Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways?

Opinion

Protein export in malaria parasites: do multiple export motifs add up to multiple export pathways? Tobias Spielmann and Tim-Wolf Gilberger Bernhard Nocht Institute for Tropical Medicine, Department of Molecular Parasitology, 20359 Hamburg, Germany

Intracellular malaria parasites export numerous proteins into their host cell, a process essential for parasite survival and virulence. Many of these proteins are defined by a short amino acid sequence motif termed PEXEL or VTS that mediates their export, suggesting a collective trafficking route. The existence of several PEXEL-negative exported proteins (PNEPs) indicates that alternative export pathways might also exist. We review recent data on the sequences mediating export of PNEPs and compare this process to PEXEL export taking into account novel findings on the function of this motif. Based on this we propose that, despite the lack of a PEXEL in PNEPs, both groups of proteins might converge in a single export pathway on their way into the host cell. Exported proteins in host cell modifications The survival and virulence of asexual blood stages of the human malaria parasite Plasmodium falciparum depend crucially on extensive host cell modifications. These modifications are based on parasite proteins exported to diverse destinations in the host cell, and require passage past the confines of the parasite and through the parasitophorous vacuole membrane (PVM) that separates the parasite from the host cell cytosol. A short amino acid motif termed the Plasmodium export element (PEXEL) or vacuolar transport signal (VTS) was found to be essential for exported proteins to reach destinations beyond the PVM both in blood stages [1,2] and liver stages [3]. This motif of the consensus RxLxE/Q/D is usually found 20 amino acids downstream of a signal peptide, and was used to predict the malaria ‘exportome’, comprising over 300 proteins in P. falciparum [1,2,4,5]. However, several welldocumented exported proteins lack the PEXEL motif [6– 9]. Although initial work indicated that PEXEL-like sequences might be involved in the export of these proteins [10], new data show that other sequence regions are also capable of mediating export [11–13]. The presence of multiple export sequences raises the question of whether Plasmodium parasites have just one export pathway into the host cell, or instead have multiple pathways. Here we address this question by comparing the sequences known to mediate export of PEXEL-positive and -negative proteins. Corresponding author: Spielmann, T. ([email protected])

6

PEXEL proteins and the role of the PEXEL motif in export Many PEXEL proteins are known to be involved in host cell modifications [14–16]. This was recently corroborated by a large-scale knockout study demonstrating that some of these proteins are involved in cytoadherence or in shaping the rheological properties of the infected red blood cell [17]. Predictions of the malaria exportome are based on the PEXEL motif and a preceding signal peptide [1,2]. However, the prediction of exported Plasmodium proteins can be problematic because their signal peptides are often recessed and are sometimes only moderately hydrophobic, and two refined methods were therefore developed to predict the malaria exportome [4,5]. A related transport motif (RxLR) was also found in oomycetes and in certain fungal plant pathogen proteins that are exported into host plant cells [18–20], and this signal was shown to be capable of driving export in P. falciparum [21]. Likewise, the malaria PEXEL motif was found to mediate protein export in plant pathogens [20,22]. However, recent data have shown that the machinery delivering fungal effectors into plant cells is derived entirely from the host cell [20], a scenario that seems unlikely in Plasmodium, therefore casting some doubt on a close resemblance between the export pathways used by fungi and by malaria parasites. This notion is supported by the recent description of a PEXEL-protein translocation machine at the P. falciparum PVM. This consists of a Plasmodium-specific ClpB ATPase orthologue (that could be involved in unfolding of the substrate; Ref. [23]), EXP2 as the suspected pore, the novel protein PTEX150, and potentially PTEX88 and thioredoxin2 [24]. Apart from thioredoxin2, these components do not appear to be present in either plants or fungi. In fact, the translocon components are absent from any other organism outside of the genus Plasmodium, including other Apicomplexans [24], in accordance with the lack of PEXEL proteins in these organisms. In the related Apicomplexan, Toxoplasma gondii, exported proteins can be injected into the host cell upon invasion [25], and this could indicate a fundamentally different mode of protein export in this parasite. Surprisingly, the PEXEL motif is not the sequence recognized by the translocon, and is instead a protease recognition site cleaved in the endoplasmic reticulum (ER) of the parasite before export [26,27]. Cleavage of the motif occurs after the L of the PEXEL motif; this generates a new

