The Experts below are selected from a list of 3138 Experts worldwide ranked by ideXlab platform
Geoffrey I Mcfadden - One of the best experts on this subject based on the ideXlab platform.
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a genetic screen in rodent malaria parasites identifies five new Apicoplast putative membrane transporters one of which is essential in human malaria parasites
Cellular Microbiology, 2018Co-Authors: Claire P. Sayers, Geoffrey I Mcfadden, Vanessa Mollard, Hayley D Buchanan, Christopher D. GoodmanAbstract:The malaria-causing parasite, Plasmodium, contains a unique non-photosynthetic plastid known as the Apicoplast. The Apicoplast is an essential organelle bound by four membranes. Although membrane transporters are attractive drug targets, only two transporters have been characterised in the malaria parasite Apicoplast membranes. We selected 27 candidate Apicoplast membrane proteins, 20 of which are annotated as putative membrane transporters, and performed a genetic screen in Plasmodium berghei to determine blood stage essentiality and subcellular localisation. Eight apparently essential blood stage genes were identified, three of which were Apicoplast-localised: PbANKA_0614600 (DMT2), PbANKA_0401200 (ABCB4), and PbANKA_0505500. Nineteen candidates could be deleted at the blood stage, four of which were Apicoplast-localised. Interestingly, three Apicoplast-localised candidates lack a canonical Apicoplast targeting signal but do contain conserved N-terminal tyrosines with likely roles in targeting. An inducible knockdown of an essential Apicoplast putative membrane transporter, PfDMT2, was only viable when supplemented with isopentenyl diphosphate. Knockdown of PfDMT2 resulted in loss of the Apicoplast, identifying PfDMT2 as a crucial Apicoplast putative membrane transporter and a candidate for therapeutic intervention.
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isolating the plasmodium falciparum Apicoplast using magnetic beads
Methods of Molecular Biology, 2018Co-Authors: Cyrille Y Botté, Geoffrey I Mcfadden, Yoshiki YamaryobotteAbstract:Plastids are key organelles in both photosynthetic and nonphotosynthetic organisms. In photosynthetic organisms, plastids can be readily purified using differential centrifugations due to the high density of photosynthetic membranes or thylakoids. The apicomplexan plastid (the Apicoplast) is an essential nonphotosynthetic plastid that lacks thylakoid and was not readily purified using conventional methods. Here, we describe a tractable method to purify intact Apicoplasts from Plasmodium falciparum blood stages using magnetic beads and affinity purification.
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Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Taher Uddin, Geoffrey I Mcfadden, Christopher D. GoodmanAbstract:Malaria parasites contain a relict plastid, the Apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the Apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the Apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from Apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonApicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported Apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have Apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the Apicoplast intact, whereas the others eventually result in Apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its Apicoplast target. Parasites treated with putative Apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the Apicoplast, which agrees with the dispensability of the Apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the Apicoplast and can be used to screen novel compounds for mode of action.
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Targeting of a Transporter to the Outer Apicoplast Membrane in the Human Malaria Parasite Plasmodium falciparum.
PloS one, 2016Co-Authors: Liting Lim, Christopher D. Goodman, Claire P. Sayers, Geoffrey I McfaddenAbstract:Apicoplasts are vestigial plastids in apicomplexan parasites like Plasmodium, the causative agent of malaria. Apicomplexan parasites are dependant on their Apicoplasts for synthesis of various molecules that they are unable to scavenge in sufficient quantity from their host, which makes Apicoplasts attractive drug targets. Proteins known as plastid phosphate translocators (pPTs) are embedded in the outer Apicoplast membrane and are responsible for the import of carbon, energy and reducing power to drive anabolic synthesis in the organelle. We investigated how a pPT is targeted into the outer Apicoplast membrane of the human malaria parasite P. falciparum. We showed that a transmembrane domain is likely to act as a recessed signal anchor to direct the protein into the endomembrane system, and that a tyrosine in the cytosolic N-terminus of the protein is essential for targeting, but one or more, as yet unidentified, factors are also essential to direct the protein into the outer Apicoplast membrane.
