Evolving Insights into Protein Trafficking to the Multiple Compartments of theApicomplexan Plastid1

MARILYN PARSONS, ANURADHA KARNATAKI and AMY E. DEROCHER

Seattle Biomedical Research Institute, Seattle, Washington 98109

ABSTRACT. The apicoplast is a relict plastid found in many medically important apicomplexan parasites, such as Plasmodium andToxoplasma. Phylogenetic analysis and the presence of four bounding membranes indicate that the apicoplast arose from a secondaryendosymbiosis. Here we review what has been discovered about the complex journey proteins take to reach compartments of the api-coplast. The targeting sequences for luminal proteins are well-defined, but those routing proteins to other compartments are only beginningto be studied. Recent work suggests that the trafficking mechanisms involve a variety of molecules of different phylogenetic origins. Wehighlight some remaining questions regarding protein trafficking to this divergent organelle.

Key Words. Chloroplast, endosymbiosis, malaria, Plasmodium, Toxoplasma.

THE double-membraned plastids of most algae and land plantsare the result of an ancient endosymbiosis between a photo-

synthetic bacterium and an early eukaryote. Eons later, an alga setup house in an early progenitor of modern day apicomplexan par-asites and its alveolate relatives. The lineage leading to Apicom-plexa jettisoned the entire algal cell except for some of its genesand its plastid. The non-photosynthetic apicomplexan plastid isnow called the apicoplast and is bounded by four membranes(Kohler et al. 1997; McFadden et al. 1996) (Fig. 1–3). The innertwo of these membranes are thought to be derived from the orig-inal plastid envelope and hence are of cyanobacterial origin. Incontrast, the outer two membranes are likely of eukyarotic origin,possibly derived from the algal plasma membrane and anapicomplexan membrane such as an endosomal membrane orthe plasma membrane (Cavalier-Smith 2003). Although the algalnucleus has been lost, its past presence can be deduced from theexistence of genes in the apicomplexans that are phylogeneticallyrelated to those of alga, including many that encode proteins thattypically reside in plastids (Fast et al. 2001; Ralph et al. 2004b).The non-photosynthetic apicoplast is phylogenetically related tothe photosynthetic plastid of the alveolate Chromera velia (Mooreet al. 2008). Apicomplexa and other alveolates are proposed tobelong to a superfamily of protists, the chromalveolates, whichincludes several other photosynthetic species (Harper and Keeling2003; van Dooren et al. 2001). Remarkably, some of these, such asthe cryptophytes, still have a functional remnant algal nucleuscalled the nucleomorph between the second and third membraneof the plastid, substantiating the secondary endosymbiosis theory(Douglas et al. 2001; Wastl and Maier 2000).

The apicoplast genome itself has been reduced to just 35 kb. Itencodes some genes required for gene expression (such as ribo-somal RNAs and tRNAs), plus a few protein coding genes (Wil-son et al. 1996). Hence, almost all proteins required for themetabolic pathways of the apicoplast have been transferred tothe nucleus. These metabolic steps include reactions involved inbiosynthesis of fatty acids (Mazumdar et al. 2006; Waller et al.1998), isoprenoids (Jomaa et al. 1999), heme (Varadharajan et al.2004), and iron–sulfur clusters (Seeber 2002). As expected fromthe lack of photosynthetic pathways, the apicoplast also lacks themembranous thylakoids where photosynthesis occurs. Nonethe-less, the apicoplast is essential for the survival of both Plasmodi-

um falciparum and Toxoplasma gondii (Dahl et al. 2006; Ficheraand Roos 1997). Because the human host lacks this organelle, theapicoplast-localized prokaryotic-like pathways are consideredpotential targets for development of novel anti-parasitic drugs(Goodman and McFadden 2007; Wiesner and Jomaa 2007;Wiesner and Seeber 2005). Most of the apicomplexan parasitesthat are important to the health of humans and farm animals[which include Eimeria (Cai et al. 2003) and Theileria (Gardneret al. 2005) in addition to the parasites mentioned above], containan apicoplast. An important exception is Cryptosporidium (Zhu,Marchewka, and Keithly 2000). The large majority of work onprotein targeting to the apicoplast has been conducted in the morewell-developed experimental systems afforded by P. falciparumand T. gondii.