1471-4922/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2009.10.001 Available online 29 October 2009

Opinion

Trends in Parasitology

Vol.26 No.1

Figure 1. Protein regions promoting export in P. falciparum. Proteins are schematically represented by coloured bars, fading from the N-terminus to the C-terminus (COOH) with hatched boxes representing repeat regions. Hydrophobic regions (dark blue) depict either signal peptides (SP) or transmembrane (TM) domains. Protein length is not drawn to scale. The PEXEL TM domain is in brackets because this group of proteins comprises both soluble and TM domain proteins. The bar representing PEXEL proteins is in red, PNEPs are in green, and the bar corresponding to plant pathogen proteins is in blue. The positions of known processing sites are marked by scissors. The background of the mature exported form of PEXEL proteins is shaded differently to the part removed in the ER. Both processed forms detected for REX2 are shown. Amino acids essential for export are indicated, with ‘x’ denoting any amino acid. Regions involved in export are shown by solid lines above the protein; solid lines with asterisks indicate regions promoting export when placed N-terminally; the dotted line marks a region suspected to affect export of episomally expressed constructs through interaction with the endogenous protein; the filled black circle represents N-terminal acetylation.

N-terminus that commences with xE/Q/D, and the terminal residue bears an acetyl group at its N-terminus [26,27] (Figure 1). Processing of the PEXEL is not equivalent to the cleavage of the signal peptide that occurs prior to PEXEL cleavage [27]. Hence, in the parasite ER, even before export, only a single residue of the PEXEL motif remains with the mature protein. Because mutation of this residue abolishes export but not processing [27], PEXEL cleavage inside the parasite is not sufficient for export, and the new N-terminus must play an essential role in mediating further trafficking. However, there is remarkably little sequence specificity in this newly created N-terminus (Figure 1): (i) the remaining PEXEL residue is the least conserved of the motif, most frequently E, Q, or D [1,2,4,5,21]. Exactly which residues are tolerated at this position remains to be determined by systematic mutagenesis, and constructs containing the fungal export motif RxLR tested in P. falciparum even indicate that it might not be essential in certain proteins [21]; (ii) the new Nterminus is acetylated [26,27], although this is not sufficient for export, and at least one example was detected where a PEXEL was cleaved without acetylation [27], suggesting that this might not be strictly necessary for export; (iii) a short region immediately downstream of the remaining PEXEL residue is required as a spacer separating the PEXEL from discrete folded structures such as green fluorescent protein. None of the sequences tested for this spacer interfered with export, including oligo A [21,28– 30]. Taken together, this suggests that beyond the remaining PEXEL residue there is no (or limited) sequence specificity required in the new N-terminus of the polypeptide that distinguishes it from non-exported proteins. For this reason a model was favoured where PEXEL recognition inside of the parasite already channels these proteins into a pathway solely destined for export, for example by

