Protein Traffic to the Plasmodium falciparum Apicoplast: Evidence for a Sorting Branch Point at the Golgi

Plasmodium falciparum, similar to many other apicomplexan parasites, contains an apicoplast, a plastid organelle of secondary endosymbiotic origin. Nuclear‐encoded proteins are targeted to the apicoplast by a bipartite topogenic signal consisting of (i) an endoplasmic reticulum (ER)‐type N‐terminal secretory signal peptide, followed by (ii) a plant‐like transit peptide. Although the signals responsible for transport of most proteins to the apicoplast are well described, the route of trafficking from the ER to the outermost apicoplast membrane is still a matter of debate. Current models of trafficking to the apicoplast suggest that proteins destined for this organelle are, on entry into the lumen of the ER, diverted from the default secretory pathway to a specialized vesicular system which carries proteins directly from the ER to the outer apicoplast membrane. Here, we have re‐examined this trafficking pathway. By titrating wild‐type and mutant apicoplast transit peptides against different ER retrieval sequences and studying protein transport in a brefeldin A‐resistant parasite line, we generated data which suggest a direct involvement of the Golgi in traffic of soluble proteins to the P. falciparum apicoplast.     

Phosphatidylinositol 3-monophosphate is involved in toxoplasma apicoplast biogenesis

Apicomplexan parasites cause devastating diseases including malaria and toxoplasmosis. They harbour a plastid-like, non-photosynthetic organelle of algal origin, the apicoplast, which fulfils critical functions for parasite survival. Because of its essential and original metabolic pathways, the apicoplast has become a target for the development of new anti-apicomplexan drugs. Here we show that the lipid phosphatidylinositol 3-monophosphate (PI3P) is involved in apicoplast biogenesis in Toxoplasma gondii. In yeast and mammalian cells, PI3P is concentrated on early endosomes and regulates trafficking of endosomal compartments. Imaging of PI3P in T. gondii showed that the lipid was associated with the apicoplast and apicoplast protein-shuttling vesicles. Interference with regular PI3P function by over-expression of a PI3P specific binding module in the parasite led to the accumulation of vesicles containing apicoplast peripheral membrane proteins around the apicoplast and, ultimately, to the loss of the organelle. Accordingly, inhibition of the PI3P-synthesising kinase interfered with apicoplast biogenesis. These findings point to an unexpected implication for this ubiquitous lipid and open new perspectives on how nuclear encoded proteins traffic to the apicoplast. This study also highlights the possibility of developing specific pharmacological inhibitors of the parasite PI3-kinase as novel anti-apicomplexan drugs.     

Protein targeting to the malaria parasite plastid

The relict plastid, or apicoplast, of the malaria parasite Plasmodium falciparum is an essential organelle and a promising drug target. Most apicoplast proteins are nuclear encoded and post-translationally targeted into the organelle using a bipartite N-terminal extension, consisting of a typical endomembrane signal peptide and a plant-like transit peptide. Apicoplast protein targeting commences through the parasite's secretory pathway. We review recent experimental evidence suggesting that the apicoplast resides in the mainstream endomembrane system proximal to the Golgi. Further, we explore possible mechanisms for translocation of nuclear-encoded apicoplast proteins across the four bounding membranes. Recent insights into the composition of the transit peptide and how it is cleaved and degraded after use are also examined. Characterization of apicoplast targeting has not only shed light on how this group of parasites mediate intracellular protein trafficking events but also it has helped identify new targets for therapeutics. The distinctive leader sequences of apicoplast proteins make them readily identifiable, allowing assembly of a virtual organelle metabolome from the genome. Such analysis has lead to the identification of several biochemical pathways that are absent from the human host and thus represent novel therapeutic targets for parasitic infection.     

Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption

Toxoplasma gondii and malaria parasites contain a unique and essential relict plastid called the apicoplast. Most apicoplast proteins are encoded in the nucleus and are transported to the organelle via the endoplasmic reticulum (ER). Three trafficking routes have been proposed for apicoplast membrane proteins: (i) vesicular trafficking from the ER to the Golgi and then to the apicoplast, (ii) contiguity between the ER membrane and the apicoplast allowing direct flow of proteins, and (iii) vesicular transport directly from the ER to the apicoplast. Previously, we identified a set of membrane proteins of the T. gondii apicoplast which were also detected in large vesicles near the organelle. Data presented here show that the large vesicles bearing apicoplast membrane proteins are not the major carriers of luminal proteins. The vesicles continue to appear in parasites which have lost their plastid due to mis-segregation, indicating that the vesicles are not derived from the apicoplast. To test for a role of the Golgi body in vesicle formation, parasites were treated with brefeldin A or transiently transfected with a dominant-negative mutant of Sar1, a GTPase required for ER to Golgi trafficking. The immunofluorescence patterns showed little change. These findings were confirmed using stable transfectants, which expressed the toxic dominant-negative sar1 following Cre-loxP mediated promoter juxtaposition. Our data support the hypothesis that the large vesicles do not mediate the trafficking of luminal proteins to the apicoplast. The results further show that the large vesicles bearing apicoplast membrane proteins continue to be observed in the absence of Golgi and plastid function. These data raise the possibility that the apicoplast proteome is generated by two novel ER to plastid trafficking pathways, plus the small set of proteins encoded by the apicoplast genome.     

Protein targeting to destinations of the secretory pathway in the malaria parasite Plasmodium falciparum

The secretory pathway in the malaria parasite Plasmodium falciparum has many unique aspects in terms of protein destinations and trafficking mechanisms. Recently, several exciting insights into protein trafficking within this intracellular parasite have been unveiled: these include signals that are required for targeting of proteins to the red blood cell and the relict plastid (known as the apicoplast); and the elucidation of the pathways of the haemoglobin proteases targeted to the food vacuole. Protein-targeting to the apical organelles in P. falciparum, however, is still not very well understood, but available research offers a tantalising glimpse of the system.     

Toxoplasma gondii Toc75 Functions in Import of Stromal but not Peripheral Apicoplast Proteins

Apicomplexa are unicellular parasites causing important human and animal diseases, including malaria and toxoplasmosis. Most of these pathogens possess a relict but essential plastid, the apicoplast. The apicoplast was acquired by secondary endosymbiosis between a red alga and a flagellated eukaryotic protist. As a result the apicoplast is surrounded by four membranes. This complex structure necessitates a system of transport signals and translocons allowing nuclear encoded proteins to find their way to specific apicoplast sub‐compartments. Previous studies identified translocons traversing two of the four apicoplast membranes. Here we provide functional support for the role of an apicomplexan Toc75 homolog in apicoplast protein transport. We identify two apicomplexan genes encoding Toc75 and Sam50, both members of the Omp85 protein family. We localize the respective proteins to the apicoplast and the mitochondrion of Toxoplasma and Plasmodium. We show that the Toxoplasma Toc75 is essential for parasite growth and that its depletion results in a rapid defect in the import of apicoplast stromal proteins while the import of proteins of the outer compartments is affected only as the secondary consequence of organelle loss. These observations along with the homology to Toc75 suggest a potential role in transport through the second innermost membrane.     

Evidence for Golgi-independent transport from the early secretory pathway to the plastid in malaria parasites

The malaria parasite Plasmodium falciparum harbours a relict plastid (termed the apicoplast) that has evolved by secondary endosymbiosis. The apicoplast is surrounded by four membranes, the outermost of which is believed to be part of the endomembrane system. Nuclear-encoded apicoplast proteins have a two-part N-terminal extension that is necessary and sufficient for translocation across these four membranes. The first domain of this N-terminal extension resembles a classical signal peptide and mediates translocation into the secretory pathway, whereas the second domain is homologous to plant chloroplast transit peptides and is required for the remaining steps of apicoplast targeting. We explored the initial, secretory pathway component of this targeting process using green fluorescent reporter protein constructs with modified leaders. We exchanged the apicoplast signal peptide with signal peptides from other secretory proteins and observed correct targeting, demonstrating that apicoplast targeting is initiated at the general secretory pathway of P. falciparum. Furthermore, we demonstrate by immunofluorescent labelling that the apicoplast resides on a small extension of the endoplasmic reticulum (ER) that is separate from the cis-Golgi. To define the position of the apicoplast in the endomembrane pathway in relation to the Golgi we tracked apicoplast protein targeting in the presence of the secretory inhibitor Brefeldin A (BFA), which blocks traffic between the ER and Golgi. We observe apicoplast targeting in the presence of BFA despite clear perturbation of ER to Golgi traffic by the inhibitor, which suggests that the apicoplast resides upstream of the cis-Golgi in the parasite's endomembrane system. The addition of an ER retrieval signal (SDEL) - a sequence recognized by the cis-Golgi protein ERD2 - to the C-terminus of an apicoplast-targeted protein did not markedly affect apicoplast targeting, further demonstrating that the apicoplast is upstream of the Golgi. Apicoplast transit peptides are thus dominant over an ER retention signal. However, when the transit peptide is rendered non-functional (by two point mutations or by complete deletion) SDEL-specific ER retrieval takes over, and the fusion protein is localized to the ER. We speculate either that the apicoplast in P. falciparum resides within the ER directly in the path of the general secretory pathway, or that vesicular trafficking to the apicoplast directly exits the ER.     

