stable transfectants. By contrast, treatment of the
transfectants with 0.09% saponin released the bulk of the S-antigen but did not release PfERC (Fig. 4, B and
C). Interestingly, saponin released the higher molecular
mass (28-kDa) GFP species but not the lower molecular mass
(26-kDa) species (Fig. 4A). This indicates that the
correctly processed Exp1-GFP is present as a soluble protein in the PV;
however, the more extensively processed product is present in an
intraparasitic compartment.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Release of EXP1-(1-35)-GFP by
sub-fractionation of the transfectants. Highly synchronized
transfectants, grown to trophozoite stage after synchronization
(~0.5 × 108 cells), were solubilized in either 450 units (4HU) or 900 units (8HU) of SLO or 0.09%
saponin and separated into supernatant (S) and pellet
(P) fractions by centrifugation. These fractions were
separated by SDS-PAGE (12% acrylamide), blotted onto polyvinylidene
difluoride membrane, and probed with antibodies recognizing GFP (1:1000
dilution), S-antigen (1:500 dilution), and PfERC (1:500
dilution).
|
|
Trafficking of Exp1-GFP into the PV is BFA-sensitive--
Previous
studies (19, 36-38) have shown that the export of proteins into the
extraparasitic compartments of P. falciparum-infected erythrocytes is inhibited by BFA. In this investigation, young (ring
stage) parasites were treated with BFA to determine whether the
trafficking of Exp1-GFP is sensitive to this drug. As shown in Fig.
5, BFA treatment resulted in trapping of
the expressed chimeric GFP protein within the parasite, whereas the
control parasites exhibited the expected ring-like PV fluorescence
pattern.

View larger version (84K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of brefeldin A treatment of ring stage
parasites on the export of Exp1-(1-35)-GFP. Erythrocytes infected
with tightly synchronized (6-10 h) ring stage transfectants were
incubated for 18 h in the presence of 5 µg/ml BFA
(lower panels) or an equivalent volume of
methanol (control; upper panels) and examined by
fluorescence microscopy. The confocal images shown (left to
right) are as follows: DIC, GFP fluorescence, and an
overlay. Bar, 5 µm.
|
|
Some Regions or Extensions of the PV May Be Sites of Sorting of
Lipid and Protein Components--
To further examine the organization
of Exp1-(1-35)-GFP within the PV, we have labeled the transfected
parasites with BODIPY-TR-ceramide as described by Behari and Haldar
(30). The BODIPY-TR-ceramide probe is taken up from the medium into the
erythrocyte membrane and transfers to membrane structures within the
infected erythrocyte cytosol (Fig. 6).
Since the probe may have detergent-like properties, we have used very
low levels (1 µM) of the BODIPY-TR-lipid probe (i.e. ~1 probe molecule:400 endogenous lipid molecules) in
an effort to avoid disruption of the membranes of the parasitized erythrocytes. Under these conditions, only a very small amount of the
ceramide was converted to sphingomyelin (data not shown) as has been
reported previously (30).

View larger version (58K):
[in this window]
[in a new window]
|
Fig. 6.
Labeling of Exp1-(1-35)-GFP transfectants
with BODIPY-TR-ceramide. A-D, the confocal images in
each set (left to right) represent DIC, GFP
fluorescence, BODIPY-TR fluorescence, and an overlay of the GFP
(green) and BODIPY-TR (red) images. The
Exp1-(1-35)-transfectants often show a restricted distribution of the
GFP chimera within the PV. Some PV subcompartments are marked with
blue arrowheads. In addition, distortions,
evaginations, and loop-like extensions of the PV to form the so-called
TVN were observed. Some regions and extensions of the PV contained both
GFP and BODIPY-TR, whereas some (indicated with white
open head arrows) contained only the
BODIPY-TR probe. BODIPY-TR-labeled structures underneath the
erythrocyte membrane (probably Maurer's clefts; white
arrowheads) also do not contain GFP. Panels
A-C are collected in a plane near the middle or upper
surface of the parasite. Panel D is collected in
a plane close to the lower surface of the parasite. E,
Diagrammatic representation of the different structures in
BODIPY-TR-labeled GFP transfectants. The Exp1 chimera is translocated
into the ER and delivered to the PV. The chimera is restricted to
subcompartments of the PV, possibly due to close apposition of the PPM
and PVM (arrowed), which may prevent diffusion between
sub-regions. Some extensions of the PV form circular compartments bound
by a double membrane that can bud into the erythrocyte cytosol. Some
regions of the PV/TVN appear to exclude the GFP chimera; these may be
regions of sorting of PV resident proteins from proteins destined for
export. The Maurer's clefts may be formed by the budding and
maturation of vesicle or tubules derived from the TVN.