directing them to the translocon in the PVM [27]. Interestingly, protein translocation channels are known to transport a wide variety of protein substrates, often recognising N-terminal sequences lacking a simple consensus [31,32]. PEXEL-negative exported proteins (PNEPs) The existence of several PEXEL-negative exported proteins (PNEPs) in P. falciparum suggests that an unknown number of proteins is missing from the currently predicted exportome. Two of these PNEPs, the skeletonbinding protein 1 (SBP1) [6] and the membrane associated histidine-rich protein 1 (MAHRP1) [7] are required for the cytoadherence ligand PfEMP1 to reach the red blood cell surface [33–35]. A third – ring-exported protein 1 (REX1) [8] – was shown to be involved in maintaining the architecture of parasite-induced vesicular structures in the host cell cytoplasm termed Maurer’s clefts [36]. Ring exported protein 2 (REX2) is another well-established exported protein without a PEXEL [9]. All these PNEPs localise to Maurer’s clefts [6–9]. SBP1, MAHRP1 and REX2 share a common structure containing a single transmembrane (TM) domain but lack a signal peptide. REX1 contains a single hydrophobic stretch, but this region most probably represents a recessed signal peptide [11]. ER intermediates were detected for all of these proteins, suggesting export via the classical secretory pathway [10,11,13,37]. SURFINs and Pf332 could represent further PNEPs [38– 40], although sequences resembling modified PEXEL motifs have been detected in these proteins [39]. An unusual PNEP is the heme detoxification protein (HDP) [41]. HDP lacks any hydrophobic region that would qualify as a TM domain or signal peptide, and the polypeptide is exported in a brefeldin A-insensitive manner into the host cell cytosol, suggesting an unconventional 7

Opinion secretion pathway. After export HDP is re-internalized into the food vacuole, where it is believed to be a major factor in hemozoin formation [41]. As with P. falciparum, other malaria species also show elaborate morphological changes in their host cells, including cleft-like structures and surface alterations [42,43]. Although PEXEL proteins are expected to contribute significantly to these host cell modifications, the surprisingly small number of predicted PEXEL proteins in some malaria species [4] could indicate that PNEPs play a more prominent role in host cell remodelling in these parasites. This hypothesis is supported by evidence for PNEPs in other Plasmodium spp. For instance, about half of the P. vivax VIR surface antigen family [44], as well as their homologues in rodent malarias, lack a PEXEL motif [4,45,46]. A second family of exported proteins without a PEXEL, termed TRAg, is also present in both human and rodent malarias [47–49]. In P. knowlesi, the two largest variant antigen families – the SICA antigens expressed on the infected red blood cell surface [50] and the KIR proteins [45] – lack a typical PEXEL motif [51]. The significance of PEXEL-like sequences in several of the SICA and KIR proteins remains to be tested. Sequences involved in the export of PNEPs Many more exported PEXEL-negative proteins might be hidden in the malaria genomes because no systematic approaches are currently available to search for them. Any common property of the PNEPs, such as an export motif, would be highly useful for predicting this missing part of the Plasmodium exportome. Two conserved motifs unrelated to the PEXEL were discovered near the N termini of the P. knowlesi SICA and KIR families; it was speculated that these sequences might be involved in export [51]. However, this hypothesis remains to be verified in P. knowlesi, and experimental data on the sequences involved in export of PNEPs are so far restricted to P. falciparum proteins. Recent studies using GFP fusion proteins indicate a confusing multitude of sequences involved in PNEP export (summarized in Figure 1). In REX1, a sequence of 10 amino acids immediately downstream of the hydrophobic stretch was found to be essential for export [11]. In SBP1, MAHRP1 and REX2 the N-terminal first 35, 50 and 10 amino acids, respectively, are involved in export [12,13]. Domain-swap experiments demonstrated that the export function of these N-terminal regions was exchangeable between SBP1 and MAHRP1 [12], and between SBP1 and REX2 [13], suggesting common mechanisms of export for these PNEPs. Complicating the picture, both in SBP1 and in MAHRP1, an additional region in N-terminal proximity of the TM domain was also implicated in export [10,12]. It should be noted, however, that these internal regions were tested in truncation constructs exposing them at the very N-terminus of the protein, and it is not known if these regions can promote export when placed internally. A further region important for export is the TM domain that is essential for promoting entry into the secretory pathway [10,12,13]. In addition, data from REX2 and SBP1 indicate that not all TM domains are compatible with export and that the properties of the TM domain can affect trafficking of these 8

