Critical Steps in Protein Export of Plasmodium falciparum Blood Stages

Review

Critical Steps in Protein Export of Plasmodium falciparum Blood Stages Tobias Spielmann1,* and Tim-Wolf Gilberger1,2 Plasmodium falciparum blood stages export a large number of proteins into their host red blood cell, leading to changes to the infected cell that are pivotal for parasite survival and contribute to parasite virulence. To reach the host cell, exported proteins follow a multistep pathway that now has been revealed to be similar for different classes of exported proteins. Here we summarise the current understanding about the critical segments in protein export of P. falciparum blood stages and discuss recent findings highlighting protein export as a potential target for chemotherapeutic interventions. Transformation of Red Blood Cells by P. falciparum Parasites A major feature of malaria parasites is their ability to prosper in different types of host cells during their complex life cycle within vertebrate and mosquito hosts. The most unusual of these host cells is the red blood cell, a highly specialised cell optimised to ferry oxygen around the body. Red blood cells do not contain any organelles and are incapable of endocytosis. As a consequence, the parasite invades the red blood cell by an active process, creating a new compartment in the host cell that is delimited by a membrane formed from invaginated red blood cell membrane and parasite-derived lipids. This membrane is termed the parasitophorous vacuolar membrane (PVM) and the compartment holding the parasite is termed the parasitophorous vacuole (PV) (reviewed in [1,2]). The PV has entirely different properties compared with the hostderived compartments harbouring other pathogens. For instance, no defences are needed by the malaria blood-stage parasite to combat acidification or digestion. However, this environment also poses many challenges, as the parasite is secluded from essential nutrients in the blood stream by the PVM, the red blood cell cytosol (comprising mainly haemoglobin), and the red blood cell membrane (Figure 1, Key Figure) and cannot profit from many molecular building blocks found in metabolically more active host cells. To prosper in this environment the parasite induces changes to the host cell; for instance, to obtain nutrients. Other profound changes to the host cell are evident on the microscopic level and include protrusions on the host cell surface termed knobs and vesicular cisternae in the host cell cytoplasm termed Maurer's clefts (Figure 1A). Knobs are the sites where P. falciparum membrane protein 1 (PfEMP1), the major virulence factor of the parasite, is concentrated and mediates the adhesion of the infected cell to the endothelium of blood vessels. This prevents the clearance of infected red blood cells by the host spleen. The ensuing obstruction of blood vessels in major organs is considered to be an important contributor to the pathology associated with the most severe form of malaria caused by P. falciparum [3]. These host cell changes are mediated by a large number of exported parasite proteins. Protein export therefore forms the basis for the survival of the intracellular parasite and is a direct cause of malaria pathology, which is exclusively attributable to the development of blood-stage parasites. Besides the relevance for malaria pathology, protein export across the unique

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http://dx.doi.org/10.1016/j.pt.2015.06.010

Trends Malaria is a major public health problem caused by parasites of the genus Plasmodium. Parasite multiplication within human red blood cells is responsible for the clinical symptoms of the disease. The development of the parasite in red blood cells is accompanied by extensive host cell modifications that contribute to parasite survival and virulence. This is mediated by several hundred different proteins that are actively exported by the parasite. Emerging data revealed that all classes of exported proteins converge at a key export step mediated by PTEX. PTEX is proposed to function as a translocation machine pumping exported proteins into the host cell. Inhibition of PTEX or earlier protein export steps leads to parasite death. This might be exploitable for malaria drug development.

1 Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany 2 Center for Structural Systems Biology, 22607 Hamburg, Germany

*Correspondence: [email protected] (T. Spielmann).

Key Figure

Key Points in Blood-Stage Protein Export

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Figure 1. (A) Schematic overview of a red blood cell (red) infected with a Plasmodium falciparum parasite (blue), showing the parasitophorous vacuole (PV), the parasite plasma membrane (PPM), the PV membrane (PVM), the nucleus (N), the endoplasmic reticulum (ER), the Maurer's clefts, and the red blood cell plasma membrane (EPM). Key export steps are highlighted by yellow circles (1–3) and the general export route is depicted with arrows. A soluble ‘Plasmodium export

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vacuolar compartment into a host cell without a working protein trafficking system represents a fascinating cell biological problem. Here we review recent work that provided new insights into some of the key steps in protein export in blood stages of the most virulent malaria parasite, P. falciparum, and discuss the importance of protein export for parasite survival and as a potential drug target.