|
|
The BODIPY-TR-ceramide probe labeled the erythrocyte membrane and the
periphery of the parasite (Fig. 6). In doubly labeled cells, it is
clear that both the GFP chimera and the BODIPY-TR probe are associated
with the PV; however, the lumenal Exp1-GFP protein appears to be
restricted to subcompartments within the PV (Fig. 6, blue
arrowheads).
The BODIPY-TR-ceramide was also present in protrusions and loops
extending from the PV and in small structures attached to the
erythrocyte membrane (Fig. 6, A, B, and
D). The loop structures are presumably the double
membrane-bound extensions of the PV known as the tubulovesicular
network (30, 39) (Fig. 6E). The small structures underlying
the erythrocyte membrane are likely to be Maurer's clefts (22, 40, 41)
(Fig. 6E). The GFP chimera is present in some regions of the
PV/TVN but is absent from others. It is possible that the regions and
extensions of the PV that lack GFP (Fig. 6, white
open head arrows) represent sites of
sorting of components destined for trafficking beyond the PV. A
diagrammatic representation of the different membrane structures in
these doubly labeled cells is shown in Fig. 6E.
Organization and Dynamics of Exp1-GFP within the PV--
We have
used FRAP to examine the organization and dynamics of Exp1-(1-35)-GFP
within the PV and in protrusions and blebs adjacent to the PV (Fig.
7). A small region of a labeled infected
erythrocyte was bleached using a high power laser pulse of 1-s
duration. After the pulse, the cell was imaged immediately and at
different time intervals thereafter. As seen in Fig. 7A, a
1-s bleach pulse directed onto one of the "beads" of fluorescence
in the PV resulted in a loss of fluorescence that was localized to the
region that was pulsed. During the 120-s postbleach period, there was
50% recovery of the fluorescence signal into the bleached area (Fig.
7F). This indicates that the bleached bead is not an
isolated structure but is part of some larger structure in which
diffusion of Exp1-(1-35)-GFP can occur. The degree of connectivity
between different beads within the PV was investigated in greater
detail using the FLIP experiment shown in Fig.
8A. A small region
corresponding to one of the PV beads was exposed to a series of 1-s
bleach pulses, and the loss of fluorescence intensity in other regions
of the PV was examined. These data show that the population of mobile GFP chimeras that was able to repopulate the region of the bleached bead was quickly depleted. In addition, the B image demonstrates that
the repeated exposure of isolated beads to the high intensity laser
pulse did not produce a substantial loss of fluorescence in other
regions of the PV. This indicates that whereas there is some
connectivity between individual beads and a larger compartment, much of
the protein population is unable to diffuse from one subcompartment to
the next within the time scale examined.

View larger version (60K):
[in this window]
[in a new window]
|
Fig. 7.
FRAP measurements and analysis of GFP in
doubly labeled P. falciparum-infected erythrocytes
expressing Exp1-(1-35)-GFP. A-E, confocal images
obtained as described under "Materials and Methods." The
left panels show the DIC image of the cells. The
second panel shows the BODIPY-TR image. The
remaining panels show the GFP images obtained at
low laser power. These images were obtained immediately prior to
bleaching (Prebleach), immediately after bleaching using a
high intensity laser pulse for 1 s at the position indicated by
the arrow (Postbleach t0) and at
times t1 and t2 after the
bleach event (Postbleach t1 and
Postbleach t2, respectively).
A, trophozoite stage-infected erythrocyte with a "necklace
of beads" fluorescence pattern; t1 = 60 s, t2 = 120 s. B, trophozoite
stage-infected erythrocyte with a smooth PV fluorescence pattern;
t1 = 30 s, t2 = 60 s. C and D, trophozoite stage-infected
erythrocytes showing PV evaginations; t1 = 30 s, t2 = 60 s. E, a
trophozoite stage-infected erythrocyte showing FV fluorescence
(arrow); t1 = 30 s,
t2 = 60 s. F-H, analysis of the
images shown in A-C, respectively. The relative
fluorescence within each outlined region (numbered) is shown
as a function of time after the bleach event (data are normalized to a
value of 100 in the prebleach image). The fluorescence intensities have
been corrected for bleaching during image acquisition as described
under "Materials and Methods." Bar, 5 µm.
|
|

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 8.