Positioning of large organelles by a membrane‐ associated cytoskeleton in Plasmodium sporozoites

Cellular organelles are usually linked to the cytoskeleton, which often provides a scaffold for organelle function. In malaria parasites, no link between the cytoskeleton and the major organelles is known. Here we show that during fast, stop‐and‐go motion of Plasmodium sporozoites, all organelles stay largely fixed in respect to the moving parasite. Cryogenic electron tomography reveals that the nucleus, mitochondrion, apicoplast and the microtubules of Plasmodium sporozoites are linked to the parasite pellicle via long tethering proteins. These tethers originate from the inner membrane complex and are arranged in a periodic fashion following a 32 nm repeat. The tethers pass through a subpellicular structure that encompasses the entire parasite, probably as a network of membrane‐associated filaments. While the spatial organization of the large parasite organelles appears dependent on their linkage to the cortex, the specialized secretory vesicles are mostly not linked to microtubules or other cellular structures that could provide support for movement.     

The chaperonin TRiC forms an oligomeric complex in the malaria parasite cytosol

The malaria parasite exports numerous proteins into its host red blood cell (RBC). The trafficking of these exported effectors is complex. Proteins are first routed through the secretory system, into the parasitophorous vacuole (PV), a membranous compartment enclosing the parasite. Proteins are then translocated across the PV membrane in a process requiring ATP and unfolding. Once in the RBC compartment the exported proteins are then refolded and further trafficked to their final localizations. Chaperones are important in the unfolding and refolding processes. Recently, it was suggested that the parasite TRiC chaperonin complex is exported, and that it is involved in trafficking of exported effectors. Using a parasite‐specific antibody and epitope‐tagged transgenic parasites we could observe no export of Plasmodium TRiC into the RBC. We tested the importance of the parasite TRiC by creating a regulatable knockdown line of the TRiC‐θ subunit. Loss of the parasite TRiC‐θ led to a severe growth defect in asexual development, but did not alter protein export into the RBC. These observations indicate that the TRiC proteins play a critical role in parasite biology, though their function, within the parasite, appears unrelated to protein trafficking in the RBC compartment.     

Replication and partitioning of the apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase

Apicomplexans are the causative agents of numerous important infectious diseases including malaria and toxoplasmosis. Most of them harbour a chloroplast-like organelle called the apicoplast that is essential for the parasites’ metabolism and survival. While most apicoplast proteins are nuclear encoded, the organelle also maintains its own genome, a 35 kb circle. In this study we used Toxoplasma gondii to identify and characterise essential proteins involved in apicoplast genome replication and to understand how apicoplast genome segregation unfolds over time. We demonstrated that the DNA replication enzymes Prex, DNA gyrase and DNA single stranded binding protein localise to the apicoplast. We show in knockdown experiments that apicoplast DNA Gyrase A and B, and Prex are required for apicoplast genome replication and growth of the parasite. Analysis of apicoplast genome replication by structured illumination microscopy in T. gondii tachyzoites showed that apicoplast nucleoid division and segregation initiate at the beginning of S phase and conclude during mitosis. Thus, the replication and division of the apicoplast nucleoid is highly coordinated with nuclear genome replication and mitosis. Our observations highlight essential components of apicoplast genome maintenance and shed light on the timing of this process in the context of the overall parasite cell cycle.     