The Repertoire and Classification of Exported Proteins The sum of all parasite proteins exported into the host cell is generally referred to as the ‘exportome’. Knowing the repertoire of exported proteins is a prerequisite to understanding how P. falciparum parasites modify their host cells and the identification of these proteins from the parasite's
5500 predicted proteins is an important goal. Exported proteins are classified into two groups. The larger group comprises proteins with a five-amino-acid motif named the ‘Plasmodium export element’ (PEXEL) or ‘host targeting’ (HT) sequence with the consensus RxLxE/Q/D [4,5]. To be functional this motif must be positioned approximately 20 amino acids downstream of a signal peptide that promotes entry into the parasite endoplasmic reticulum (ER). For unknown reasons, the signal peptide of many of these proteins is recessed for up to 80 amino acids from the N terminus. The second, smaller group of exported proteins are the PEXEL-negative exported proteins (PNEPs). These proteins contain a hydrophobic region that mediates entry into the secretory pathway but otherwise are defined only by the lack of a PEXEL/HT motif [6,7]. In recent years it has started to become clear how the PEXEL motif functions in export (Figure 1B). The PEXEL sequence is a recognition site for the ER-resident protease Plasmepsin V, which cleaves the substrate between the third and fourth residues of the motif (RxL # xE/Q/D) [8–12]. Cleavage is independent of signal peptidase processing (which removes the signal peptide) and occurs during or shortly after import into the parasite's ER [13]. The newly exposed N terminus starts with the remaining two residues of the PEXEL sequence (xE/Q/D) and is Nacetylated. The Plasmepsin V cleavage event and information in the new N terminus are required for export. While some data showed that exposure of the new N terminus in the ER by means other than Plasmepsin V cleavage can be sufficient for export [14,15], other results suggested that specifically cleavage by Plasmepsin V is mandatory for export [9]. While at present this discrepancy remains, it is established that the new N terminus contributes to export through the remaining conserved PEXEL/HT residue, alternative residues further downstream, or both [14–17]. A likely scenario is that PEXEL/HT cleavage is a crucial initial step for export after

element’ (PEXEL) containing protein and a transmembrane PEXEL-negative exported protein (PNEP) are shown as these represent the best-studied examples of each group. The Golgi is not shown. Circle 1 shows export-relevant events within the parasite's ER. Circle 2 shows the phase when proteins reach the PPM and are delivered across the PVM into the host cell (see Box 1 for details). Circle 3 depicts possible sorting mechanisms in the host cell. (B) PEXEL proteins are cotranslationally inserted into the ER where the PEXEL motif (red) is cleaved by Plasmepsin V (PMV), exposing the new N terminus required for further export (blue). PNEPs of the most common type with a single transmembrane domain are inserted into the ER membrane and the export information in their N terminus (blue) equally mediates export. Hydrophobic regions (transmembrane domain or signal peptide) are shown as white boxes. (C) A vesicle containing exported proteins (assuming here that transmembrane proteins and soluble proteins arrive in the same vesicles) fuses with the PPM, releasing the soluble protein (green) into the PV with immediate access to the translocon for exported proteins (PTEX). The transmembrane protein (orange) is delivered to the PPM and requires extraction before it can be transported by the PTEX. (D) Soluble proteins (green beans) and transmembrane proteins (orange bones) are distributed in the host cell. Soluble proteins reach the target site by diffusion and binding to, for example, the red blood cell cytoskeleton (EC), the knobs (Ks), or other structures. Some, such as KAHRP, first associate with the Maurer's clefts from where they reach the knobs. Membrane proteins could travel in protein transport aggregates (PTAs) containing chaperones such as the PEXEL proteins HSP40 and HSP70 that presumably deliver them to the Maurer's clefts. Insertion of transmembrane proteins into the Maurer's cleft membrane (MCM) would require another protein translocation machine (indicated by a question mark). From the Maurer's clefts, proteins might be trafficked in electron dense vesicles (EDVs) to the knobs or the host cell surface, possibly along tethers (Ts) or actin cables. Some Maurer's clefts proteins are also found on microvesicles (MVs) released into the serum.