FLIP measurements and analysis of GFP in
P. falciparum-infected erythrocytes expressing
Exp1-(1-35)-GFP. Confocal images were obtained as described under
"Materials and Methods." Trophozoite stage-infected erythrocytes
showed a "necklace of beads" fluorescence pattern (A)
and a smooth PV fluorescence pattern (B and C).
The left panel shows the DIC images of the cells.
The second panel shows the GFP fluorescence image obtained immediately
prior to bleaching (PreFLIP). The region indicated by the
arrow was repeatedly subjected to an intense laser pulse
(1-s duration, 30-s intervals). Images were collected after the cell
had been subjected to 4, 8, and 12 of these pulses (corresponding to
the FLIP1, FLIP2, and FLIP3 images,
respectively). The B image of the PV region shown in the
right panel was constructed using the prebleach
(PreFLIP) image and the postbleach images (FLIP2
in A and B and FLIP3 in C).
Only those regions of the PV that did not undergo movement were
analyzed. The color table for the B
image is shown in Fig. 9. Bar, 5 µm.
|
|
The PV often had a smoother appearance in erythrocytes infected with
more mature parasites (Fig. 7B). As with the previous example, application of a 1-s bleach pulse resulted in some loss of
fluorescence in the region exposed to the high intensity laser pulse.
However, in this case, the loss of fluorescence occurred throughout a
larger region of the PV. After 30 s, significant recovery of
fluorescence into this bleached area was observed. The analysis shown
in Fig. 7G indicates that the increase in fluorescence in
the bleached region is associated with a small decrease in fluorescence
intensity in other regions of the PV. This suggests that the recovery
of fluorescence in the bleached region may reflect the diffusion of
Exp1-GFP from the unbleached regions of the PV. This was confirmed by
performing a FLIP analysis on parasites exhibiting a smooth PV
morphology (Fig. 8, B and C). In contrast to the
observations on parasites with a "beaded" PV, repeated bleaching of
one region of the PV resulted in a relatively uniform loss of
fluorescence throughout the entire PV (see the respective B
images). These results demonstrate that the lumen of the PV is continuous in the cells exhibiting a smooth PV appearance. It is
also worth noting that in the case of each of the cells shown in Fig.
8, there were compartments adjacent to the PV that were protected from
bleaching, indicating that these structures had blebbed from the PV or
were not in communication with it.
The localized bleaching and the partial recovery observed over the time
course of the FRAP experiment can be explained if the fusion protein
exhibits a half-time for recovery on the order of seconds to tens of
seconds. A similar apparent diffusion rate has been reported for
another PV-located GFP chimera (19). These recovery times are
significantly slower than predicted for free protein diffusion (42).
The restricted diffusion could suggest that the GFP chimera is present
in the form of aggregates or is associated in some way with the PVM or
the PPM, although the efficient release of the chimera in
saponin-treated cells argues against this interpretation.
Alternatively, the restricted diffusion may be due to close apposition
of the PPM and PVM in some regions, which could limit diffusion within
the PV lumen.
When a looped extension of the PV/TVN was subjected to a 1-s bleach
pulse (Fig. 7C) a localized loss of fluorescence was
observed followed by significant recovery of fluorescence into the
bleached area. The fluorescence signal recovered to ~50% within the
60-s postbleach period (Fig. 7H), indicating that this
TVN extension is connected to a larger compartment,
presumably the PV. Again, however, the relatively slow half-time for
recovery indicates that there are some restrictions to diffusion
through the solute continuum between the TVN and the PV proper.
Moreover, in some cells, the extensions appear to have lost the
continuum with the PV. As shown in Fig. 7D, there was no
recovery of the arrowed PV extension after the bleach pulse.
Blebbing of regions of the TVN has been reported previously (19).