Sequence determinants of trafficking to the apicoplastlumen. The presence and distinct origins of the four membranesof the apicoplast pose a challenging problem to the cell. As theprotein-coding genes are nucleus encoded, apicoplast luminalproteins themselves must find their way across four membranes.Furthermore, other proteins must also reach the differentmembranes and intermembrane spaces within the apicoplast.Specific protein import machinery must exist at each membraneto facilitate transport of proteins, while allowing others to beretained. It has been hypothesized that the import machinery ofeach membrane reflects the origin of that membrane (van Doorenet al. 2001).

When the 35 kb DNA was recognized as similar to that ofchloroplasts (as opposed to mitochondria), and as residing in amultimembraned compartment, the search began for apicomplex-an genes encoding proteins homologous to those of chloroplasts.These studies exploited the nascent genome projects in T. gondii(Ajioka et al. 1998; Gajria et al. 2007; Kissinger et al. 2003) andP. falciparum (Gardner et al. 2002). Several candidate genes wereidentified, allowing experiments to identify the determinants re-quired for targeting of those proteins to the apicoplast. Notablythese proteins had N-terminal extensions as compared with theirbacterial counterparts. The N-terminal extensions commencedwith a predicted signal peptide, presumably to route the proteinsinto the endoplasmic reticulum (ER). When these extensions werefused to the 50 end of the gene-encoding green fluorescent protein(GFP) and expressed in the parasites, the resulting proteins local-ized to the apicoplast lumen (DeRocher et al. 2000; Waller et al.1998, 2000; Yung, Unnasch, and Lang-Unnasch 2001). However,when only the signal peptide region was used, the GFP fusionproteins were secreted. Furthermore, signal peptides from otherproteins of the secretory system could be substituted for those ofapicoplast proteins without affecting targeting to the organelle(Tonkin et al. 2006b). Thus it appears that the signal peptides ofapicoplast proteins function to insert those proteins into the ER.

Corresponding Author: Marilyn Parsons, Seattle Biomedical Re-search Institute, 307 Westlake Avenue N, Seattle, Washington 98109,USA—Telephone number: 1206 256 7315; FAX number: 1206 2567229; e-mail: marilyn.parsons@sbri.org

1Presentation delivered at the symposium: Cellular Compartment-alization: Protists Do It Their Way, 21–26 July 2008, The InternationalSociety of Evolutionary Protistology and The International Society ofProtistologists, Dalhousie University, Halifax, NB, Canada.

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J. Eukaryot. Microbiol., 56(3), 2009 pp. 214–220r 2009 The Author(s)Journal compilation r 2009 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2009.00405.x

Furthermore, the signal peptide is required for apicoplast target-ing, because its deletion led to cytosolic or mitochondrial local-ization of different proteins (DeRocher et al. 2000; Harb et al.2004; Waller et al. 2000; Yung et al. 2001). Thus the initial step intargeting of proteins to the apicoplast is fundamentally differentfrom that in targeting to primary plastids, in which proteins aretypically synthesized in the cytosol and routed directly into theplastid. On the other hand, this first step, entry into the ER, seemsto be a common feature for secondary plastids, whether they arethought to be derived from red alga [e.g. Apicomplexa, dinofla-gellates (Sharples et al. 1996) and stramenopiles (heterokonts)(Bhaya and Grossman 1991)] or green alga [e.g. Euglena (Sulliand Schwartzbach 1995; Sulli et al. 1999)]. This finding reinforcesthe concept that the outermost membrane of secondary plastids isrelated to that of the secretory system. In fact, the secondaryplastids of stramenopiles bear ribosomes on their outer surface,indicating that proteins are directly imported across the outerplastid membrane during synthesis (Gibbs 1979). However, ribo-somes are not detected on the outer membrane of the apicoplast.

Interestingly, the N-terminal extension present on apicoplastluminal proteins consists of more than just a signal peptide. It isfollowed by a region of variable length and sequence, which isenriched in basic amino acids and reduced for acidic amino acids.This region is similar to sequences that route proteins to thestroma of chloroplasts, dubbed transit peptides, which are alsoenriched for basic and hydroxylated amino acids (reviewed in(Bruce 2001). In P. falciparum, the AT bias of the nuclear genomemeans that most of the basic amino acids are lysine and aspara-gine, whereas in T. gondii (which lacks such an AT bias) theyare predominantly lysine and arginine (Ralph et al. 2004a). Thisregion is also required for routing proteins to the apicoplast(DeRocher et al. 2000; Waller et al. 2000) and at least one suchregion, that of T. gondii ribosomal protein S9, has been shown toallow import of a reporter protein into pea chloroplasts (DeRocheret al. 2000). In recognition of these conserved characteristics, this