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which the newly exposed N terminus promotes further export. It is unknown whether the Nterminal acetylation also influences export, but alone this modification is not sufficient to promote export in an otherwise export-refractory N terminus [16]. It was also reported that the PEXEL/HT can bind phosphatidylinositol 3-phosphate on the luminal side of the ER membrane and it was suggested that this is a deciding factor for protein export [18], although less efficient export was also supported without this interaction [17]. This ER luminal localisation for phosphatidylinositol 3-phosphate is so far unprecedented and future work will need to address its specific role in protein export. The consensus of the PEXEL/HT motif was originally discovered by sequence comparisons of exported proteins or their export-promoting regions [4,5]. This PEXEL/HT consensus was recently further restricted with the help of a Plasmepsin V cleavage assay to test the functionality of different variants of the motif [19]. PEXEL-related sequences included in previous exportome predictions [20,21], such as the KxLxD present in the major virulence factor PfEMP1, were found not to be cleaved by Plasmepsin V [19]. The narrower PEXEL consensus was also validated in newly developed inhibitors of P. falciparum parasites that mimic the PEXEL sequence. For instance, these compounds were effective only if they contained an R but not a K in the first position of the motif [13,22]. Based on the new criteria for defining the PEXEL, PfEMP1s were reclassified as PNEPs and other proteins were excluded from the exportome [19]. Under these criteria, and taking improved gene annotations into consideration, the currently predicted PEXEL exportome comprises
450 proteins [19]. While these studies indicated that the variance of the PEXEL is limited, the unusual PEXEL of the ring-infected surface antigen (RESA) proteins, RxLxxE, is nevertheless cleaved and promotes export. In part this may be explained by the fact that only the RxL is important for Plasmepsin V cleavage [16] and there is generally lower dependence on the last conserved PEXEL residue remaining with the new N terminus in RESA [19] and other PEXEL proteins [14–16]. It was proposed that the RESA PEXEL may be specifically adapted to the particular timing of expression of this protein [19,23]. These findings are important as other unusual PEXEL motifs may exist that have to date escaped detection. It is therefore interesting to note that new data show that, depending on the sequence environment, noncanonical PEXEL/HT motifs (e.g., KxLxE) can still be functional in P. falciparum and this was used to identify such proteins in this parasite [24]. Exported proteins with noncanonical PEXEL/HT motifs are also known from other Plasmodium species [25,26], where they may be more dominant [24]. Hence, the number of proteins in the PEXEL exportome is likely to rise, although the dependence on as-yet-unknown properties of the sequences flanking the motif prevents reliable prediction of these proteins. In contrast to PEXEL proteins, PNEPs lack a defined motif required for export. However, the Nterminal region is essential for the export of many PNEPs [6,14,27–31]. This unifying PNEP export region is functionally exchangeable with the mature N terminus in PEXEL proteins [14]. Hence, PNEPs and matured PEXEL proteins may be trafficked by similar domains, raising the possibility that there is a core export domain and an at least partially overlapping export pathway [14,32]. In addition to the N terminus, the type of transmembrane (TM) domain is also crucial for the export of PNEPs, with only PNEP TM domains allowing efficient export [14,27,28]. While most of the PNEPs initially identified were of similar structure with a single internal hydrophobic region (serving in most cases as a TM domain) but no signal peptide [7,29, 33–38], they are now known to include a wider spectrum of domain structures [6,30,39]. This includes both soluble and integral membrane proteins with classical signal peptides. Although reliable prediction of the number of PNEPs is currently not possible due to a lack of clearly defined signature motifs, they appear not to be a rare exception in P. falciparum [6] and might be

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even more prominent in other Plasmodium species [25]. Several new PNEPs were also identified in P. yoelii and a motif was defined that predicted many more PNEPs in this parasite [40]. This motif cannot easily be reconciled with the PNEP export regions in P. falciparum and it will be interesting to see whether it can predict further PNEPs in this parasite. At present it is estimated that there are several dozens of PNEPs in P. falciparum [6]. Adding the 60 PfEMP1 variants and the currently predicted
450 PEXEL proteins [19], the exportome of P. falciparum parasites comprises about 550 different proteins in total, accounting for
10% of all predicted open reading frames in this parasite. While not all of these proteins are exported at all times – for instance, in the case of mutually exclusively expressed protein families or exported proteins that are expressed in gametocytes [41–44] or liver stages [45–47] – this still represents a surprisingly high proportion of the parasite's protein repertoire. This highlights the profoundness of the parasite's takeover of the host cell and can be seen as the parasite literally turning this enucleated cell into a part of itself.