We also found that the GFP chimera that is located within
the FV is not connected by a solute continuum to the PV. As shown in
Fig. 7E, bleaching of the FV compartment did not affect the fluorescence signal from the PV, and there was no recovery of signal
into the FV within the 120-s postbleach period. Interestingly, bleaching of the FV compartment revealed another compartment within the
parasite that contains the GFP chimera (Fig. 7E,
asterisk). This may represent an endocytic packet of the PV
en route to the FV or a subcompartment within the parasite secretory
system en route to the PV.
We have also used FRAP analysis to examine the organization
and dynamics of the BODIPY-TR-ceramide probe. An examination of doubly
labeled cells confirmed the observation that some of the BODIPY-TR-labeled membrane compartments had separated from the PV (Fig.
9A, upper
panel). These structures were often observed to colocalize
with the GFP chimera (Fig. 9A, arrow in
lower panel) and to exhibit mobility within the
erythrocyte cytosol when monitored over time (Fig. 9A,
lower panels). Since the GFP chimera is likely to
be present in an aqueous environment within these structures, the
colocalization indicates that these structures comprise a double
membrane that encloses two lumenal compartments, with the GFP chimera
located in the outer lumen. The inability to resolve the two bilayers
reflects the close apposition of the membranes to form a thin outer
lumen whose dimensions are below the level of resolution available
using the confocal microscope. Such structures are presumably formed by
circularization of extensions of the PV/TVN as shown in Fig.
6E. Similar structures have been observed by EM (39). The
absence of a lipid continuum between these structures and the PVM was
confirmed by the FRAP experiment shown in Fig. 9A
(upper panel). High intensity illumination of the
structure at the excitation wavelength of the BODIPY-TR probe resulted
in almost complete ablation of the BODIPY-TR fluorescence in this structure but had little effect on the fluorescence signal from the
other membranes. In addition, there was insignificant recovery of
fluorescence into the structure over a 1-min period. These results are
consistent with a loss of connectivity to the PVM and other parasite
membranes.

View larger version (53K):
[in this window]
[in a new window]
|
Fig. 9.
FRAP measurements and analysis of BODIPY-TR
in doubly labeled P. falciparum-infected erythrocytes
expressing Exp1-(1-35)-GFP. Panels on the
extreme left show DIC images. The
upper (red) and lower
(green) panels show the respective BODIPY-TR and
GFP images that were obtained during the course of the FRAP
measurement. These show the prebleach image, the image obtained
immediately after bleaching using a high intensity laser pulse (568 nm)
for 1 s at the position indicated by the arrow, and the
images obtained 30 and 60 s after the bleach event. A,
trophozoite stage-infected erythrocyte showing a double membrane-bound
structure that has budded from the PV (arrow). B,
trophozoite stage-infected erythrocyte showing PV evaginations. The
B image shown on the right was
constructed from the BODIPY-TR prebleach and bleach images. The
QR image was constructed using the prebleach, bleach, and
60-s postbleach images as described under "Materials and Methods."
Bar, 5 µm.
|
|
In other cases, some of these structures appeared to remain
connected to the PV. As shown in Fig. 9B, a high intensity
pulse of 1-s duration directed at the tip of an extension of the PV resulted in partial bleaching of the BODIPY-TR signal in this compartment. In contrast to the previous example, the degree of fluorescence loss at the bleach site was limited, and the bleached area
extended throughout a large area of the PV proximal to the illuminated
region (see B image). This is consistent with
significant diffusion of BODIPY-TR-ceramide occurring during the bleach
time and image acquisition time and is in the correct range for the diffusion of a membrane lipid (42). This is further
confirmed by the QR image shown in Fig. 9, which
shows that there is subsequent recovery into the bleached region. These
data indicate that the protrusion is connected to the PVM by a lipid
continuum. The laser pulse also caused some bleaching of an adjacent
region of the erythrocyte membrane. Diffusion of the probe within the
host cell membrane allowed recovery of fluorescence in this compartment on a similar time scale. It is important to note that the bleach pulse
had no effect on the fluorescence signal from the GFP chimera in this
doubly labeled cell (Fig. 9B, bottom
panels).