portion of the apicoplast bipartite targeting sequence is also calledthe transit peptide. Mutagenesis experiments on the transit peptideof acyl carrier protein (ACP) have shown that an overall positivecharge of the transit peptide, particularly near the N-terminus, isimportant for proper targeting to the apicoplast in both P. falcip-arum and T. gondii (Foth et al. 2003; Tonkin, Roos, and McFad-den 2006a). However, the exact location of the basic residues isnot important for proper targeting. The ACP transit peptide inP. falciparum also encodes a predicted Hsp70 binding site and pointmutagenesis in this site prevented targeting to the apicoplast sug-gesting that Hsp70 acts as a molecular chaperone during proteintrafficking to the apicoplast (Foth et al. 2003). The apicomplexantransit peptides lack the hydrophobic stop-transfer sequences oftenseen on proteins destined for the three-membraned secondary plast-ids of Euglena and dinoflagellates (Patron et al. 2005; Sulli andSchwartzbach 1995; Sulli et al. 1999). The stop-transfer sequencesarrest import into the ER, so that only the transit peptide is imported.Hence these proteins traffic as single pass transmembrane proteins.In contrast proteins destined for the apicoplast lumen appear to befully imported into the ER before trafficking.

In most examples studied thus far, the predicted bipartitetrafficking sequence is sufficient to route a reporter to the apicop-last lumen. However, in the case of superoxide dismutase 2, thisbipartite sequence routes a reporter to the mitochondrion (Brydgesand Carruthers 2003). Only when the full-length protein is fusedto GFP is the protein also localized to the apicoplast (Pino et al.2007). Additional proteins, such as the thioredoxin-dependentperoxidase, aconitase, and a pyruvate kinase are also dually lo-calized to the mitochondrion and apicoplast (Pino et al. 2007;Saito et al. 2008). The apicoplast and mitochondrial pyruvate kin-ase are encoded by the same nuclear gene, but initiate translationfrom different sites (Saito et al. 2008). In other cases alternativesplicing or alternative translation start sites have been ruled out(Pino et al. 2007), suggesting that the same initial translationproduct can be routed either to the mitochondrion or to the api-

Fig. 1–3. Toxoplasma gondii and the apicoplast. 1. Transmission EM of a T. gondii tachyzoite showing the apicoplast (A), a dense granule (DG),endoplasmic reticulum (ER), Golgi (G), inner membrane complex (IMC), micronemes (Mc), mitochondrion (Mt), nucleus (N) nuclear envelope (NE),plasma membrane (PM), parasitophorous vacuole (PV), and parasitophorous vacuole membrane (PVM). Scale bar 5 500 nm. The inner membrane com-plex is homologous to the alveolae in other chromalveolates. 2. A cartoon of the apicoplast pointing out the membranes derived from the plastid (PL innerand PL outer), periplastid membrane, and outermost membrane. The proteins Tic20 and PfiTPT, which reside in the innermost membrane, are shown.Although TgATrx1, TgAPT1, and TgFtsH1 all appear to inhabit multiple compartments, their precise location within the apicoplast is not known; thereforepossible locations are indicated by gray arrows. 3. Enlargement of the apicoplast from panel 1. Bar 5 200 nm. The apicoplast in this cell is somewhatsmaller than is typically seen and may be a transverse section of an elongated apicoplast.

215PARSONS ET AL.—PROTEIN TRAFFICKING TO APICOPLAST COMPARTMENTS

coplast via the ER. The proposed model is that the signal sequenceof these proteins binds relatively poorly to the ER signal recog-nition particle, allowing some of the molecules to be fully trans-lated in the cytosol and then imported into the mitochondrion.Conversely, although some apicoplast transit peptides resemblemitochondrial-targeting sequences, the presence of a strong signalpeptide routes the proteins effectively into the ER, precludingtargeting of those proteins to the mitochondrion. This is clearlydifferent from the situation with primary plastids, where organ-elle-specific targeting is accomplished by the transit peptide itself.