Delivering Exported Proteins to the Host Cell Export of proteins out of the parasite can be divided into two general phases (Figure 1): first, transport within the parasite; and second, the passage of the protein from the parasite into the host cell. The first phase comprises traversal of the protein through the parasite's secretory system. It starts with the entry of the protein into the parasite's ER, after which it follows a vesicular pathway towards the parasite boundary involving classical secretory components as indicated by sensitivity of this phase to brefeldin A or ER retention signals [48]. It is the initial step at the ER where the PEXEL motif takes effect, but how processing of the motif influences export is unknown. One possibility is that Plasmepsin V cleavage already directs the protein to a specific vesicular trafficking route designated for export. In this respect it is interesting to note that the PEXEL protein RESA is found in dense granules before export and that in Toxoplasma gondii a motif resembling a PEXEL was found in several dense granule proteins [49]. Although the motif in T. gondii is also cleaved, it promotes, not export, but association with the inner face of the PVM. By contrast, only one of two dense granule proteins exported across the PVM in T. gondii harbours a PEXEL-like sequence [50,51]. As the dense granules in P. falciparum are discharged only around the time of invasion and other dense granule proteins are not exported, they are unlikely to be a general export organelle. Interestingly, recent work identified PEXEL-like sequences in other apicomplexans such as Cryptosporidium parvum and Babesia bovis. The authors provided evidence for a role of these motifs in the timed secretion of proteins from organelles known as ‘spherical bodies’ into Babesia-infected red blood cells [52]. It may therefore be that PEXEL-like sequences are signals to proteolytically unmask different types of transport signals in secretory trafficking in apicomplexan parasites. In the second phase, the exported protein leaves the parasite and enters the host cell, a process that requires passage across two membranes: first the parasite plasma membrane (PPM) and second the PVM. Recent data showed that components of the translocon for exported proteins (PTEX), a complex proposed to form a protein conducting channel at the PVM [53], are essential for the passage of all known types of exported proteins into the host cell. This includes both soluble and TM proteins as well as PEXEL proteins and PNEPs, highlighting this complex as a key step where protein export converges at the parasite boundary [54,55] (Figure 1C). PTEX contains at least five different proteins, forming a macromolecular complex of >1.2 MDa [53,56]. The complex includes heat shock protein 101 (HSP101), exported protein 2 (EXP2), thioredoxin 2 (TRX2), and two Plasmodium-specific proteins termed PTEX88 and PTEX150. HSP101 is a ClpA/B-like ATPase from the AAA+ superfamily, commonly associated with protein unfolding [57]. EXP2, a small peripheral membrane protein found on the inner side of the PVM [58] forms a
600-kDa

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apparently homo-oligomeric complex and was suggested to constitute the putative membranespanning pore to which the remainder of the PTEX complex is attached [53,56]. TRX2 is a member of the redox-active thioredoxin family and might be involved in the unfolding of substrates with disulfide bridges to enable passage through the PTEX. This might be required in the oxidising environment of the PV [59,60]. HSP101 and PTEX150 are both suspected to be present as oligomers [52,55] and are mandatory for PTEX function, whereas TRX2 and PTEX88 have less central roles [55,61–63] and are present in lower stoichiometries [56]. Notably, protein export in PTEX88-deficient Plasmodium berghei parasites was not measurably decreased but sequestration in infected mice was reduced by an unknown mechanism [62]. So far, no functional data are available for the predicted pore component EXP2 in P. falciparum. However, very recent data showed that PfEXP2 can complement the role of T. gondii GRA17, a related protein implicated in the long-sought nutrient pore activity of apicomplexan PVMs [64]. As one explanation for this finding, it was proposed that PfEXP2 may be a part of both a nutrient and a protein-conducting pore [64]. Despite the clear evidence for the role of PTEX in protein export, there is so far no direct evidence that PTEX has protein translocation activity. Independent work mechanistically supports active protein translocation at the parasite boundary for different types of exported proteins but to date did not specifically link this with PTEX [6,14,65,66]. It is intriguing that besides soluble proteins TM proteins are also substrates of PTEX. While soluble exported proteins will be directly released into the PV after fusion of transport vesicles with the PPM and thus have direct access to PTEX at the PVM, membrane-embedded exported proteins will end up in the PPM (Figure 1C). It is therefore mechanistically unclear how PTEX transports these proteins. There is evidence for a first translocation step of TM proteins out of the PPM [14], but the molecular basis for this is unknown. In contrast to the situation in organellar outer membranes, such as in mitochondria, which translocate TM proteins directly from the cytoplasm [67,68], proteins arriving at the PPM are already integral to the membrane and require extraction rather than full translocation (Figure 1B). This may be reminiscent of the TM protein dislocation system present in the ER-associated protein degradation machinery (ERAD) or the inner nuclear membrane, where integral membrane proteins are removed from the membrane [69–71]. Typically, chaperones are needed on the distal side of the membrane to help the extraction [69]. It might be that PTEX already contributes to the PPM extraction, either directly or, for instance, through disassociated HSP101 [54]. Alternatively, PPM extraction might be achieved by independent factors in this membrane and the PTEX dependence for TM protein export is a manifestation of a post-PPM step. In mitochondria and chloroplasts, the translocons of the outer and inner membranes interact transiently during substrate passage [72,73]. It will be crucial to dissect the translocation steps at the PPM and PVM of malaria parasites to understand how the different types of proteins cross the two membranes at the parasite boundary to reach the host cell. Possible scenarios are shown in Box 1. It is at present unclear which sequence features mediate substrate recognition for translocation. However, as all types of exported proteins are now known to converge at PTEX [54,55] and the mature N terminus of PEXEL proteins and PNEP N termini were found to contain an exchangeable core export domain [14], the N termini may act at this step [7]. Interestingly, the proteins requiring PPM extraction and translocation at the PVM also have a requirement for a specific TM domain [14,27,28], which might indicate that this process depends on specific properties of this domain or is actively promoted by specific TM sequences. It is also interesting to note that exported proteins with more than two TM domains have not been described. This could indicate restrictions imposed by the membrane translocation/extraction steps during export.