 |
DISCUSSION |
Proteins destined for extraparasitic locations in malaria
parasite-infected erythrocytes are thought to transit through the ER
and the PV prior to transfer to the erythrocyte cytosol (19, 43, 44);
however, the nature of the polypeptide secretory signals that direct
proteins to their correct destinations is currently the subject of some
debate. An analysis of the sequences of exported parasite proteins
reveals some rather unusual motifs. Proteins that are destined for
sites within the confines of the PVM have classical hydrophobic
N-terminal signal sequences (i.e. a stretch of 10-15
hydrophobic amino acids close to the N terminus). By contrast, many
proteins that are directed past the PVM to the erythrocyte cytosol do
not have classical N-terminal signals. Proteins such as KAHRP, the
mature parasite-infected erythrocyte surface antigen and the
ring-infected erythrocyte surface antigen have a hydrophobic stretch of
amino acids starting 20-80 amino acids from the N terminus (see Table
I in Ref. 43). An exception to this rule is HRP2, which has a classical
hydrophobic signal and yet is largely located in the erythrocyte
cytosol (45, 46). It is possible that information determining the final
destination of the protein is contained within the N-terminal signal.
Alternatively, both classical and recessed hydrophobic signal sequences
may function interchangeably to direct the translocation of proteins
into the ER (with the PV as the default destination), with additional
trafficking information encoded elsewhere in the polypeptide sequence.
Work from Wickham et al. (19) indicated that the
noncanonical signal sequence of KAHRP is sufficient for transport to
the PV but that additional signal information is needed for export
beyond this point. By contrast, the work of Burghaus and Lingelbach
(22) suggested that the classical signal sequence of Exp1 directed a
chimera to the erythrocyte cytosol. They proposed that the erythrocyte cytosol is the default destination and that additional signal information is needed to retain proteins in the PV.
In this work, we have reexamined the trafficking signals within the
exported protein, Exp1. Exp1 was originally described as an antigen of
P. falciparum that is transported from the parasite to the
PVM and, to some extent, to membrane-bound compartments in the
erythrocyte cytosol (14, 23, 24, 33). Exp1 is an integral membrane
protein with the C-terminal domain facing the erythrocyte cytosol (14).
We prepared a chimeric gene construct encoding an N-terminal fragment
of Exp1 linked to the reporter protein, GFP. The construct was
successfully used to prepare stably transfected P. falciparum. The transfectants were readily maintained in culture
and continued to express the fluorescent protein even under very low
drug pressure. This suggests that the expression of the GFP chimera did
not compromise the growth of the parasites. Western analysis indicated
that part of the population of Exp1-(1-35)-GFP was processed to a
28-kDa product, presumably by removal of the N-terminal signal by a
signal peptidase in the ER, while part of the population was further
processed to a 26-kDa species.
Using confocal fluorescence microscopy, we have shown that the
Exp1-(1-35)-GFP chimera is present in the parasite PV and in extensions of the PV as well as in a compartment within the parasite cytoplasm. There appears to be little or no GFP released into the
erythrocyte cytosol. To confirm the location of the chimera, we have
subfractioned the parasitized erythrocytes using selective permeabilization protocols. The pore-forming toxin, SLO, inserts into
the erythrocyte membrane and releases the soluble erythrocyte components but leaves the PV intact (21, 28, 29). By contrast, saponin,
a detergent that interacts with cholesterol, disrupts the erythrocyte
membrane and the PVM but leaves the PPM intact (21, 47). Saponin lysis
of the transfectants released the 28-kDa form of the GFP chimera but
did not release the 26-kDa form. This indicates that the 28-kDa product
is present in the PV but that the 26-kDa product is located in a site
within the confines of the PPM. When the fractionation data are taken
together with the fluorescence microscopy data (which show that some of the GFP is localized near the hemozoin crystal) and with previous electron microscopy (EM) studies (19), the data indicate that some of
the chimera is correctly processed and trafficked to the PV, while a
proportion of the population is diverted to (or endocytosed into) the
FV, where it is further degraded. Previous studies have shown that the
core of GFP forms a highly stable
-barrel structure that may resist
proteolytic degradation (48). Western analysis of the relative levels
of the 26- and 28-kDa species indicates that a significant proportion
of the GFP chimera (~50%) is present in the FV. However, the
fluorescence from this compartment was usually less than that from the
PV. This may reflect the larger volume of the FV and/or be due to the
acidic environment of the FV (49) quenching the fluorescence of the GFP
(50).