Apicomplexan parasites have several unique secretory organ-elles including dense granules, micronemes, and rhoptries (seeFig. 1). Apicoplast proteins must be distinguished from proteinstargeted to these organelles, and for luminal proteins the transitpeptide fulfills that function. It is probable that the transit peptidesof apicoplast proteins have distinct functional domains (Foth et al.2003; Harb et al. 2004), reflecting their required actions at differ-ent steps of the protein trafficking process. An extensive study ofthe T. gondii ferredoxin reductase transit peptide indicated thateven though several deletions had no discernable effect onapicoplast targeting, larger deletions from the transit peptide ledto accumulation at the periphery of the apicoplast, mislocalizationto the rhoptries, or secretion. Like the transit peptides of chlorop-lasts, the apicomplexan transit peptides are cleaved to yield themature protein. A protease related to the plastid stromal process-ing peptidase has been identified in P. falciparum and this prote-ase also bears a signal and transit sequence, indicating that itresides in the apicoplast lumen (van Dooren et al. 2002).

The similarities between various apicoplast targeting sequencesfrom P. falciparum led to the development of algorithms that pre-dict whether a protein is likely to reside in the apicoplast (Fothet al. 2003; Zuegge et al. 2001). Application of one of thesealgorithms to the P. falciparum genome, coupled with knowledgeabout chloroplast metabolic pathways, led to the identification ofover 500 candidate apicoplast proteins including those participat-ing in the pathways noted earlier (Ralph et al. 2004b). As yet,relatively few have been experimentally verified to reside in theplastid. Furthermore many of the proteins predicted to be targetedto the plastid are of unknown function (i.e. hypothetical), raisingthe possibility that additional pathways may map to the organelle.

Proteins targeted to the outer compartments of the apicop-last. Very few proteins localized to the apicoplast membranes orintermembrane spaces have been identified thus far. The traffick-ing of these proteins as compared with luminal proteins is sum-marized in Table 1. Interestingly, only two of the identifiedproteins contain a typical bipartite targeting sequence: Oneis a predicted transporter of sugar phosphates and related mole-cules dubbed the P. falciparum inner membrane triose phosphatetransporter (PfiTPT) (Mullin et al. 2006). This molecule was so-named because of its similarity to translocators that reside in thechloroplast inner membrane and exchange phosphorylated C3,C5, and C6 compounds for inorganic phosphate (although the

specificity of PfiTPT has not been demonstrated). P. falciparuminner membrane triose phosphate transporter, which has multipletransmembrane domains, is processed to remove the transit pep-tide, indicating that the N-terminus of the molecule was exposedto a peptidase, most likely the stromal processing peptidase men-tioned above. This bipartite targeting sequence was able to escortGFP to the apicoplast lumen in T. gondii (Karnataki et al. 2007b).Furthermore, PfiTPT is not sensitive to exogenous protease, ar-guing that the protein is not exposed to the cytosol. The authorspropose therefore that this protein is confined to the inner mem-brane of the apicoplast (Mullin et al. 2006). The other protein isTgTic20 a homolog of a chloroplast inner membrane transloconcomponent which was identified in T. gondii (van Dooren et al.2008). The C-terminus of TgTic20 was demonstrated to be ex-posed to the lumen of the apicoplast using the split GFP system(Cabantous, Terwilliger, and Waldo 2005). Here, the C-terminalsegment of GFP was fused to the C-terminus of TgTic20, and thebulk of GFP was directed to the apicoplast lumen using theapicoplast targeting sequence of ferredoxin reductase. Althoughneither partial protein alone is fluorescent, if they are in the samecompartment they will interact to yield a fluorescent protein. Thefluorescent signal obtained indicates that the C-terminus ofTgTic20 lies within the apicoplast lumen. Because the signaland transit regions of PfiTPT and TgTic20 can confer localiza-tion of a reporter to the apicoplast lumen, these proteins arethought to traffic similarly to luminal proteins, although they areof course embedded in the membrane (Mullin et al. 2006).