15-deoxyspergualin primarily targets the trafficking of apicoplast proteins in Plasmodium falciparum

15-Deoxyspergualin, an immunosuppressant with tumoricidal and antimalarial properties, has been implicated in the inhibition of a diverse array of cellular processes including polyamine synthesis and protein synthesis. Endeavoring to identify the mechanism of antimalarial action of this molecule, we examined its effect on Plasmodium falciparum protein synthesis, polyamine biosynthesis, and transport. 15-Deoxyspergualin stalled protein synthesis in P. falciparum through Hsp70 sequestration and subsequent phosphorylation of the eukaryotic initiation factor eIF2alpha. However, protein synthesis inhibition as well as polyamine depletion were invoked only by high micromolar concentrations of 15-deoxyspergualin, in contrast to the submicromolar concentrations sufficient to inhibit parasite growth. Further investigations demonstrated that 15-deoxyspergualin in the malaria parasite primarily targets the hitherto underexplored process of trafficking of nucleus-encoded proteins to the apicoplast. Our finding that 15-deoxyspergualin kills the malaria parasite by interfering with targeting of nucleus-encoded proteins to the apicoplast not only exposes a chink in the armor of the malaria parasite, but also reveals new realms in our endeavors to study this intriguing biological process.     

Molecular identification of a malaria merozoite surface sheddase

Proteolytic shedding of surface proteins during invasion by apicomplexan parasites is a widespread phenomenon, thought to represent a mechanism by which the parasites disengage adhesin-receptor complexes in order to gain entry into their host cell. Erythrocyte invasion by merozoites of the malaria parasite Plasmodium falciparum requires the shedding of ectodomain components of two essential surface proteins, called MSP1 and AMA1. Both are released by the same merozoite surface "sheddase," but the molecular identity and mode of action of this protease is unknown. Here we identify it as PfSUB2, an integral membrane subtilisin-like protease (subtilase). We show that PfSUB2 is stored in apical secretory organelles called micronemes. Upon merozoite release it is secreted onto the parasite surface and translocates to its posterior pole in an actin-dependent manner, a trafficking pattern predicted of the sheddase. Subtilase propeptides are usually selective inhibitors of their cognate protease, and the PfSUB2 propeptide is no exception; we show that recombinant PfSUB2 propeptide binds specifically to mature parasite-derived PfSUB2 and is a potent, selective inhibitor of MSP1 and AMA1 shedding, directly establishing PfSUB2 as the sheddase. PfSUB2 is a new potential target for drugs designed to prevent erythrocyte invasion by the malaria parasite.     

Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii

The apicoplast is a distinctive organelle associated with apicomplexan parasites, including Plasmodium sp. (which cause malaria) and Toxoplasma gondii (the causative agent of toxoplasmosis). This unusual structure (acquired by the engulfment of an ancestral alga and retention of the algal plastid) is essential for long-term parasite survival. Similar to other endosymbiotic organelles (mitochondria, chloroplasts), the apicoplast contains proteins that are encoded in the nucleus and post-translationally imported. Translocation across the four membranes surrounding the apicoplast is mediated by an N-terminal bipartite targeting sequence. Previous studies have described a recombinant "poison" that blocks plastid segregation during mitosis, producing parasites that lack an apicoplast and siblings containing a gigantic, nonsegregating plastid. To learn more about this remarkable phenomenon, we examined the localization and processing of the protein produced by this construct. Taking advantage of the ability to isolate apicoplast segregation mutants, we also demonstrated that processing of the transit peptide of nuclear-encoded apicoplast proteins requires plastid-associated activity.     

Traffic jams: protein transport in Plasmodium falciparum

Protein targeting in malaria parasites is a complex process, involving several cellular compartments that distinguish these cells from more familiar systems, such as yeast or mammals. At least a dozen distinct protein destinations are known. The best studied of these is the vestigial chloroplast (the apicoplast), but new tools promise rapid progress in understanding how Plasmodium falciparum and related apicomplexan parasites traffic proteins to their invasion-related organelles, and how they modify the host by trafficking proteins into its cytoplasm and plasma membrane. Here, Giel van Dooren and colleagues discuss recent insights into protein targeting via the secretory pathway in this fascinating and important system. This topic emerged as a major theme at the Molecular Approaches to Malaria conference, Lorne, Australia, 2-5 February 2000.     