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endosymbiosis undone by stepwise elimination of the plastid in a parasitic dinoflagellate
Proceedings of the National Academy of Sciences of the United States of America, 2015Co-Authors: James I Macrae, Sebastian G Gornik, Andrew Cassin, Abhinay Ramaprasad, Zineb Rchiad, Malcolm J Mcconville, Antony Bacic, Geoffrey I McfaddenAbstract:Organelle gain through endosymbiosis has been integral to the origin and diversification of eukaryotes, and, once gained, plastids and mitochondria seem seldom lost. Indeed, discovery of nonphotosynthetic plastids in many eukaryotes—notably, the Apicoplast in apicomplexan parasites such as the malaria pathogen Plasmodium—highlights the essential metabolic functions performed by plastids beyond photosynthesis. Once a cell becomes reliant on these ancillary functions, organelle dependence is apparently difficult to overcome. Previous examples of endosymbiotic organelle loss (either mitochondria or plastids), which have been invoked to explain the origin of eukaryotic diversity, have subsequently been recognized as organelle reduction to cryptic forms, such as mitosomes and Apicoplasts. Integration of these ancient symbionts with their hosts has been too well developed to reverse. Here, we provide evidence that the dinoflagellate Hematodinium sp., a marine parasite of crustaceans, represents a rare case of endosymbiotic organelle loss by the elimination of the plastid. Extensive RNA and genomic sequencing data provide no evidence for a plastid organelle, but, rather, reveal a metabolic decoupling from known plastid functions that typically impede organelle loss. This independence has been achieved through retention of ancestral anabolic pathways, enzyme relocation from the plastid to the cytosol, and metabolic scavenging from the parasite’s host. Hematodinium sp. thus represents a further dimension of endosymbiosis—life after the organelle.
Boris Striepen - One of the best experts on this subject based on the ideXlab platform.
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Replication and partitioning of the Apicoplast genome of Toxoplasma gondii is linked to the cell cycle and requires DNA polymerase and gyrase.
International journal for parasitology, 2021Co-Authors: Érica S. Martins-duarte, Lilach Sheiner, Sarah B Reiff, Wanderley De Souza, Boris StriepenAbstract:Abstract 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.
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A Plastid Protein That Evolved from Ubiquitin and Is Required for Apicoplast Protein Import in Toxoplasma gondii.
mBio, 2017Co-Authors: Justin D. Fellows, Swati Agrawal, Michael J. Cipriano, Boris StriepenAbstract:Apicomplexan parasites cause a variety of important infectious diseases, including malaria, toxoplasma encephalitis, and severe diarrhea due to Cryptosporidium Most apicomplexans depend on an organelle called the Apicoplast which is derived from a red algal endosymbiont. The Apicoplast is essential for the parasite as the compartment of fatty acid, heme, and isoprenoid biosynthesis. The majority of the approximate 500 Apicoplast proteins are nucleus encoded and have to be imported across the four membranes that surround the Apicoplast. Import across the second outermost membrane of the Apicoplast, the periplastid membrane, depends on an Apicoplast-specific endoplasmic reticulum-associated protein degradation (ERAD) complex and on enzymes of the associated ubiquitination cascade. However, identification of an Apicoplast ubiquitin associated with this machinery has long been elusive. Here we identify a plastid ubiquitin-like protein (PUBL), an Apicoplast protein that is derived from a ubiquitin ancestor but that has significantly changed in its primary sequence. PUBL is distinct from known ubiquitin-like proteins, and phylogenomic analyses suggest a clade specific to apicomplexans. We demonstrate that PUBL and the AAA ATPase CDC48AP both act to translocate Apicoplast proteins across the periplastid membrane during protein import. Conditional null mutants and genetic complementation show that both proteins are critical for this process and for parasite survival. PUBL residues homologous to those that are required for ubiquitin conjugation onto target proteins are essential for this function, while those required for polyubiquitination and preprotein processing are dispensable. Our experiments provide a mechanistic understanding of the molecular machinery that drives protein import across the membranes of the Apicoplast.IMPORTANCE Apicomplexan parasites are responsible for important human diseases. There are no effective vaccines for use in humans, and drug treatment faces multiple challenges, including emerging resistance, lack of efficacy across the lifecycle, and adverse drug effects. The Apicoplast is a promising target for novel treatments: this chloroplast-like organelle is derived from an algal symbiont, is absent from the host, and is essential for parasite growth and pathogenesis. We use Toxoplasma gondii as a model to study the Apicoplast due to its strong genetic tools and established functional assays. We identify a plastid ubiquitin-like protein (PUBL) which is a novel ubiquitin-like protein and demonstrate its importance and that of the motor protein CDC48AP for Apicoplast protein import. These findings broaden our understanding of the evolution and mechanistic workings of a unique parasite organelle and may lead to new opportunities for treatments against important human pathogens.
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Lipid kinases are essential for Apicoplast homeostasis in Toxoplasma gondii.