Other non-luminal proteins of the apicoplast lack the typical N-terminal bipartite targeting sequence. Two examples are the pu-tative orthologues P. falciparum outer membrane triose phosphatetransporter PfoTPT (Mullin et al. 2006) and T. gondii apicoplastphosphate translocator TgAPT1 (Karnataki et al. 2007b), both ofwhich are related to PfiTPT. Like PfiTPT, the specificity of thesetranslocators has not been determined. Neither protein undergoesprocessing at the N-terminus. PfoTPT appears to be in the outerapicoplast membrane based on its sensitivity to exogenous prote-ase following recovery of organelles after hypotonic lysis ofthe parasites (Mullin et al. 2006). In contrast, TgAPT1 appearsto localize to multiple membranes of the T. gondii apicoplast, asshown by immunoelectron microscopy, although the close spac-ing of the membranes makes it difficult to determine whether itresides in all four membranes (Karnataki et al. 2007b). Althoughthe permeability of the individual membranes of the apicoplasthas not yet been explored, all of the membranes from which theyare putatively derived, with the exception of the outer envelopemembrane of the chloroplast, are impermeable to small chargedmolecules. The predicted metabolic pathways of the apicoplastindicate that several charged molecules must be imported acrossall four membranes to serve as substrates for luminal enzymes.Hence it is likely that TgAPT1 populates multiple membranes toprovide the transport functions that are accomplished by the tworelated transporters in P. falciparum.

We identified another apicoplast integral membrane protein,TgFtsH1, in T. gondii and a related FtsH in P. falciparum(Karnataki et al. 2007a). FtsH family members are membrane-bound zinc metalloproteases that are found in bacteria, mi-tochondria, and chloroplasts and degrade mis-folded membraneproteins (the abbreviation Fts is derived from the filamentoustemperature sensitive phenotype of bacterial mutants). LikeTgAPT1 and PfoTPT, TgFtsH1 lacks the typical bipartite target-ing sequence; however, unlike those proteins, TgFtsH1 undergoesprocessing at both the N- and C-termini (Karnataki et al. in press),and has only a single transmembrane domain. Immunoelectronmicroscopy studies of this integral membrane protease show thatTgFtsH1 also likely populates multiple membranes. These find-ings raise the question: how are some molecules targeted to mul-

Table 1. Sorting motifs and trafficking of apicoplast proteins.

Luminalproteins

Innermembraneproteins

Proteinsof outercompartments

Signal sequence N-terminal N-terminal InternalTransit peptide Yes Yes NoSeen in vesicles Unknown Unknown Yes (T. gondii)Traffics through Golgi No Not tested Not testedTrafficking modulated

during plastid cycleYes

(P. falciparum)Not tested Yes (T. gondii)

T. gondii, Toxoplasma gondii.

216 J. EUKARYOT. MICROBIOL., 56, NO. 3, MAY–JUNE 2009

tiple membranes whereas others appear to be restricted to specificmembranes?

Recent studies from our laboratory show that a thioredoxin-likeprotein resides in multiple intermembrane spaces of the apicoplast(DeRocher et al. 2008) (Fig. 4). Thioredoxins are involved in re-dox homeostasis in multiple cellular compartments and inchloroplasts they have been shown to bind proteins involved ina variety of biochemical pathways including fatty acid, is-oprenoid, and heme biosynthesis (Balmer et al. 2003). In the pho-tosynthetic cyanobacterium Synechocystis, the major thioredoxintargets appear to be membrane proteins (Mata-Cabana, Florencio,and Lindahl 2007). The T. gondii apicoplast thioredoxin(TgATrx1), is in part soluble and in part peripherally associatedwith membranes (DeRocher et al. 2008). Although TgATrx1 lacksthe canonical N-terminal bipartite targeting sequence, it does havea predicted N-terminal signal anchor sequence, which could routethe protein into the ER. Indeed, the region containing the signalanchor sequence is required for targeting to the apicoplast. Dele-tion analysis shows that sequences within a 160 aa region down-stream of the signal anchor are also required for proper targeting.This region does not closely resemble a transit peptide, so furtherexperimental work will be required to determine the specific mo-tifs that provide for apicoplast localization. Nonetheless, the tworegions together, when fused to GFP, route the fusion protein to adonut-shaped region around the apicoplast lumen; the sameimmunofluorescence pattern seen for the native TgATrx1. Thispattern is therefore unlike that seen when GFP is fused to the sig-nal and transit peptides of luminal proteins. Hence this targetingsequence is functionally distinct from those of apicoplast luminalproteins.