An Apicoplast Localized Ubiquitylation System Is Required for the Import of Nuclear-encoded Plastid Proteins

Apicomplexan parasites are responsible for numerous important human diseases including toxoplasmosis, cryptosporidiosis, and most importantly malaria. There is a constant need for new antimalarials, and one of most keenly pursued drug targets is an ancient algal endosymbiont, the apicoplast. The apicoplast is essential for parasite survival, and several aspects of its metabolism and maintenance have been validated as targets of anti-parasitic drug treatment. Most apicoplast proteins are nuclear encoded and have to be imported into the organelle. Recently, a protein translocon typically required for endoplasmic reticulum associated protein degradation (ERAD) has been proposed to act in apicoplast protein import. Here, we show ubiquitylation to be a conserved and essential component of this process. We identify apicoplast localized ubiquitin activating, conjugating and ligating enzymes in Toxoplasma gondii and Plasmodium falciparum and observe biochemical activity by in vitro reconstitution. Using conditional gene ablation and complementation analysis we link this activity to apicoplast protein import and parasite survival. Our studies suggest ubiquitylation to be a mechanistic requirement of apicoplast protein import independent to the proteasomal degradation pathway.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Toxoplasma gondii myosin F,an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so‐called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin‐based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation.     

An apicoplast‐resident folate transporter is essential for sporogony of malaria parasites

Malaria parasites are fast replicating unicellular organisms and require substantial amounts of folate for DNA synthesis. Despite the central role of this critical co‐factor for parasite survival, only little is known about intraparasitic folate trafficking in Plasmodium. Here, we report on the expression, subcellular localisation and function of the parasite's folate transporter 2 (FT2) during life cycle progression in the murine malaria parasite Plasmodium berghei. Using live fluorescence microscopy of genetically engineered parasites, we demonstrate that FT2 localises to the apicoplast. In invasive P. berghei stages, a fraction of FT2 is also observed at the apical end. Upon genetic disruption of FT2, blood and liver infection, gametocyte production and mosquito colonisation remain unaltered. But in the Anopheles vector, FT2‐deficient parasites develop inflated oocysts with unusual pulp formation consisting of numerous single‐membrane vesicles, which ultimately fuse to form large cavities. Ultrastructural analysis suggests that this defect reflects aberrant sporoblast formation caused by abnormal vesicular traffic. Complete sporogony in FT2‐deficient oocysts is very rare, and mutant sporozoites fail to establish hepatocyte infection, resulting in a complete block of parasite transmission. Our findings reveal a previously unrecognised organellar folate transporter that exerts critical roles for pathogen maturation in the arthropod vector.     

The Suf Iron-Sulfur Cluster Synthesis Pathway Is Required for Apicoplast Maintenance in Malaria Parasites

The apicoplast organelle of the malaria parasite Plasmodium falciparum contains metabolic pathways critical for liver-stage and blood-stage development. During the blood stages, parasites lacking an apicoplast can grow in the presence of isopentenyl pyrophosphate (IPP), demonstrating that isoprenoids are the only metabolites produced in the apicoplast which are needed outside of the organelle. Two of the isoprenoid biosynthesis enzymes are predicted to rely on iron-sulfur (FeS) cluster cofactors, however, little is known about FeS cluster synthesis in the parasite or the roles that FeS cluster proteins play in parasite biology. We investigated two putative FeS cluster synthesis pathways (Isc and Suf) focusing on the initial step of sulfur acquisition. In other eukaryotes, these proteins can be located in multiple subcellular compartments, raising the possibility of cross-talk between the pathways or redundant functions. In P. falciparum, SufS and its partner SufE were found exclusively the apicoplast and SufS was shown to have cysteine desulfurase activity in a complementation assay. IscS and its effector Isd11 were solely mitochondrial, suggesting that the Isc pathway cannot contribute to apicoplast FeS cluster synthesis. The Suf pathway was disrupted with a dominant negative mutant resulting in parasites that were only viable when supplemented with IPP. These parasites lacked the apicoplast organelle and its organellar genome – a phenotype not observed when isoprenoid biosynthesis was specifically inhibited with fosmidomycin. Taken together, these results demonstrate that the Suf pathway is essential for parasite survival and has a fundamental role in maintaining the apicoplast organelle in addition to any role in isoprenoid biosynthesis.