Cellular microbiology, 2014Co-Authors: Wassim Daher, Boris Striepen, Juliette Morlon-guyot, Lilach Sheiner, Gaelle Lentini, Laurence Berry, Lina Tawk, Jean-françois Dubremetz, Kai Wengelnik, Maryse LebrunAbstract:Summary Phosphoinositides regulate numerous cellular processes by recruiting cytosolic effector proteins and acting as membrane signalling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3 kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the Apicoplast, an endosymbiotic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P-binding proteins and identified in total six PX and FYVE domain-containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2. Although depletion of putative PI(3)P-binding proteins shows that they are not essential for parasite growth and Apicoplast biology, conditional disruption of PIKfyve induces enlarged Apicoplasts, as observed upon loss of PI(3)P. A similar defect of Apicoplast homeostasis was also observed by knocking down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the Apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2. Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species that lack an Apicoplast, supporting a direct role of PI(3,5)P2 in Apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.
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An Apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins
PLoS Pathogens, 2013Co-Authors: Swati Agrawal, Giel G Van Dooren, Duk-won D Chung, Nadia Ponts, Jacques Prudhomme, Carrie F. Brooks, Elisadra M. Rodrigues, John C. Tan, Michael T. Ferdig, Boris StriepenAbstract: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.
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Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast
The Journal of biological chemistry, 2012Co-Authors: Stephanie Glaser, Geoffrey I Mcfadden, Boris Striepen, Giel G Van Dooren, Swati Agrawal, Carrie F. Brooks, Matthew K. HigginsAbstract:Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the Apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an Apicoplast-localized protein, essential for parasite survival and protein import into the Apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.
David S Roos - One of the best experts on this subject based on the ideXlab platform.
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multiple functionally redundant signals mediate targeting to the Apicoplast in the apicomplexan parasite toxoplasma gondii
Eukaryotic Cell, 2004Co-Authors: Omar S Harb, Martin Fraunholz, Bithi Chatterjee, Michael Crawford, Manami Nishi, David S RoosAbstract:Most species of the protozoan phylum Apicomplexa harbor an endosymbiotic organelle—the Apicoplast—acquired when an ancestral parasite engulfed a eukaryotic plastid-containing alga. Several hundred proteins are encoded in the parasite nucleus and are posttranslationally targeted to the Apicoplast by a distinctive bipartite signal. The N-terminal 20 to 30 amino acids of nucleus-encoded Apicoplast targeted proteins function as a classical signal sequence, mediating entry into the secretory pathway. Cleavage of the signal sequence exposes a transit peptide of variable length (50 to 200 amino acids) that is required for directing proteins to the Apicoplast. Although these peptides are enriched in basic amino acids, their structural and functional characteristics are not well understood, which hampers the identification of Apicoplast proteins that may constitute novel chemotherapeutic targets. To identify functional domains for a model Apicoplast transit peptide, we generated more than 80 deletions and mutations throughout the transit peptide of Toxoplasma gondii ferredoxin NADP+ reductase (TgFNR) and examined the ability of these altered transit peptides to mediate proper targeting and processing of a fluorescent protein reporter. These studies revealed the presence of numerous functional domains. Processing can take place at multiple sites in the protein sequence and may occur outside of the Apicoplast lumen. The TgFNR transit peptide contains at least two independent and functionally redundant targeting signals, each of which contains a subdomain that is required for release from or proper sorting within the endoplasmic reticulum. Certain deletion constructs traffic to multiple locations, including the Apicoplast periphery, the rhoptries, and the parasitophorous vacuole, suggesting a common thread for targeting to these specialized compartments.
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tropical infectious diseases metabolic maps and functions of the plasmodium falciparum Apicoplast
Nature Reviews Microbiology, 2004Co-Authors: Stuart A. Ralph, Ross F. Waller, Christopher J. Tonkin, David S Roos, Bernardo J. Foth, Giel G Van Dooren, Michael J Crawford, Martin Fraunholz, Geoffrey I McfaddenAbstract:Discovery of a relict chloroplast (the Apicoplast) in malarial parasites presented new opportunities for drug development. The Apicoplast – although no longer photosynthetic – is essential to parasites. Combining bioinformatics approaches with experimental validation in the laboratory, we have identified more than 500 proteins predicted to function in the Apicoplast. By comparison with plant chloroplasts, we have reconstructed several anabolic pathways for the parasite plastid that are fundamentally different to the analogous pathways in the human host and are potentially good targets for drug development. Products of these pathways seem to be exported from the Apicoplast and might be involved in host-cell invasion.
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Targeting and Processing of Nuclear-encoded Apicoplast Proteins in Plastid Segregation Mutants of Toxoplasma gondii
The Journal of biological chemistry, 2001Co-Authors: Boris Striepen, Charles H Pletcher, John M. Murray, David S RoosAbstract: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.