Although the signal and transit peptide of PfiTPT and TgTic20are presumably required for localization of the molecule to theplastid and appear to be functionally the same as luminal targetingsequences, the data obtained thus far suggest that the sequencesthat targets protein to the outer membranes of the apicoplast aretotally different. The algorithms designed to identify the apicop-last proteins do not recognize these proteins as being apicoplast-targeted. Motifs that are sufficient for proper trafficking ofTgAPT1 and its homologue PfoTPT, as well as TgFtsH1, havenot been identified. Both the single transmembrane domain andthe peptidase domain of TgFtsH1 are required for proper local-ization, because their deletion leads to cytosolic or ER localiza-tion, respectively (Karnataki et al. 2007a), but there is no evidencethat they are sufficient. There are no obvious similarities between

the transporters and the protease that enable sequence alignmentor suggest a common motif for targeting. This is perhaps not sur-prising given the very different predicted structures and propertiesof the molecules.

Proteins associated with apicoplast membranes are presentin vesicles. Immunoelectron microscopy experiments with anti-bodies directed against epitope-tagged TgFtsH1, TgAPT1, andTgATrx1 (expression was driven by the cognate promoters)showed labeling not only of the peripheral compartments of theapicoplast, but also of spherical vesicles (DeRocher et al. 2008;Karnataki et al. 2007a, b). In particular, C-terminally taggedTgATrx1 identifies an abundant set of vesicles on immunoelec-tron microscopy (Fig. 5). The preponderance of the gold particlesmarking the locations of TgAPT1 and TgFtsH1 were localizedclose to membranes in the vesicles, suggesting that are routed asintegral membrane proteins (Karnataki et al. 2007a, b). Further-more TgATrx1, the protein that eventually is localized to inter-membrane spaces, is also predominantly adjacent to membraneswhile residing in the vesicles (DeRocher et al. 2008). This sug-gests that although TgATrx1 ultimately behaves as a soluble orperipheral membrane protein, in vesicles it is closely associatedwith the vesicle membrane. In thin sections, the vesicles took upapproximately 3% of the surface area of the parasite. Morpholog-ically similar vesicles can be seen in untransfected cells, indicat-ing they are not an artifact of overexpression. In retrospect, thevesicles were readily observed on immunofluorescence analysesas apparent tubules and vesicles extending from the apicoplast atthe time of apicoplast elongation. We have suggested that theseare apicoplast-specific transport vesicles. However, no immuno-fluorescence or immunoelectron microscopy evidence has beenpublished that shows the association of luminal proteins withvesicles.

Mechanisms of trafficking: from the ER to apicoplast. Aswith luminal proteins, the first step in trafficking proteins local-ized to the outer compartments of the apicoplast appears to beentry into the ER. The data supporting this contention include thefact that certain deletion mutants of TgFtsH1 and TgAPT1 areretained in the ER (Karnataki et al. 2007a) (ADR., unpubl. data).Full-length epitope-tagged versions of these proteins also showsome ER localization, the extent of which varies during the plastiddivision cycle in T. gondii. Furthermore, as mentioned above, de-letion of the sole transmembrane domain of FtsH1 leads tocytosolic localization, indicating that the transmembrane domainlikely functions as an internal signal sequence.

Fig. 4,5. Immunoelectron microscopy of TgATrx1. Cells transfected with TgATrx1 tagged with HA epitope tags at the C-terminus were analyzedwith anti-HA monoclonal antibody followed by protein A gold. 4. Apicoplast showing labeling on multiple membranes. Scale bar 5 100 nm. 5. TgATrx1present on apicoplast (a) and abundant vesicles (v). Arrows indicate site where vesicle appears to be fusing with outer membrane of the apicoplast. Scalebar 5 200 nm. This figure is reproduced from (DeRocher et al. 2008) r American Society for Microbiology.

