Download Protein trafficking in Plasmodium falciparum

Review
TRENDS in Parasitology
Vol.20 No.12 December 2004
Protein trafficking in Plasmodium
falciparum-infected red blood cells
Brian M. Cooke1, Klaus Lingelbach2, Lawrence H. Bannister3 and Leann Tilley4
1
Department of Microbiology, Monash University, Victoria 3800, Australia
Fachbereich Biologie und Parasitologie, Philipps-Universität Marburg, D-35032 Marburg, Germany
3
Centre for Neuroscience Research, Hodgkin Building, Guy’s, King’s and St. Thomas’ School of Biomedical Sciences,
Kings College London, Guy’s Campus, London, SE1 1UL, UK
4
Department of Biochemistry, La Trobe University, Victoria 3086, Australia
2
Plasmodium falciparum inhabits a niche within the most
highly terminally differentiated cell in the human body –
the mature red blood cell. Life inside this normally
quiescent cell offers the parasite protection from the
host’s immune system, but provides little in the way of
cellular infrastructure. To survive and replicate in the red
blood cell, the parasite exports proteins that interact
with and dramatically modify the properties of the host
red blood cell. As part of this process, the parasite
appears to establish a system within the red blood cell
cytosol that allows the correct trafficking of parasite
proteins to their final cellular destinations. In this
review, we examine recent developments in our understanding of the pathways and components involved in the
delivery of important parasite-encoded proteins to their
final destination in the host red blood cell. These complex
processes are not only fundamental to the survival of
malaria parasites in vivo, but are also major determinants
of the unique pathogenicity of this parasite.
The normal functioning of eukaryotic cells requires the
transport of newly synthesized polypeptides to their
appropriate cellular location, either within the cell’s
cytoplasm, within an organelle, at the cell membrane or
out into the external milieu. The classical pathway for the
secretion of proteins in eukaryotic cells requires a series of
sequential and highly regulated steps that involve budding and fusion of small vesicles. Proteins destined for
export are directed into the endoplasmic reticulum (ER)
then transit through the Golgi apparatus before release,
by exocytosis, from the cell. In most eukaryotes, the signal
that directs proteins to the secretory pathway is a
sequence of variable length, which is located near the
N-terminus of the protein, and is characterized by a stretch
of hydrophobic amino acids. Additional targeting signals are
used to divert proteins to other compartments, such as
lysosomes, or into the regulated secretory pathway.
For Plasmodium falciparum, protein trafficking
involves an additional level of sophistication in that the
parasite spends part of its life cycle within a parasitophorous vacuole (PV) inside the red blood cell (RBC) of its
host (Figure 1 and 2a). The mature human RBC has been
Corresponding author: Brian M. Cooke ([email protected]).
Available online 12 October 2004
referred to as a ‘floating corpse’ [1] because it has no
nucleus, no protein synthesis capability and no proteintrafficking machinery. Nonetheless, the parasite manages
to export a relatively large number of proteins beyond the
confines of its own plasma membrane (PM) and the PV
membrane (PVM), across the RBC cytoplasm to the host
cell membrane, where a subset of these proteins ultimately interact with the RBC membrane skeleton or are
exposed on the RBC surface (for reviews, see Refs [2,3]).
Some of these exported proteins assemble into unique
membranous structures within the RBC cytosol
(Figures 2a–e). These observations raise several obvious
questions. What is the machinery that facilitates the
transport of proteins across the PVM, across the RBC
cytoplasm and their insertion into the host RBC membrane? What are the signals that direct proteins to
particular extracellular destinations? Do these trafficking
pathways represent novel mechanisms and molecules that
have evolved in adaptation to life inside a RBC or does the
parasite exports proteins involved in intracellular proteintrafficking, thus using biologically conserved mechanisms
and molecules? These are important questions because
the exported proteins are crucial to the pathogenicity of
P. falciparum. For example, the parasite-derived knobassociated histidine-rich protein (KAHRP) [4,5] is trafficked to the RBC membrane where it is a major
component of the knobs that anchor one of a large family
of polymorphic integral membrane proteins, known as
P. falciparum erythrocyte membrane protein-1 (PfEMP-1).
These proteins are essential for the adherence of parasitized RBCs (pRBCs) to the vascular endothelium that
causes many of the frequently fatal syndromes associated
with human malaria (for review, see Ref. [3]). Indeed,
PfEMP-1 and KAHRP are just two of a diverse range of
exported parasite proteins that interact with the RBC
membrane. Many of these have been well characterized at
the molecular level, but the functions of most remain
unknown or incompletely described [2] (Table 1).