Sorting of Exported Proteins in the Host Cell The last step of the journey of exported proteins is their targeting within the host cell (Figure 1D). This post-export process is equally important and may be the most challenging for the parasite,

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Box 1. Possible Scenarios for TM Proteins to Become Substrates of PTEX Several lines of evidence indicate that exported TM proteins initially travel as integral membrane proteins from the ER to the PPM via the classical secretory pathway [48], which will result in delivery to the PPM in an integral form (Figure 1C). For instance, this was supported by the finding that ER-retained exported TM proteins in brefeldin A-treated parasites showed a solubility typical for integral membrane proteins [14,102] and by the fact that these proteins were properly inserted into mammalian microsomes [14]. In principle, the now-integral PPM proteins could then be trafficked by vesicles from the PPM to the PVM to become substrates of PTEX (by lateral insertion into PTEX once in the PVM). However, experimental evidence does not support this mode of transport: fusion of exported TM proteins to a domain that can be conditionally prevented from unfolding resulted in an arrest of these proteins in the PPM, not the PVM, when unfolding was prevented [14]. This is indicative of a membrane extraction step at the PPM before the TM protein can reach the PVM translocon, as vesicular trafficking, in contrast to many translocation processes, is insensitive to folding. These findings also suggested that exported TM proteins in the PPM have their N terminus (containing the export-promoting domain) facing the PV lumen. Taking into consideration a first extraction step at the PPM, there are several theoretical possibilities for how exported TM proteins can reach the host cell (Figure I). At the PPM there are three basic possibilities: (1) there is an active extractor contributing to membrane extraction similar to the situation with ERAD; (2) there are factors (e.g., PTEX at the PVM) that assist extraction by others but do not alone extract the protein from the PPM; and (3) the PPM does not contribute. At the PVM a role for PTEX has already been demonstrated [54,55]. There are again three possibilities: (a) PTEX receives the TM protein that is already present in the PV; (b) parts of PTEX, for instance HSP101 [54], disassociate to either extract or contribute to the extraction of the protein from the PPM and lead it back to the pore at the PVM for delivery to the host cell; and (c) PTEX directly connects to the PPM or the factors in the PPM with the inserted TM protein and contributes to pulling it from the membrane. In principle each of the three PPM scenarios (1)–(3) would be possible with each of the three PVM scenarios (a)–(c). For instance, the protein could be extracted from the PPM by an active mechanism in the PPM (1), reach the PV, and then be translocated by PTEX [(1) + (a)] or, alternatively, a disassociated part of PTEX could help with extraction [(1) + (b)] or PTEX could associate with the extractor to form a single pore [(1) + (c)]. It should also be noted that, for both (1) and (2), the factor contributing to extraction could already associate with the TM protein in the ER. This might be especially relevant in case (2) where, for instance, a chaperone might prevent full insertion into the membrane core to make possible later extraction. Such a scenario would explain the mixed solubilities observed with some exported TM proteins.