217PARSONS ET AL.—PROTEIN TRAFFICKING TO APICOPLAST COMPARTMENTS

Several potential trafficking routes from the ER have been pro-posed for proteins destined for the apicoplast; these differ in themechanism of trafficking from the ER and the location of sortingfrom other proteins of the secretory system. In brief, they include(1) trafficking via a direct connection with the ER, with proteinsorting at the apicoplast; (2) vesicular trafficking directly to theGolgi apparatus, followed by sorting; (3) vesicular trafficking di-rectly to the apicoplast, followed by sorting; and (4) sorting at theER, followed by vesicular trafficking to the apicoplast. The firstpossibility proposes that the outermost apicoplast membrane isdistinct yet contiguous with the ER, as it is in the secondary plast-ids of several alga (Gibbs 1979). In this model, apicoplast proteinswould be retained at the apicoplast while other secretory proteinswould move on to the Golgi. In Apicomplexa, the lack of ribo-somes and the presence of a plastid-specific transporter protein onthe outermost membrane of the apicoplast (e.g. PfoTPT) clearlyindicate a functional segregation of this membrane from the ER.No physical connections between the ER and the apicoplast havebeen observed upon electron microscopy, although it is possiblethese connections are rare and/or transient. The presence of thespherical vesicles bearing TgATrx1 or the apicoplast membraneproteins is harder to reconcile with this model, but it could be thatthey represent enlarged, specialized regions of the ER. It is alsopossible they represent protein being trafficked from the apicop-last to some unidentified hydrolytic compartment. In this model,as well as the third model, which also proposes sorting of proteinsat the apicoplast, one might predict that COPII vesicles, whichtransport proteins to the Golgi, would be formed in the vicinity ofthe apicoplast. Although this has not been studied, COPI, whichmediates retrograde trafficking, and the retrograde traffickingreceptor ERGIC2, are detected at the Golgi and ER, but not atthe apicoplast (Hager et al. 1999; Pfluger et al. 2005). This pro-vides some circumstantial evidence that trafficking from the ER tothe Golgi bypasses the apicoplast.

The last three models all involve vesicular trafficking from theER but differ as to the location of sorting of apicoplast-targetedproteins from those addressed to other locations in the secretorysystem. Hence, the vesicles bearing the apicoplast membrane pro-teins would provide for transport to the apicoplast. A priori, sort-ing at the level of the Golgi would seem most likely because thatis where most sorting of newly synthesized proteins of the secre-tory system occurs. However, the trafficking of apicoplast luminalproteins is insensitive to the Golgi inhibitor brefeldin A in both T.gondii (DeRocher et al. 2005) and P. falciparum (Tonkin et al.2006b). In T. gondii, it is also insensitive to low temperature,which blocks trafficking to the Golgi apparatus (DeRocher et al.2005). Although similar experiments on membrane proteins havenot yet been published, these studies argue against sorting in theGolgi apparatus (model 2). One caveat is that all of the studiesluminal protein trafficking were performed using heterologouspromoters, which might alter the timing of expression, and hencethe availability of particular trafficking pathways to the newlysynthesized proteins.

In the third model, the vesicles bearing apicoplast membraneproteins are not apicoplast specific, but rather represent the firststep in trafficking of all secretory molecules (Tonkin, Kalanon,and McFadden 2007). The insensitivity of luminal proteintrafficking to brefeldin A may argue against this model, as maythe observed location of retrograde trafficking moleculesdescribed above. However, as yet direct experimental evidencedoes not distinguish this model from the fourth model where pro-tein sorting occurs in the ER via the formation of apicoplast-specific and Golgi-specific vesicles.

With respect to all of the models invoking vesicular trafficking,it should be noted that vesicles bearing proteins targeted to thelumen or innermost membrane of the apicoplast have not been

reported. Furthermore, the tubular/vesicular staining so easilyseen for membrane proteins in immunofluorescence experimentsis not observed for luminal proteins (although most studies usedheterologous promoters). It is also possible that there is more thanone way to reach the apicoplast. Nonetheless, if the vesicles bear-ing membrane proteins are indeed targeting to the apicoplast,these vesicles must bear distinct molecules that allow the vesiclesand target membranes to identify one another and fuse. Such mol-ecules could include the small GTP-binding proteins (Rabs),and SNARES, which facilitate membrane fusion. Although it isnot certain that similar molecules will be involved in the traffick-ing of the identified vesicles, it appears to be a good place to startlooking.

Endoplasmic reticulum to apicoplast trafficking of membraneproteins is regulated during the cell cycle in T. gondii, increasingat the time of apicoplast elongation. Furthermore, we observed alarger proportion of ER-localized TgAPT1 when the protein wasexpressed from the TgDHFR promoter as opposed to its own pro-moter, suggesting that timing of expression could be important(the levels of protein expression were somewhat lower than withthe TgAPT1 promoter) (Karnataki et al. 2007b). An earlier studyin P. falciparum using promoters differentially regulated duringthe erythrocytic cell cycle showed that GFP bearing an apicoplasttargeting sequence was secreted in early stages (rings) but local-ized to the apicoplast in the later stages (trophozoites) (Chereshet al. 2002). Furthermore, in P. falciparum, transcripts encodingapicoplast targeted proteins are coordinately expressed during thetrophozoite stage (Bozdech et al. 2003). Together these datasuggest the capacity to transport proteins to the apicoplast is reg-ulated during the plastid division cycle in Apicomplexa. However,these studies address somewhat different steps in trafficking,because the membrane protein TgAPT1 was retained in theER whereas the luminal protein escaped the ER but was mislo-calized. This difference could reflect a difference in species, inhow membrane and soluble proteins are handled by the parasites,or in the pathways the individual proteins need to follow. Forexample, the ability of proteins to unfold or fold could be impor-tant in trafficking.