Trafficking of proteins to the parasitophorous vacuole
Trafficking of proteins within the confines of the parasite
appears to involve most of the elements of a classical
vesicle-mediated secretory pathway. Brefeldin A (BFA) is
a drug that specifically blocks classical vesicle-mediated
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Review
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Vol.20 No.12 December 2004
RBC membrane
TVN
MSP1
PfEMP-1
Other
organelles
(e.g. FV,
apicoplast)
KAHRP
Knob
Exp
RP
AH
M
ABC
transporter
1
BP
S
1
P-
EM
Pf
Pf
1p
s3 S F
c
Se f N
Pf P
p p
r1 2 3
Sa S e c
f
P Pf
TVN
-1
S-antigen
Storage
organelles
(e.g.
rhoptries)
PfRab6
ER
MC
PfERD2
PfERC
?
PVM
COP1
COPII
DMV
Golgi-like
complex
PV
PfGRP
Nuclear
envelope
Parasite
nucleus
PPM
TRENDS in Parasitology
Figure 1. Putative trafficking pathways in a Plasmodium falciparum-infected red blood cell. Soluble proteins destined for export are directed into the ER by recognition of a
classical or recessed N-terminal hydrophobic signal sequence. The signal peptide is cleaved and the proteins pass through a rudimentary Golgi associated with the nuclear
envelope en route to the PV. Markers for the ER include PfERC and PfGRP, whereas PfERD2, PfRab6, COPI and COPII represent proteins needed for vesicle formation and
transport to and from the Golgi complex (see Ref. [7]). KAHRP, S-antigen and MSP-1 might be transported in distinct vesicles to different sub-compartments of the PV, or
might be co-transported to the PV in mixed cargo vesicles, and sorted into resident and forward-destined proteins within this compartment. Some proteins are retrieved from
the PM or diverted from the ER or Golgi to intracellular organelles such as the FV and the apicoplast, or to regulated secretory compartments such as the rhoptries and
micronemes. Proteins destined for a location beyond the PVM are first released into the PV. It has been proposed that recognition of a translocation motif downstream from
the proteolytic cleavage site results in translocation of proteins such as KAHRP across the PVM, via a putative ATP-dependent transporter (ABC transporter). The exported
soluble proteins diffuse across the RBC cytosol and could interact with the external surface of MCs or with the RBC membrane skeleton. Some (e.g. KAHRP) are important in
surface knob formation. Exported integral membrane proteins, such as PfEMP-1, are believed to be trafficked via a vesicle-mediated pathway. There is ultrastructural
evidence for the presence of DMVs in the parasite cytosol, which might be involved in the delivery of membrane-associated proteins to the PVM. PfEMP-1 is then transferred
to MCs, which represent an intermediate parasite-derived compartment en route to the RBC membrane. The MCs are distinguished by the presence of markers such as
PfSBP-1, PfEMP-3 and MAHRP. Three components of the COPII coat, namely Sar1p, Sec31p and Sec23p, have been reported to be exported to MCs, along with PfNSF, which
is involved in SNARE-mediated membrane fusion. Abbreviations: COP, coat protein; DMV, double membrane-bound vesicle; ER, endoplasmic reticulum; EXP, exported
protein; FV, food vacuole; KAHRP, knob-associated histidine-rich protein; MAHRP, membrane-associated histidine-rich protein; MCs, Maurer’s clefts; MSP, merozoite surface
protein; PfEMP, P. falciparum erythrocyte membrane protein; PfERC, P. falciparum ER-related calcium-binding protein; PfERD, P. falciparum ER-retention defective; PfGRP,
P. falciparum glucose-regulated protein; PfNSF, P. falciparum N-ethylmaleimide sensitive factor; PfRab, P. falciparum Ras protein from rat brain; PfSBP, P. falciparum
skeleton-binding protein; PM, plasma membrane; PPM, parasite PM; PV, parasitophorous vacuole; PVM, PV membrane; RBC, red blood cell; S-antigen, heat-stable antigen;
SNARE, soluble NSF attachment protein receptor; TVN, tubovesicular network.
trafficking by interfering with the Plasmodium ADP
ribosylation factor GDP–GTP exchange protein [a key
molecule required for the assembly of coat protein (COP),
which coats the transport vesicles] [6] and prevents
secretion of most exported proteins examined so far.
Moreover, structural components of the ER and Golgi
complex, and the coat components, in addition to those of
the budding and fusion machinery of the COPI- and
COPII-mediated pathways for protein trafficking, have
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been identified (Figure 1) (for reviews, see Refs [7–9]).