Our data show that the PV is the default destination for export of
proteins from the malaria parasite. Taken together with the work of
Wickham et al. (19), the data indicate that both classical
and recessed N-terminal signal sequences of malaria proteins contain
information only for entry into the ER (and hence default secretion to
the PV), whereas additional sequence information is needed to direct
proteins past this point. This is in direct contrast to the data of
Burghaus and Lingelbach (22), who reported that an Exp1-luciferase
chimera was largely directed to the erythrocyte cytosol. This
discrepancy may be explained by the different approach taken by
Burghaus and Lingelbach (22). For example, they used a transient
transfection system and used luciferase as a reporter. Since protocols
for the transfection of P. falciparum are very inefficient
(about 1 in 100,000 for transient transfection and 1 in a million for
stable transfection (see Refs. 51 and 52), Burghaus and Lingelbach (22)
relied on a cell fractionation protocol and the very high sensitivity
of the luciferase reporter to detect the chimeric product in parasites
3 days after the transfection protocol. In our study, we used a dual
cassette transfection construct that, in addition to the transgene,
encodes human DHFR, which confers resistance to WR99210. We cultured
the parasites for several months in the presence of drug and thus
obtained a transfectant stably expressing the chimeric protein from an
episome. It is possible that, in the transient transfection system, the
electroporation protocols used to introduce the plasmid or initial high
copy numbers of the plasmid in some cells compromised the integrity of
the parasite membranes, allowing release of the fusion protein into the
erythrocyte cytosol. Also, it is interesting to note that luciferase
carries a SKL sequence at its C terminus that functions as a
peroxisomal import sequence in higher eukaryotes (53). Thus, it is
possible that this motif or another cryptic motif within the luciferase
reporter is responsible for translocation of the chimera into the
erythrocyte cytosol.
The export of Exp1-(1-35)-GFP is sensitive to brefeldin A. This
indicates that the protein transits through the ER before secretion
into the PV. The Western blot analysis and the selective permeabilization studies are consistent with the suggestion that the
N-terminal signal has been removed during trafficking of
Exp1-(1-35)-GFP through the ER. Since the Exp1-GFP chimeric construct
lacks the transmembrane domain, it is likely that it exists as a
soluble protein in the lumen of the PV. Nonetheless, the GFP chimera
often appeared to be restricted to subdomains of the PV. For example, the fusion protein often appeared to reside in bead-like
subcompartments forming a "necklace" around the parasite. To
examine the nature of the PV subcompartments, we have used
BODIPY-TR-ceramide, which appears to preferentially label membrane
components of the PVM/TVN. The dual labeling of live cells showed that
some regions and extensions of the PV/TVN contained both GFP and the
BODIPY-TR lipid probe; however, in other regions there appeared to be a
segregation of the lipid and protein probes.
Photobleaching studies allow an analysis of the organization and
dynamics of fluorescent components in living cells (42). In this work,
we have used photobleaching protocols to examine the diffusional
properties of GFP in the PV. Theoretical considerations predict that
unrestricted diffusion of soluble Exp1-(1-35)-GFP molecules would be
sufficiently rapid to allow complete equilibration of the protein
throughout the PV compartment within the 1-s bleach time employed in
this study. This would result in an even loss of fluorescence
throughout a continuous compartment the size of the PV (42). The
observation that a bleach pulse at the PV produces a local loss of
fluorescence that subsequently recovers shows that the diffusion of the
GFP chimera in the PV is considerably slower than in free solution. In
GFP-labeled cells that exhibited a smooth PV appearance, these slowly
diffusing GFP molecules were free to equilibrate throughout the entire
PV lumen. This was not the case in GFP-labeled cells that exhibited a
PV with a beaded morphology. In these cells, it appeared that the
Exp1-GFP was located in subcompartments within the PV and that there
was little or no diffusion between these subcompartments on the time
scale of the measurements. This provides a direct demonstration of
unlinked subcompartments within the PV. We propose that the barrier to GFP diffusion arises from a constriction of the PV lumenal space due to
the close apposition of the PPM and PVM in some regions (Fig.
6E). These constrictions are presumably responsible for the
"string of beads" appearance of the PV.