Trends in Parasitology Vol.26 No.1

proteins downstream of ER entry [12,13]. Thus, the TM domain might add crucial specificity to the other export domains. To assess the relation of PNEP to PEXEL export, the most important question is whether the various regions involved in export of PNEPs contain cryptic PEXEL motifs. PfEMP1, for instance, shares a similar protein structure to PNEPs, but contains a functional modified PEXEL in its Nterminus [1]. A motif termed ‘PEXEL- like’ was detected in the TM-proximal region involved in MAHRP1 export [10]. To date, this motif has not been analysed by mutagenesis. Because there is no evidence for processing in this region (the processing expected for a functional PEXEL would be easily detectable because it would halve the size of MAHRP1), and the N-terminal region of MAHRP1 can also promote export [12], it is unlikely that this motif represents a genuine PEXEL. In both REX2 and SBP1, the N-terminal region involved in export contains the sequence LAE overlapping with a partial PEXEL [13]. However, a mutational analysis showed that only the E (and not the L) of the LAE sequence in REX2 was essential for export [13]. In SBP1, a detailed analysis of the Nterminus using alanine scans showed that the LAE was dispensable, but revealed a different region important for export that coincidentally contains the related sequences LAD and LQD [12]. At odds with the importance of the single E in REX2, changing all negative charges in the SBP1N-terminus did not abolish export, although it was reduced [12]. Finally, a functional PEXEL would be expected to be processed. REX2 is the only PNEP for which there is evidence for processing, and this occurs two residues upstream of the E that is essential for export, creating an N-terminus similar but not identical to that generated by cleavage of PEXEL proteins (Figure 1). However, no causal link between processing and export has yet been established in REX2 [13]. Neither the region required for the export of REX1, nor the N-terminal 50 amino acids of MAHRP1, contain any sequence resembling a PEXEL motif. There are also no other obvious sequence motifs shared among all PNEP export regions. Taken together these data indicate that there is no unifying simple motif, and certainly no genuine PEXEL motif, to be found in the PNEP export regions. Because the N-termini of SBP1, MAHRP1 and REX2 are exchangeable it is likely that some other, less clear-cut property is shared between these domains. It has been proposed that a difference in the isoelectric point (pI) of the N-terminal domain versus the C-terminus of SBP1 and MAHRP1 might be a feature required for export [10,12]. However, as previously noted [12], this might simply be required for the protein to assume the correct orientation in the membrane, a function that is known to be affected by charge [52], and so might indirectly affect export by placing export domains on the wrong side of the membrane. Alternatively the pI might reflect the presence of specific amino acids that are required for export. Do multiple export motifs equal multiple export pathways? The sequences promoting export of PNEPs appear remarkably unspecific because they appear to lack a common motif