Furthermore, the malaria genome project has also
revealed genes that encode other components such as
v- and t-soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), some clathrin components, adaptin and dynamin. Electron microscopy (EM)
has revealed an ER that proliferates extensively throughout the parasite cytoplasm [10] and is continuous with the
nuclear envelope [11]. The parasite also possesses a
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(a)
(d)
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Vol.20 No.12 December 2004
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(b)
(e)
(c)
(f)
(g)
Figure 2. Transmission electron micrographs of structures associated with export from the parasite into the host red blood cell. A RBC containing a late trophozoite or early
schizont, a cluster of MCs and a LM of the TVN is shown in (a). At higher magnification, (b) shows a group of MCs and the dense material surrounding them. (c) An example of
the LM of the PVM, continuous with the TVN. (d) High-power view of a MC showing patches of dense material (arrows) associated with its exterior. Elsewhere in the cleft, the
typical dense coating is absent, suggesting that this might represent an early stage in cleft maturation. (e) This electron micrograph shows a typical MC with an external
coating of dense material. A small (25 nm) VES is also visible. (f) A section through a KN at the surface of a pRBC showing electron-dense material beneath its membrane.
(g) A ‘vesicle in a vesicle’ at the surface of a ring-stage parasite PM represents a potential means of exporting membrane-associated material into the PVM and into the RBC
cytoplasm. Note the presence of two membranes surrounding the vesicle, and a possible point of fusion with the PVM. Scale barsZ100 nm (b,c,d,e,g); 1 mm (a), and 10 nm (f).
Abbreviations: FP, fusion point of inner membrane of the DMV with the PVM; KN, knob; LM, loop-like membranous extension; MCs, Maurer’s clefts; MM, the membranes of
the DMV; PAR, parasite; PM, plasma membrane; PVM, parasitophorous vacuolar membrane; RBC, red blood cell; TVN, tubulovesicular network; VES, vesicle.
rudimentary Golgi-like complex, which appears as a collection of one or a few tubular or flat cisternae surrounded by
vesicles of various sizes [11–13]. The complex lies close to a
cluster of coated vesicles formed by budding from the outer
nuclear membrane or (in rings and trophozoites) an
extension of it (Figure 1), a relation reminiscent of the cisGolgi network in other eukaryotes [11].
All proteins analyzed to date that are transported into
the cytoplasm of pRBCs traverse the PV [14–17]. Soluble
proteins are delivered into the PV by the fusion of
transport vesicles with the parasite PM [8,11]. Some
proteins, such as the heat-stable antigen (S-antigen) and
the serine-rich antigen (SERA) family, remain in this
compartment, whereas other proteins, such as KAHRP,
the mature parasite-infected erythrocyte surface antigen
(MESA) and PfEMP-3, are directed outwards across the
PVM.
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It has been suggested that proteins destined for transit
to the host cell cytosol might be segregated into a specialized secretory compartment within the parasite [18,19].
However, recent data suggest that both PV-resident
proteins and proteins destined for forward transit are
released into the PV as the obligate intermediate depot
[14,17,20,21]. There is evidence for sub-compartments
within the PV [16,22], thus the possibility remains that
PV-resident and forward-destined proteins might be
separated into different compartments.