Part of the population of chimeric GFP molecules appears to be
directed to the FV. This may represent an overflow pathway for proteins
en route through the secretory system. Alternatively, it is possible
that the GFP fusion proteins are taken up during the parasite's
feeding process. The parasite uses a cytostome (mouth) to ingest small
packets of hemoglobin from the host cytoplasm. These endocytic
compartments are surrounded by a double membrane originating from the
PPM and the PVM and thus are likely to contain proteins from the PV as
well as the erythrocyte cytoplasm (Fig. 6E). These vesicles
are transported to and fuse with the FV, which would deliver the fusion
proteins to this compartment. In some cells, bleaching of the
FV-located GFP chimera revealed the presence of other structures within
the parasite cytosol; these may represent endocytic or secretory vesicles.
In more mature stage parasites, the GFP chimera was often present in
looped extensions of the PV. Bleaching of a PV extension was often
followed by gradual recovery of the signal, which indicates that these
protrusions remain connected to the PV. However, some of the
evaginations appeared to be closed compartments; photobleaching of
these blebs did not cause a loss of fluorescence signal throughout the
PV, and no recovery was observed. This indicates a physical barrier to
diffusion of the chimera between the evaginated region and the PV. In
some cases, the GFP-Exp1 chimera is packaged into double membrane-bound
compartments that are released from the PV. Membrane-bound structures
containing endogenous Exp1 have been observed in the erythrocyte
cytosol using immunofluorescence microscopy (14, 15). These budded
double membrane-bound compartments were quite frequently observed in
trophozoite stage parasites; however, they did not connect or fuse with
the erythrocyte membrane. This suggests that that these PV
lumen-containing structures probably do not play a direct role in
delivery of proteins to the erythrocyte membrane. This is in agreement
with previous studies (19, 36). However, we also observed regions of
the PV and protrusions emanating from the PV that were labeled with the
BODIPY-TR-ceramide probe but that excluded the GFP chimera. These
regions may represent sites for sorting of PV-resident proteins from
proteins destined for transport to the erythrocyte membrane. PV
resident-free structures may bud from the PV as vesicles or tubules and
mature to form the Maurer's clefts.
The photobleaching data provide information about the organization of
the membranous structures in the erythrocyte cytosol that add to
previous EM and fluorescence microscopy studies. Previous studies have
revealed the presence of large circular double membrane-bound compartments and large multiple membrane whorls (the so-called TVN)
that remain connected to the PV (30, 54, 55). Our data support at least
partial connectivity between the PV and the TVN, although blebbing of
the TVN also appears to occur. Maurer's clefts have been reported to
be interconnected subcompartments of the TVN complex (30, 54). However,
our data suggest that they are separate compartments.
Taken together with previous studies, our data allow us to build a
picture of protein export in infected erythrocytes. Proteins with
either classical or recessed signal sequences are routed through the ER
to the PV, with concomitant cleavage of the signal peptide. Proteins
that lack additional sequence signals remain in the PV, whereas
recognition of a "translocation motif" in exported proteins, such
as KAHRP, results in translocation of these proteins across the PVM,
probably via an ATP-dependent transporter (21). Integral
membrane proteins such as PfEMP1 that are destined for the erythrocyte
membrane are presumably incorporated into vesicles or tubules that bud
from specialized regions of the PV that exclude PV-resident proteins.
The PfEMP1-containing structures are presumably trafficked to (or
mature to become) the Maurer's clefts, and then PfEMP1 is transferred
to the erythrocyte membrane. The Maurer's clefts contain homologues of
components of the classical secretory pathway such as Sar1p (13, 19),
Sec31p (26), and a component SNARE-mediated fusion process, NSF
(56), which may be involved in the sorting of proteins destined for the
erythrocyte membrane. Therefore, it appears likely that trafficking
between these compartments is vesicle-mediated. Indeed, vesicle-like
structures that may be involved in the trafficking of proteins from the
PVM (44) and the Maurer's
clefts2 have been identified
at the EM level.
In summary, we have shown that by tagging a parasite protein of
interest at the gene level with GFP it is possible to decipher the
signals that target proteins to specific compartments of the infected
erythrocyte. In addition, the GFP transfectants were used to monitor
the location and organization of the labeled component in live
parasites during the course of the cell cycle. Use of photobleaching
techniques enabled us to identify novel subcompartments within the PV.
This has allowed an increased understanding of the nature of the
pathway for protein trafficking in parasitized erythrocytes. The
unusual characteristics of the external sector of this pathway make it
a potential target for new antimalarial strategies.