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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 www.sciencedirect.com 1471-4922/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2004.09.008 Review 582 TRENDS in Parasitology 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 www.sciencedirect.com 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 Review (a) (d) TRENDS in Parasitology Vol.20 No.12 December 2004 583 (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. www.sciencedirect.com 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 584 Review TRENDS in Parasitology Vol.20 No.12 December 2004 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 www.sciencedirect.com 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 Review 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 www.sciencedirect.com Vol.20 No.12 December 2004 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, 586 Review TRENDS in Parasitology 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 www.sciencedirect.com 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 www.sciencedirect.com 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. 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