Membrane-associated proteins destined for onward
transport could have a novel and unusual mechanism
for delivery. Recent EM studies have revealed evidence
for the presence of a distinctive class of doublemembrane-bound vesicles (DMVs) with low-density
contents (called ‘vesicles within vesicles’) that appear
to bud from the ER and fuse with the parasite PM
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Table 1. Characteristics of some exported proteins of Plasmodium falciparuma
Proteins exported to the PVM and beyond
Protein (kDa)b
Location
Predicted signal sequencec
Putative function
Refs
Soluble antigens and structural proteins
GBP-130 (96)
RBC cytoplasm
KAHRP (71)
RBCMS
Recessed
Recessed
[75]
[17,73,74,76]
MESA and PfEMP-2 (168)
RESA and Pf155 (127)
PfEMP-3 (274)
HRP2 and SHARP (32)
RBCMS
RBCMS
MC, RBCMS
RBC cytoplasm, might be
secreted from pRBC
MC, RBCMS
RBC cytoplasm, possibly
associated with MC
MC
Recessed
Recessed
Recessed
N-terminal
NK
Essential for knob formation; binds to RBC
spectrin, actin and the cytoplasmic tail of PfEMP-1
Binds to RBC protein 4.1
Binds to RBC spectrin; might stabilize RBCMS
Appears to be involved in trafficking PfEMP-1
Binds heme; promotes heme detoxification
None
None
NK
NK
[62]
[83]
None
Weak Bet3p homologue; might be involved in
vesicle fusion
[57]
RBC cytoplasm
N-terminal
Lipid remodeling
[84,85]
PVM, RBC cytoplasm
MC, RBCMS
None
None
Membrane protein; transporter component
Putative serine-threonine kinase
[38]
[86]
MC
MC
MC
RBC cytoplasm, MC
None
None
None
None
Small GTPase
COPII coat protein
COPII coat protein
Vesicle fusion component
[53]
[33]
[54]
[55]
N-terminal
N-terminal
None
NK
NK
Cytoadherence ligand; involved in antigenic
variation
Binds heme; might protect MC
NK
Possibly involved in antigenic variation
[87]
[88]
[89,90]
PfMC–2TM (w35)
PVM and TVN
PVM and TVN
MC, RBCMS and RBC
surface
MC
MC and RBCMS
RBC cytoplasm and RBC
membrane surface
MC
[70,71]
ETRAMP and SEP (w11)
REX (83)
PfAARP1 (213)
PVM, TVN and MC
MC
RBCMS
N-terminal
N-terminal
None
Sub-family of STEVOR; possibly involved in
antigenic variation
NK
NK
Multiple transmembrane domains; unknown
function
NK
Proteases involved in parasite invasion
NK
NK
NK
[92]
[57,93,94]
[95]
[96]
[97,98]
Pf332 and Ag332 (689)
PfAARP2 (166)
41–2 (22)
Enzymes and transporters
Fatty acyl-CoA synthetase-1 (95)
PfGCN20 (95)
FEST and Pf255 (318)
Trafficking machinery
PfSar1p (22)
PfSec31p (163)
PfSec23p (86)
PfNSF (89)
Membrane proteins
EXP1 and CRA (17)
EXP2 (33)
PfEMP-1 (w246)
MAHRP (29)
PfSBP1 (36)
RIFIN and STEVOR (w37)
Proteins exported to the PV
ABRA (87)
SERA 1–5 (81–125)
FIRA (190)
GLURP (80)
S-Antigen (63)
None
None
N-terminal
N-terminal
N-terminal
N-terminal
N-terminal
N-terminal
N-terminal
[77]
[78,79]
[56,80,81]
[29,82]
[63]
[46]
[65,66]
[22,67]
[69]
[91]
a
Note that the table is not an exhaustive list of parasite proteins exported to the PVM or beyond. Whereas several investigators have independently confirmed the cellular
localization of some of these proteins, others have been detected by fluorescence microscopy using a single antiserum and have not been verified by independent groups or
different experimental approaches. Abbreviations: ABRA, acid-basic repeat antigen; Ag332, antigen 332; CRA, circumsporozoite protein-related antigen; ETRAMP, earlytranscribed membrane protein; EXP, exported protein; FEST, falciparum-exported serine-threonine kinase; FIRA, falciparum interspersed-repeat antigen; GBP, glycophorinbinding protein; GLURP, glutamic acid-rich protein; HRP, histidine-rich protein; KAHRP, knob-associated histidine-rich protein; MAHRP, membrane-associated histidine-rich
protein; MESA, mature parasite-infected erythrocyte surface antigen; MC, Maurer’s cleft; NK, not known; PfMC-2TM, Maurer’s cleft two transmembrane protein; Pf155,
P. falciparum 155kDa antigen; Pf255, P. falciparum 255kDa antigen; PfAARP, P. falciparum asparagine- and aspartate-rich protein; PfEMP, P. falciparum erythrocyte
membrane protein; PfGCN, P. falciparum gonial cell neoplasm protein; PfNSF, P. falciparum N-ethylmaleimide-sensitive factor; PfSBP, P. falciparum skeleton-binding
protein; pRBC, parasitized red blood cell; PfSar1p, P. falciparum secretion-associated Ras-related protein; PVM, parasitophorous vacuolar membrane; RBC, red blood cell;
RBCMS, red blood cell membrane skeleton; RESA, ring-infected erythrocyte surface antigen; REX, ring-exported protein; RIFIN, repetitive interspersed family; S-antigen,
heat-stable antigen; SDS, sodium dodecyl sulfate; SHARP, small histidine- and alanine-rich protein; STEVOR, subtelomeric variable open-reading frame; TVN, tubovesicular
network.
b
Predicted molecular weight for parasite line 3D7 (http://www.expasy.org/tools/pi_tool.html), which could vary between different parasite lines and/or isolates. Note that the
frequent occurrence of highly charged regions of low-complexity sequence in many malaria proteins is associated with non-uniform binding of SDS and consequent
anomalous size migration in SDS-polyacrylamide gels. Typically, such proteins run much larger than their predicted molecular weight [3].
c
Signal sequence predicted by MalSig (http://bioserve.latrobe.edu.au/cgi-bin/pfsigseq.py).