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Figure I. Protein translocation scenarios at the PPM and PVM.

as the host red blood cell contains no protein sorting system of its own. Only a few studies have specifically analysed sorting in the host cell. Soluble proteins can directly reach target structures by diffusion via specific interaction domains. For instance, the proteins of the Plasmodium helical interspersed subtelomeric (PHIST) family [20] are targeted to different sites of the host cell, interacting with the host cell cytoskeleton components such as spectrin, or parasite proteins at specific sites such as the acidic terminal sequence of PfEMP1 molecules at the knobs [74–76]. Interestingly, several of these proteins appear to contain more than one interaction domain,

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thereby interlinking different structures in the host cell [74]. This may be a reason for the altered rigidity of the infected cells caused by some of these proteins [74,77]. Other proteins, such as the major knob component KAHRP, first attach to the Maurer's clefts where they may be assembled into multiprotein complexes destined for the red blood cell periphery [78,79]. Exported TM proteins permanently or transiently appear at the Maurer's clefts, highlighting these structures as a sorting hub in the host cell. However, how TM proteins reach the clefts is unclear. Translocation at the PVM could either insert the protein laterally into the membrane followed by vesicular trafficking to the Maurer's clefts or translocate the protein through the membrane, delivering the protein in a soluble form into the host cell cytoplasm. Several lines of evidence are in favour of a non-vesicular pathway towards the Maurer's clefts: full-length soluble TM proteins were detected in the host cell cytoplasm [14,80], no vesicular pathway was evident in time-lapse imaging of an integral Maurer's cleft protein [81], and the TM protein PfEMP1 was found in complexes with soluble chaperones termed J dots that may represent trafficking structures [30,82]. However, how these proteins are inserted into the Maurer's cleft membrane remains obscure. Also still tentative is the trafficking of proteins from the Maurer's clefts to the host cell surface [32]. Remodelled host cell actin, connecting the Maurer's clefts with the knobs, was implicated in successful trafficking of PfEMP1 to the host cell surface. In red blood cells from patients with haemoglobinopathies (genetic defects in haemoglobin such as the sickle cell haemoglobin HbS or HbC that protect against severe malaria [83]) and in the knockout of a parasite protein termed PTP1, this remodelling was impaired and PfEMP1 failed to reach the host cell surface and the cytoadherence of infected red blood cells was reduced [78,84–86]. In addition, vesicle-like structures positive for PfEMP1 were seen both on these actin filaments and on structures termed ‘tethers’ that were observed to attach Maurer's clefts to the red blood cell periphery [29,84,87,88]. However, newer work also showed impaired trafficking across the PVM in parasites growing in HbC or HbS red blood cells and this might be a further reason for the reduced cytoadherence of these red blood cells [89]. Interestingly, some integral Maurer's cleft proteins were also found in microvesicles, exosomelike vesicles originating from infected red blood cells that are involved in cell-to-cell communication [90,91]. The presence of Maurer's cleft proteins in these vesicles might indicate that the corresponding trafficking processes originate at these structures, raising the possibility that Maurer's clefts are the source of different vesicular pathways. Overall, the sorting in the host cell appears to be rather rudimentary and in part based on diffusion and interaction. The Maurer's clefts appear to play a central role for many proteins and may be the source of a vesicular system for trafficking to the host cell surface and beyond.

Importance of Protein Export and Suitability as a Drug Target The export of roughly 10% of all proteins encoded by the parasite genome is a massive investment by the parasite. It underlines the fundamental role of protein export in parasite biology and therefore represents a target for experimental drug design. Somewhat unexpectedly, inactivation of the PTEX component HSP101 did not lead to rapid parasite death in vitro but to an arrest in development at the transition between the ring and the trophozoite stage [54,55]. This arrest was reversible for up to 48 h if HSP101 function was reinstated [54]. Later-stage parasites were not affected. This is a rather mild phenotype considering that the inactivation of HSP101 inhibited the export of all classes of exported proteins, preventing a large number of effectors from reaching the host cell. The PTEX complex might therefore not seem to be the most optimal of drug targets, as specific inhibitors would require pharmacokinetic properties or treatment regimens to uphold drug levels past the 48 h. However, as many of the affected