Opinion and, as such, stand in clear contrast to PEXEL proteins. Nevertheless, we argue that this does not necessarily mean that PNEPs use a different export pathway from PEXEL proteins. In fact, after processing of the PEXEL (as outlined above) only ill-defined export-relevant sequence information is contained within the novel N-terminus, and this region might be functionally similar to the Nterminal domains that promote the export of PNEPs. The similarity is most striking in the case of REX2 for which a processed form with an essential E at position 2 or 3 was detected [13] (Figure 1). Given the known residues tolerated at position 5 of the PEXEL, Q, in addition to E and D, would also be compatible with export. Furthermore, there is evidence from constructs with the fungal RxLR motif tested in P. falciparum that, in the absence of the E/Q/D residue present in the PEXEL, negative charges further downstream are essential for export [21]. Assuming RxLbased processing of these constructs, this suggests that negative charges (and, potentially, Q residues) might be functional at other than the penultimate position. Taken together, this might explain why different regions of SBP1 and MAHRP1, when placed N-terminally, were found to allow for export, and this could account for the low pI of the PNEP export domains. Hence, it is possible that the PNEP N-termini are comparable to the N termini of processed PEXEL proteins, suggesting that the PNEP pathway converges with the PEXEL pathway either in the secretory system of the parasite or further downstream as a substrate of the same translocon. If export depends on the proposed PEXELmediated pre-selection necessary to guide proteins into the correct pathway for export [27], it will be interesting to find out how PNEPs bypass this step to reach the translocon. The idea of a translocon for all exported proteins does not solve the dilemma of how the limited sequence specificity found in the PNEP export regions or the mature PEXEL Nterminus mediates export. It is possible that these N-termini bear some so far unrecognised specificity such as a common fold, chaperone or trafficking factor binding site that might not be apparent at the primary sequence level. The diversity of the N-termini of translocated proteins could also reflect additional trafficking information needed to reach different destinations in the host cell. Alternatively, similar to protein translocation in the outer chloroplast membrane [53,54], diverse translocon substrates could be targeted by different accessory factors or translocons of different compositions. With a translocon revealed in P. falciparum [24] this can now be experimentally tested. At present, other (translocon-independent) possibilities for PNEP export cannot be excluded: the lack of a clear consensus in the PNEP export regions could also be due to a multitude of export signals that contribute to export, similar to the situation found in dense secretory granule sorting [55]. By analogy, this could manifest itself in bulk flow export through the proposed formation of nascent Maurer’s clefts from the PVM [10]. Conclusions We believe that, despite a lack of recognisable export motifs in PNEPs, there are similarities to mature PEXEL

Trends in Parasitology

Vol.26 No.1

proteins, suggesting that downstream of PEXEL cleavage the export pathway might accommodate both types of proteins. Clearly, experimental data are now required to provide evidence for this hypothesis. If the translocon should turn out to be the only way into the host cell, its specificity will define all exported proteins and this could be exploited to identify the entire malaria exportome. It will be interesting to see whether an expanded exportome will provide candidates for functions so far underrepresented in exported proteins, such as nutrient acquisition, signalling and waste disposal. Acknowledgements We thank Andreas Kru¨ger, Arlett Heiber and Christof Gru¨ring for critical reading of the manuscript. Work in our laboratory is supported by grants SP1209/1, GI312/4 and GRK1459 of the Deutsche Forschungsgemeinschaft. T.S. was supported by a Humboldt Fellowship.

References 1 Marti, M. et al. (2004) Targeting malaria virulence and remodeling proteins to the host erythrocyte. Science 306, 1930–1933 2 Hiller, N.L. et al. (2004) A host-targeting signal in virulence proteins reveals asecretome in malarial infection. Science 306, 1934–1937 3 Singh, A.P. et al. (2007) Plasmodium circumsporozoite protein promotes thedevelopment of the liver stages of the parasite. Cell 131, 492–504 4 Sargeant, T.J. et al. (2006) Lineage-specific expansion of proteins exported toerythrocytes in malaria parasites. Genome Biol. 7, R12 5 van Ooij, C. et al. (2008) The malaria secretome: from algorithms to essentialfunction in blood stage infection. PLoS Pathog. 4, e1000084 6 Blisnick, T. et al. (2000) Pfsbp1, a Maurer’s cleft Plasmodium falciparum protein, is associated with the erythrocyte skeleton. Mol. Biochem. Parasitol. 111, 107–121 7 Spycher, C. et al. (2003) MAHRP-1, a novel Plasmodium falciparum histidine-rich protein, binds ferriprotoporphyrin IX and localizes to the Maurer’s clefts. J. Biol. Chem. 278, 35373–35383 8 Hawthorne, P.L. et al. (2004) A novel Plasmodium falciparum ring stage protein, REX, is located in Maurer’s clefts. Mol. Biochem. Parasitol. 136, 181–189 9 Spielmann, T. et al. (2006) A cluster of ring stage-specific genes linked to a locus implicated in cytoadherence in Plasmodium falciparum codes for PEXEL-negative and PEXEL-positive proteins exported into the host cell. Mol. Biol. Cell 17, 3613–3624 10 Spycher, C. et al. (2006) Genesis of and trafficking to the Maurer’s clefts of Plasmodium falciparum-infected erythrocytes. Mol. Cell. Biol. 26, 4074–4085 11 Dixon, M.W. et al. (2008) Targeting of the ring exported protein 1 to the Maurer’s clefts is mediated by a two-phase process. Traffic 9, 1316– 1326 12 Saridaki, T. et al. (2009) Export of PfSBP1 to the Plasmodium falciparum Maurer’s clefts. Traffic 10, 137–152 13 Haase, S. et al. (2009) Sequence requirements for the export of the Plasmodium falciparum Maurer’s clefts protein REX2. Mol. Microbiol. 71, 1003–1017 14 Cooke, B.M. et al. (2001) The malaria-infected red blood cell: structural and functional changes. Adv. Parasitol. 50, 1–86 15 Cooke, B.M. et al. (2004) Protein trafficking in Plasmodium falciparum-infected red blood cells. Trends Parasitol. 20, 581–589 16 Maier, A.G. et al. (2009) Malaria parasite proteins that remodel the host erythrocyte. Nature Rev. 7, 341–354 17 Maier, A.G. et al. (2008) Exported proteins required for virulence and rigidity of Plasmodium falciparum-infected human erythrocytes. Cell 134, 48–61 18 Whisson, S.C. et al. (2007) A translocation signal for delivery of oomycete effector proteins into host plant cells. Nature 450, 115–118 19 Rehmany, A.P. et al. (2005) Differential recognition of highly divergent downy mildew avirulence gene alleles by RPP1 resistance genes from two Arabidopsis lines. Plant Cell 17, 1839–1850 9