(Figure 2g). The release of vesicles into the PV has
been reported previously [23].
The origin of these structures is not clear, but they
could form as a result of infolding of the ER or of the Golgi
membranes before budding. Following budding into the
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PV, the released vesicles could fuse with the PVM and
then re-bud into the RBC cytosol, thus providing a
possible mechanism for the export of entire membrane
compartments from the parasite. These structures could
play a role in generating the novel membrane structures
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TRENDS in Parasitology
that appear in the RBC cytosol, although further characterization of these structures is clearly needed [11].
Trafficking of proteins across the PVM
Attempts to analyze the signal sequences of exported
parasite proteins have revealed some rather unusual
motifs. Proteins destined for sites in the ER, parasite PM,
PV, PVM and apical organelles appear to have classical
hydrophobic N-terminal signal sequences (i.e. a stretch of
w15 hydrophobic amino acids commencing three to 17
amino acids from the N-terminus) [24–26]. By contrast,
several proteins that are directed past the PVM to the
RBC cytosol have a longer (up to 30 amino acids)
hydrophobic stretch that is recessed (up to 80 amino
acids) from the N-terminus [24,26]. Similar internal signal
sequences are found in some proteins of higher eukaryotes, most notably ovalbumin [27].
There are, however, exceptions to the general rule that
exported proteins have recessed signals. For example,
proteins destined for export to the parasite food vacuole
have a recessed hydrophobic region but, in this case, the
hydrophobic segment functions as a membrane anchor
and is cleaved only after transit to the PM [28]. A further
exception is the histidine-rich protein-2 (HRP-2), which
has a classical hydrophobic signal, and yet is exported to
the RBC cytosol [29–31]. Because secretion of proteins
containing classical and recessed signal sequences can be
inhibited with BFA [17,32,33], both types of signal
sequences can direct translocation across the ER membrane. The recessed hydrophobic signals of exported
P. falciparum proteins are not easily recognized by
algorithms designed to predict classical signals in higher
eukaryotes, but an algorithm trained on Plasmodium
proteins, MalSig (http://bioserve.latrobe.edu.au/cgi-bin/
pfsigseq.py), recognizes both classical and non-canonical
sequences [34]. Analysis of exported parasite proteins
usually reveals recognizable proteolytic cleavage sites just
after the hydrophobic region [34]. It remains to be shown,
however, whether the recessed signals are cleaved in the
lumen of the ER by signal peptidases or whether the
extended N-terminal domains serve additional, as yet
unknown, functions.
Recent advances in our knowledge of trafficking pathways in pRBCs have come from studies using reporters
such as the green fluorescent protein (GFP) or luciferase
to monitor the fates of proteins [15–17,35,36]. With the
advent of transfection procedures for the asexual stages of
P. falciparum, it has become possible to follow the
transport of proteins in live cells and to dissect the signals
that direct proteins to particular destinations. For
example, constructs have been generated in which gene
fragments encoding the N-terminal regions of a series of
exported proteins have been appended to reporter proteins. These constructs are introduced into P. falciparum
using either transient or stable transfection systems, and
the locations of the proteins are assessed by fluorescence
microscopy or biochemical assays [15–17,35,36]. When
GFP is used as a reporter protein, the studies generally
confirm that the classical signal sequences of the acylcarrier protein (ACP), exported protein-1 (EXP-1) and
HRP-2, as well as the non-canonical signal sequence of
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585
KAHRP, can direct default transport to the PV, but that
additional sequences are needed for export of GFP beyond
this point. However, it should be pointed out that different
data might be obtained when different reporter proteins
are employed. Thus, transport of luciferase beyond the
vacuolar membrane does not require additional sequences
from parasite proteins [35], indicating that we still do not
completely understand the protein-sorting motifs for
translocation across the vacuolar membrane. Transfection
experiments further suggest that an element in the
histidine-rich region of KAHRP and HRP-2 (within a
region just downstream from the hydrophobic region) is
needed for forward transit of GFP beyond the confines of
the parasite [17]. The studies of Lopez-Estrano [36]
indicate that basic amino acids in this region form an
important part of the signal that directs both HRP-2 and
KAHRP across the PV. However, it is unlikely that basic
amino acid residues are obligatory components of a PVM
translocation signal because some proteins reported to be
transported across the vacuolar membrane [e.g. the ringinfected erythrocyte surface antigen (RESA) [37] and MESA]
lack basic residues downstream of the signal sequence.