Protein trafficking within apicoplast. Once proteins havepassed the first membrane of the apicoplast, they now must crossthree membranes to reach the apicoplast lumen. According to thesecondary endosymbiosis hypothesis, the two inner membraneswould contain homologues to the translocons of the inner andouter membranes of the chloroplast [Tic and Toc, respectively,reviewed in (Soll and Schleiff 2004)]. As noted above, a homo-logue of Tic20 has been identified in T. gondii and regulatedknockouts of Tic20 show that the protein is required for import ofproteins to the lumen of the apicoplast (van Dooren et al. 2008). Acandidate Tic22 is also present in the genomes of both T. gondiiand P. falciparum, suggesting the presence of a Tic-related com-plex in the inner membrane of the apicoplast. The Toc complexincludes receptors for the transit peptides, which contain a char-acteristic GTP-binding motif and a trans-membrane pore com-prised of the beta-barrel protein Toc75. Thus far no homologuesof proteins of the Toc apparatus have been identified from thecompleted genome sequences of T. gondii or P. falciparum, leav-ing open the question of how proteins traverse this membrane.Indeed proteins of the Toc complex remain to be discovered inother secondary plastids (McFadden and van Dooren 2004).

How then do proteins cross the periplastid and plastid outermembranes of the apicoplast? One set of candidates are homo-logues of the ER-associated degradation (ERAD) pathway, whichtransports misfolded proteins from the ER back to the cytosol forproteasomal degradation. Chromalveolates, cousins of apicom-plexans, have duplicated, diverged copies of much of their ERADmachinery. Several of these duplicate proteins are in the peri-

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plastid compartment of the red alga Phaeodactylum tricornuntumsuggesting that they comprise the translocon across the third, peri-plastid membrane (Sommer et al. 2007). Furthermore, some ofthese proteins, including Der1- and Cdc48- related proteins, havehomologues in apicomplexans where they have been proposed toact as translocons across both the second and third apicoplastmembranes (Sommer et al. 2007; Tonkin et al. 2007).

Perspectives. Plastids have been co-opted by other organismsmany times during evolution. The apicoplast falls near the end ofthe continuum from endosymbiosis to organelle to genome-freerelict. In the dozen years since the apicoplast was identified, stud-ies conducted in several laboratories have significantly contrib-uted to our understanding of this organelle, which is essential forseveral human and veterinary pathogens. Deciphering the signalsthat route proteins to the apicoplast allowed the identificationof candidate proteomes for the plastids of P. falciparum, andT. gondii, and hence potential drug targets. Much work is yet to bedone to fully understand trafficking to this organelle. For example,all proteins destined for the apicoplast are first imported into theER, but how they are directed from the ER to the plastid remainsunsolved. Several models have been proposed but understandingwhat really happens awaits experimental evidence. A transitpeptide domain marks proteins destined for the apicoplast lumenbut what signals direct membrane proteins to the apicoplast?Characterization of such sequences might lead to bioinformaticidentification of proteins involved in metabolic pathways or pro-cesses unique to the organelle. Next, how are these signals rec-ognized? We are now getting glimpses of how luminal proteinsare escorted across multiple membranes to their final destination,but still do not understand how proteins are routed to specificmembranes. A related question is: to what extent do the four api-coplast membranes differ in protein composition, and, by exten-sion, function? All of the protein transport processes are expectedto be essential to the parasite. Some may be shared between api-coplasts and the chloroplasts of chromists, so cross-fertilizationwill surely continue. Even though we have made excellent prog-ress in furthering our understanding of this important organellethere are still several secrets enclosed within the four membranesof the apicoplast.

ACKNOWLEDGMENT

This work was supported by NIH R01AI50506. The content issolely the responsibility of the authors.

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Received: 10/07/08, 01/31/09; accepted: 02/01/09

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