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exported proteins, such as PfEMP1 and its trafficking factors, play a role in virulence [92], the in vivo effect of inhibiting export might be much more profound. Although PTEX knock down also affects gametocytes, it acts only on stages I and II, limiting the antitransmission effect for potential drugs. By contrast, the liver stage could be a target as PTEX components were detected in this life cycle phase [93]. A previous large-scale knockout study estimated that
25% of the P. falciparum exported proteins are essential [77]. At least some of these proteins could be expected to have functions leading to parasite death rather than growth arrest when prevented from reaching the host cell. Hence, the observed growth arrest after the general inhibition of protein export might raise the exciting prospect that there is a checkpoint for parasite growth that depends on the completion of host cell modifications. Only if host cell modifications have reached a stage to permit PfEMP1 surface expression and hence cytoadherence is the rapid parasite growth seen in trophozoites possible without risking clearance of the infected cell by the spleen. The timing for such a checkpoint at the transition to the trophozoite stage would therefore be fitting. Interestingly, the Plasmodium surface anion channel (PSAC) at the surface of the infected red blood cell that mediates access of the parasite to serum nutrients was inactive when PTEX was not functional [54]. However, the export of CLAG3, a protein clearly linked to PSAC activity [94], was unaffected [54]. This unexpected result suggested that export of other proteins via PTEX is also required for PSAC activity. Lack of nutrients may be another reason for the growth arrest observed after HSP101 inactivation. In contrast to the global inhibition of protein export through the abolition of PTEX function, inhibition of Plasmepsin V irreversibly killed the parasites. This was despite a much milder phenotype on general protein export without strongly visible retention of exported proteins in the parasite's ER or in the parasite periphery [13]. It is possible that accumulation of precursors in the ER has a more detrimental effect than in the parasitophorous vacuole, the probable site of action of the PTEX knock down. Future work will show the suitability of these targets for drug development. A detailed characterisation of factors affecting the activity properties of the present Plasmepsin V inhibitors has already been conducted, setting the stage for further optimisation to obtain leads for drug development [22].

Concluding Remarks We expect to see further improvements in the identification of exported proteins in P. falciparum but also in other Plasmodium species. The next important goal is the functional characterisation of these proteins (see Outstanding Questions Box), which has so far predominantly yielded data on functions in host cell rigidity and PfEMP1 transport and display [77,78,95–99]. There are likely to be many other functions of exported proteins that have so far remained obscure, such as the recently discovered functions in intercellular communication [90,91] and lipid biology [100], or for which the molecular basis was previously unknown, such as in the rosetting of infected red blood cells [101]. A further exciting topic are the translocation steps in the parasite periphery that so far have not been resolved and, given the number of substrates, may be more complex than currently known. For instance, if there is no vesicular pathway from the PPM to the PVM, PVMresident TM proteins would also require translocation to become inserted into the PVM. However, these proteins would then need to be laterally released into the PVM, for instance by a stop transfer sequence, to distinguish them from exported proteins that are translocated through the PVM into the host cell. It is therefore possible that this machinery has a level of complexity comparable with that found in the membranes of mitochondria and chloroplasts.

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Outstanding Questions How does PEXEL cleavage mediate protein export? At which step and how do the mature PEXEL N termini and PNEP N-termini promote export? How are exported transmembrane proteins extracted from the parasite plasma membrane? Are there further components involved in protein translocation at the parasite periphery? Are different accessory factors needed for different types of exported proteins? What is the role of EXP2 in protein transport? How many proteins are missing from the currently predicted malaria exportomes and is there a way to predict all exported proteins? How are transmembrane proteins inserted into the Maurer's clefts? By which mechanism are proteins trafficked from the Maurer's clefts to the host cell membrane? Can clinically relevant inhibitors acting on protein export be generated?

A key finding of recent work is the experimental confirmation that protein export, as expected, is essential for parasite survival and that there are ways to globally interfere with this process. It would be gratifying if this long-studied process now enabled the design of clinically relevant inhibitors. Acknowledgments The authors thank Michael Kolbe and the members of their laboratories for critically reading the manuscript. The authors gratefully acknowledge the funding of the DFG (SP1209/1-2; SP1209/2-1), the research training group GRK1459 (Sorting and Interactions between Proteins of Subcellular Compartments), and the priority program SPP1580 (SP1209/3-1, Intracellular Compartments as Places of Pathogen–Host Interaction).

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