Ansorge et al. [14] showed that translocation of soluble
parasite-encoded proteins across the vacuolar membrane
requires ATP. Thus, it is likely that an ATP-dependent
translocator is involved in transfer of proteins across the
PVM. An ATP-binding cassette (ABC) protein, P. falciparum gonial cell neoplasm protein 20 (PfGCN-20), has
been localized to multiple regions of pRBCs, including
membranous compartments in the host RBC cytosol. It
has been suggested that PfGCN-20 might function as part
of a multimeric ABC transporter involved in protein
translocation across the PVM [38]. Clearly, identification
and characterization of the transporter for exported
proteins would greatly advance our understanding of
this process. Consistent with the requirement for protein
unfolding as a prerequisite for membrane translocation,
chaperone-like proteins have been identified within the
PV (J. Nyalwidhe and K. Lingelbach, unpublished).
Exported membrane structures in the host RBC cytosol
The mature human RBC has no architecture of intracellular membranes; thus the parasite needs to set up its
own transport system. Proteins, such as KAHRP, HRP-2
and MESA, have been observed in large membrane-free
aggregates in the RBC cytosol [30,39,40]. These proteins
could transit the PV as unfolded polypeptides, then refold
and form complexes that diffuse across the host RBC
compartment, and spontaneously assemble at the appropriate destinations as a result of interactions with
RBC proteins. However, proteins such as PfEMP-1 are
presumably delivered to the RBC membrane via a vesiclemediated pathway if they are synthesized as integral
membrane proteins [8,41].
This has led to intense interest in the membrane-bound
compartments that appear in the RBC cytosol as the
parasite matures. In ring-stage parasites, short finger-like
extensions of the PVM are observed and these structures
can bud to release small membrane-bound vesicles or
tubules [42]. As the parasite matures, structures known
as Maurer’s clefts (MCs) appear in the RBC cytosol,
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characterized by a translucent lumen and an electrondense coat (Figures 2b,d,e) of variable thickness. Larger
aggregates of dense material, similar to that seen in the
surface knobs, are often attached to their exterior
(Figure 2d) [43–45]. Initially, MCs appear as twisted and
branched structures located near the PV, but they
gradually relocate closer to the RBC membrane and, in
more-mature stage parasites, appear to be tethered by
fibrous connections to the RBC membrane skeleton
[43–46]. These structures do not actually fuse with the
host RBC membrane and, clearly, are not formed as
indentations of its surface [44]. The degree of elaboration
of MCs appears to vary between different strains of
parasite. For example, in some parasite lines, such as
K1, the MCs form extensive stacks, whereas in other lines,
such as 3D7 and ITO4, they are usually less complex and/
or present largely as single cisternae (Figures 2d,e). In
some preparations [45], including three-dimensional EM
reconstructions [47], small vesicles (20–25 nm in diameter) were observed in the vicinity of MCs (Figure 2e) and
close to the RBC membrane. These might be involved in
trafficking of proteins to or from the MCs, although it
should be noted that they are distinct in size and
appearance from the coated vesicles involved in ER-toGolgi transport in the parasite cytoplasm.
A second type of membranous structure present in the
pRBC cytosol is the circular cleft or tubulovesicular network (TVN) [10,44,48]. In section, this is usually seen as a
circular or elliptical double-membrane loop (Figures 2a,c)
wrapping a pocket of RBC cytosol. In some sections, the
loops can be traced back to the PVM (Figure 2c). The unit
membranes limiting the TVN and the PVM lack the
electron-dense coat associated with MCs, and have
distinctive antigen profiles [e.g. exported proteins-1 and
-2 (Exp-1 and Exp-2) are present in the PVM and TVN, but
not in MCs] [49]. The TVN appears to have a role in the
uptake of components from the RBC PM and surrounding
medium [50], and the PVM and TVN could function as an
intermediate compartment in the trafficking to the MCs
[17,36]. Some studies [47,48] have used fluorescent lipid
probes to examine the structures in the RBC cytosol and
have concluded that MCs and TVN form part of a
continuous network. There is also evidence from serial
EM sectioning that the clefts might be continuous with the
PVM [47]. However, a three-dimensional confocal fluorescence image reconstruction from a Z-section series
taken through a pRBC transfected with KAHRP–GFP
provides an image of a live cell that shows MCs as distinct
entities positioned close to the periphery of the host cell
(see Ref. [17]). Consistent with this view is the observation
that reporter proteins that are transported to the PV do
not diffuse into the lumen of MCs [16,17]. Further studies
are needed to define the connectivity or otherwise between
the TVN and MCs.