Opinion 20 Dou, D. et al. (2008) RXLR-mediated entry of Phytophthora sojae effector Avr1b into soybean cells does not require pathogen-encoded machinery. Plant Cell 20, 1930–1947 21 Bhattacharjee, S. et al. (2006) The malarial host-targeting signal is conserved in the Irish potato famine pathogen. PLoS Pathog. 2, e50 22 Grouffaud, S. et al. (2008) Plasmodium falciparum and Hyaloperonospora parasitica effector translocation motifs are functional in Phytophthora infestans. Microbiology 154, 3743–3751 23 Gehde, N. et al. (2009) Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum. Mol. Microbiol. 71, 613–628 24 de Koning-Ward, T.F. et al. (2009) A newly discovered protein export machine in malaria parasites. Nature 459, 945–949 25 Ravindran, S. and Boothroyd, J.C. (2008) Secretion of proteins into host cells by Apicomplexan parasites. Traffic 9, 647–656 26 Chang, H.H. et al. (2008) N-terminal processing of proteins exported by malaria parasites. Mol. Biochem. Parasitol. 160, 107–115 27 Boddey, J.A. et al. (2009) Role of the Plasmodium export element in trafficking parasite proteins to the infected erythrocyte. Traffic 10, 285–299 28 Lopez-Estrano, C. et al. (2003) Cooperative domains define a unique host cell-targeting signal in Plasmodium falciparum-infected erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 100, 12402–12407 29 Knuepfer, E. et al. (2005) Function of the Plasmodium export element can be blocked by green fluorescent protein. Mol. Biochem. Parasitol. 142, 258–262 30 Przyborski, J.M. et al. (2005) Trafficking of STEVOR to the Maurer’s clefts in Plasmodium falciparum-infected erythrocytes. EMBO J. 24, 2306–2317 31 Schnell, D.J. and Hebert, D.N. (2003) Protein translocons: multifunctional mediators of protein translocation across membranes. Cell 112, 491–505 32 Schatz, G. and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519–1526 33 Spycher, C. et al. (2008) The Maurer’s cleft protein MAHRP1 is essential for trafficking of PfEMP1 to the surface of Plasmodium falciparum-infected erythrocytes. Mol. Microbiol. 68, 1300–1314 34 Cooke, B.M. et al. (2006) A Maurer’s cleft-associated protein is essential for expression of the major malaria virulence antigen on the surface of infected red blood cells. J. Cell Biol. 172, 899–908 35 Maier, A.G. et al. (2007) Skeleton-binding protein 1 functions at the parasitophorous vacuole membrane to traffic PfEMP1 to the Plasmodium falciparum-infected erythrocyte surface. Blood 109, 1289–1297 36 Hanssen, E. et al. (2008) Targeted mutagenesis of the ring-exported protein-1 of Plasmodium falciparum disrupts the architecture of Maurer’s cleft organelles. Mol. Microbiol. 69, 938–953 37 Saridaki, T. et al. (2008) A conditional export system provides new insights into protein export in Plasmodium falciparum-infected erythrocytes. Cell. Microbiol. 10, 2483–2495