Small vesicles that could be involved in trafficking from
the PVM to MCs (Figure 2e) are difficult to visualize in the
RBC cytosol. Ring-stage pRBCs labelled with acridine
orange have been reported to contain small mobile vesicles
[51], and a small number of membrane-limited vesicles
have been observed in this stage by EM [11]. Trelka et al.
[52] used aluminium fluoride (a compound that, in other
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Vol.20 No.12 December 2004
systems, activates small GTP-binding proteins and prevents ADP ribosylation factor-mediated coatomer shedding following vesicle formation) to block protein
trafficking across the RBC cytosol. Following aluminium
fluoride treatment, strings of vesicles with diameters of
60–100 nm were observed [41,52]. Immunoelectron
microscopy suggests that these vesicles contain PfEMP-1,
are coated with COPII proteins, and are associated with
actin and myosin [41,52].
Trafficking machinery in the host RBC cytosol
The membranous structures in the RBC cytosol are
assumed to be involved in trafficking of membraneassociated proteins. However, the mechanics of the
trafficking process still need to be defined. Given that
the RBC lacks the endogenous coat proteins needed for
vesicle-mediated transport, the parasite must set up its
own budding and fusion machinery. In this context, it is of
some interest that components of the Plasmodium COPII
complex have been reported to be exported to the host cell
cytosol. Immunofluorescence and immunoelectron
microscopy studies indicate that the coat proteins Sar1p,
Sec31p and Sec23p are exported to the host RBC cytoplasm where they are associated with MCs [17,33,41,53,54].
A Plasmodium homolog of P. falciparum N-ethylmaleimidesensitive factor (PfNSF), which is involved in SNAREmediated membrane fusion, also appears to be transported
to the RBC cytosol [55]. Moreover, another parasite
protein that is exported to MCs, PfEMP-3, could show
structural similarity to USO1p, a tethering protein
involved in ER-to-Golgi traffic in yeast [56]. Similarly,
the exported antigen 41–2 shares some homology to BET3,
a Golgi-associated protein that might play a role in protein
trafficking to the Golgi [57]. This suggests that the
malaria parasite is capable of elaborating components of
the classical vesicle-mediated trafficking machinery outside the boundaries of its own PM – that is, it exports a
secretory system.
There is some uncertainty as to how these transport
proteins might be exported to the parasite cytosol, given
that, with the exception of PfEMP-3, they have no
obvious secretory signal sequences. Hence, the recent
report of the export of ‘vesicles within vesicles’ from the
parasite to the PV is of some interest here. This novel
pathway provides a possible means of relocating components of the parasite’s trafficking machinery to the
RBC cytosol (Figures 1 and 2g) [11]. There is apparently
no retrograde transport of membrane vesicles from the
RBC membrane [58,59], although a novel pathway
seems to allow uptake of raft-associated proteins from
the RBC membrane [60].
The precise nature of the interaction between MCs and
the RBC membrane skeleton remains unclear, but there is
limited evidence to suggest that they might interact
specifically with proteins of the RBC membrane skeleton
[44,46,61]. The docking molecule(s) on the surface of the
cleft have not been identified, but P. falciparum skeletonbinding protein-1 (PfSBP-1) has been suggested to play a
role [46].
Review
TRENDS in Parasitology
The role of Maurer’s clefts in protein trafficking
MCs are thought to be an important transit depot for
PfEMP-1 en route to the RBC membrane. Transit of newly
synthesized protein to the pRBC surface requires about
nine hours, and a significant proportion of the PfEMP-1
population appears to remain in an as yet poorly identified
intracellular pool (most likely associated with MCs) [45].
PfEMP-1 is inserted into the MC membrane with the
C-terminal domain exposed to the RBC cytoplasm and
the N-terminal domain buried inside the cleft [45]. The
reorganization of other parasite proteins probably controls
the transfer of PfEMP-1 to the RBC membrane. It is
noteworthy that several soluble proteins that are destined
for the RBC membrane skeleton appear to be transiently
recruited onto MCs where they could either be assembled
into the cytoadhesion complex and/or participate in the
process of its assembly before insertion of PfEMP-1 into
the RBC membrane (Table 1) [9,17,45,56,62].
Until recently, PfSBP-1 was the only integral membrane protein known to be resident in MCs [46], but
additional markers for MCs have now been described. One
of these, membrane-associated histidine-rich protein
(MAHRP) [63], resembles both PfSBP-1 and PfEMP-1 in
that a N-terminal secretory signal is absent, it has a long
phenylalanine-rich transmembrane domain and might be
inserted into MCs with the C-terminal region facing out to
the RBC cytosol [45,46,63]. It is possible that MAHRP
(along with PfSBP-1) is one of several MC ‘coat proteins’
that plays an important structural and functional role in
protein trafficking. Future experiments to purify and
analyze the complete proteome of MCs will be extremely
helpful to further our understanding of these structures
and define their precise composition and role in pRBCs.
Interestingly, MAHRP and PfSBP-1 do not appear to
have any close paralogues in P. falciparum. By contrast,
PfEMP-1 is a member of a protein family with 75 members (http://www.wehi.edu.au/MalDB-www/genome.tab),
although there is cell-surface predominant expression of
a single PfEMP-1 type in a given pRBC [64]. Several other
multicopy gene families encoding membrane-associated
proteins also appear to be exported to the RBC cytosol. The
repetitive interspersed family (RIFIN) and subtelomeric
variable open-reading frame (STEVOR) (276 members), in
addition to early-transcribed membrane protein (ETRAMP)
and small exported-protein (SEP) (13 members) families of
proteins possess a N-terminal hydrophobic signal (which
appears to be cleaved in most members of the families),
and one or two transmembrane domains [22,65–67]. There
are reports of exposure of at least one RIFIN at the RBC
surface [65]; however, other members of this family are
associated with membranous structures in the RBC
cytosol [68]. Some members of the RIFIN–STEVOR and
ETRAMP–SEP families also appear to be associated with
MCs [22,66,67]. REX is a further, recently described,
MC-associated protein with a single transmembrane
spanning domain. Although the precise function of this
protein has not been determined, it shows significant
homology to the ER–Golgi vesicle-tethering protein p115
[69]. By contrast, the P. falciparum MC two transmembrane protein (PfMC–2TM) family (11 members) has a
signal sequence, two closely spaced transmembrane
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Vol.20 No.12 December 2004
587
segments in the C-terminal region of the protein and a
basic C-terminal domain and at least one member is
reported to be MC-associated [70,71].
PfEMP-1, MAHRP, PfSBP-1, REX and the ETRAMP–
SEP family are all transcribed in ring-stage parasites [72]
(http://plasmoDB.org), and some of these proteins could be
involved in generating the TVN and MCs, whereas
members of the RIFIN–STEVOR, and PfMC–2TM
families are produced in the trophozoite stages and could
be involved in modifying the host compartment to facilitate transfer of PfEMP-1 to the pRBC surface. Whereas
PfEMP-1 has a highly acidic C-terminal (cytoplasmic)
domain, other membrane proteins exported to the RBC
cytosol tend to have more basic-domains predicted or
shown to be facing the RBC cytoplasm. It is not known
whether these domains are involved in electrostatic
interactions, but it is interesting to speculate that these
might be involved in stabilizing knob complexes [73,74].
Future directions
Over the past few years, our understanding of the cell
biological processes underlying host RBC modification by
P. falciparum has advanced considerably. This is largely
because of the complete sequencing and annotation of the
P. falciparum genome, the development of transfection
systems for asexual-stage parasites, highly sophisticated
morphological analyses, and the identification of new
parasite proteins exported into the RBC and their interactions with other RBC and parasite proteins. However,
despite several exciting new developments, it is clear that
our molecular understanding of this complex situation
remains far from complete. In particular, the current view
that a eukaryotic cell establishes and maintains, outside
the confines of its PM, a highly complex machinery
required for protein and membrane trafficking is without
precedence and raises several conceptual questions, some
of which have been addressed in this review. The future
challenge will be to complete the pieces of this important
puzzle and to integrate them into a mechanistic model
that can be experimentally verified.
Acknowledgements
We acknowledge funding from the National Health and Medical Research
Council of Australia (B.M.C. and L.T.), The National Institutes of Health
(B.M.C.), The Wellcome Trust (L.H.B.) and the Deutsche Forschungsgemeinschaft (K.L.). We thank John Hopkins for electron micrographic
assistance.
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