10

Trends in Parasitology Vol.26 No.1 38 Mattei, D. and Scherf, A. (1992) The Pf332 gene of Plasmodium falciparum codes for a giant protein that is translocated from the parasite to the membrane of infected erythrocytes. Gene 110, 71–79 39 Winter, G. et al. (2005) SURFIN is a polymorphic antigen expressed on Plasmodium falciparum merozoites and infected erythrocytes. J. Exp. Med. 201, 1853–1863 40 Hodder, A.N. et al. (2009) Analysis of structure and function of the giant protein Pf332 in Plasmodium falciparum. Mol. Microbiol. 71, 48–65 41 Jani, D. et al. (2008) HDP-a novel heme detoxification protein from the malaria parasite. PLoS Pathog. 4, e1000053 42 Bray, R.S. and Garnham, P.C. (1982) The life-cycle of primate malaria parasites. Br. Med. Bull. 38, 117–122 43 Mackenstedt, U. et al. (1989) Comparative morphology of human and animal malaria parasites. I. Host–parasite interface. Parasitol. Res. 75, 528–535 44 del Portillo, H.A. et al. (2001) A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax. Nature 410, 839– 842 45 Janssen, C.S. et al. (2004) Plasmodium interspersed repeats: the major multigene superfamily of malaria parasites. Nucl. Acids Res. 32, 5712– 5720 46 Carlton, J.M. et al. (2008) Comparative genomics of the neglected human malaria parasite Plasmodium vivax. Nature 455, 757–763 47 Burns, J.M., Jr et al. (1999) Protective immunization with a novel membrane protein of Plasmodium yoelii-infected erythrocytes. Infect. Immun. 67, 675–680 48 Burns, J.M. et al. (2000) An unusual tryptophan-rich domain characterizes two secreted antigens of Plasmodium yoelii-infected erythrocytes. Mol. Biochem. Parasitol. 110, 11–21 49 Jalah, R. et al. (2005) Identification, expression, localization and serological characterization of a tryptophan-rich antigen from the human malaria parasite Plasmodium vivax. Mol. Biochem. Parasitol. 142, 158–169 50 al-Khedery, B. et al. (1999) Antigenic variation in malaria: a 3’ genomic alteration associated with the expression of a P. knowlesi variant antigen. Mol. Cell 3, 131–141 51 Pain, A. et al. (2008) The genome of the simian and human malaria parasite Plasmodium knowlesi. Nature 455, 799–803 52 von Heijne, G. (1992) Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J. Mol. Biol. 225, 487–494 53 Ivanova, Y. et al. (2004) Members of the Toc159 import receptor family represent distinct pathways for protein targeting to plastids. Mol. Biol. Cell 15, 3379–3392 54 Agne, B. and Kessler, F. (2009) Protein transport in organelles: the Toc complex way of preprotein import. FEBS J. 276, 1156–1165 55 Dikeakos, J.D. and Reudelhuber, T.L. (2007) Sending proteins to dense core secretory granules: still a lot to sort out. J. Cell Biol. 177, 191–196