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a plastid segregation defect in the protozoan parasite toxoplasma gondii
The EMBO Journal, 2001Co-Authors: Michael K Shaw, Boris Striepen, Charles H Pletcher, Lewis G Tilney, David S RoosAbstract:Apicomplexan parasites—including the causative agents of malaria (Plasmodium sp.) and toxoplasmosis (Toxoplasma gondii)—harbor a secondary endosymbiotic plastid, acquired by lateral genetic transfer from a eukaryotic alga. The Apicoplast has attracted considerable attention, both as an evolutionary novelty and as a potential target for chemotherapy. We report a recombinant fusion (between a nuclear-encoded Apicoplast protein, the green fluorescent protein and a rhoptry protein) that targets to the Apicoplast but grossly alters its morphology, preventing organellar segregation during parasite division. Apicoplast-deficient parasites replicate normally in the first infectious cycle and can be isolated by fluorescence-activated cell sorting, but die in the subsequent host cell, confirming the ‘delayed death’ phenotype previously described pharmacologically, and validating the Apicoplast as essential for parasite viability.
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nuclear encoded proteins target to the plastid in toxoplasma gondii and plasmodium falciparum
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: Ross F. Waller, Alan F Cowman, Boris Striepen, Patrick J Keeling, Robert G K Donald, Emanuela Handman, Naomi Langunnasch, Gurdyal S Besra, David S Roos, Geoffrey I McfaddenAbstract:A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or “Apicoplast,” is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii Apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the Apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the Apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), β-ketoacyl-ACP synthase III (FabH), and β-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively Apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green fluorescent protein reporter to the Apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the Apicoplast implicates fatty acid biosynthesis as a likely function of the Apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for Apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid/bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.
Giel G Van Dooren - One of the best experts on this subject based on the ideXlab platform.
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The Dark Side of the Chloroplast: Biogenesis, Metabolism and Membrane Biology of the Apicoplast
Advances in Botanical Research, 2017Co-Authors: Giel G Van Dooren, Sanduni V. HapuarachchiAbstract:Abstract Members of the phylum Apicomplexa contain plastids, termed Apicoplasts, that were derived by secondary endosymbiosis. Unlike most of their sun-loving cousins, apicomplexans are parasites that live in the dark recesses of the animal hosts they infect. As a consequence, Apicoplasts are not photosynthetic, but nevertheless carry out essential metabolic processes. In this chapter, we examine the evolution, biogenesis and functions of the Apicoplast. In particular, we focus on the biology of the membranes that surround this organelle, which play key roles in the biogenesis of the organelle, and link the metabolic functions of the Apicoplast with the rest of the cell.
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An Apicoplast localized ubiquitylation system is required for the import of nuclear-encoded plastid proteins
PLoS Pathogens, 2013Co-Authors: Swati Agrawal, Giel G Van Dooren, Duk-won D Chung, Nadia Ponts, Jacques Prudhomme, Carrie F. Brooks, Elisadra M. Rodrigues, John C. Tan, Michael T. Ferdig, Boris StriepenAbstract: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.
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Tic22 Is an Essential Chaperone Required for Protein Import into the Apicoplast
The Journal of biological chemistry, 2012Co-Authors: Stephanie Glaser, Geoffrey I Mcfadden, Boris Striepen, Giel G Van Dooren, Swati Agrawal, Carrie F. Brooks, Matthew K. HigginsAbstract:Most plastids proteins are post-translationally imported into organelles through multisubunit translocons. The TIC and TOC complexes perform this role in the two membranes of the plant chloroplast and in the inner two membranes of the Apicoplasts of the apicomplexan parasites, Toxoplasma gondii and Plasmodium falciparum. Tic22 is a ubiquitous intermembrane translocon component that interacts with translocating proteins. Here, we demonstrate that T. gondii Tic22 is an Apicoplast-localized protein, essential for parasite survival and protein import into the Apicoplast stroma. The structure of Tic22 from P. falciparum reveals a fold conserved from cyanobacteria to plants, which displays a non-polar groove on each side of the molecule. We show that these grooves allow Tic22 to act as a chaperone. General chaperones are common components of protein translocation systems where they maintain cargo proteins in an unfolded conformation during transit. Such a chaperone had not been identified in the intermembrane space of plastids and we propose that Tic22 fulfills this role.
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The Toxoplasma Apicoplast Phosphate Translocator Links Cytosolic and Apicoplast Metabolism and Is Essential for Parasite Survival
Cell host & microbe, 2009Co-Authors: Carrie F. Brooks, Giel G Van Dooren, Hanne Risan Johnsen, Mani Muthalagi, San San Lin, Wolfgang Bohne, Karsten Fischer, Boris StriepenAbstract:Apicomplexa are unicellular eukaryotic pathogens that carry a vestigial algal endosymbiont, the Apicoplast. The physiological function of the Apicoplast and its integration into parasite metabolism remain poorly understood and at times controversial. We establish that the Toxoplasma Apicoplast membrane-localized phosphate translocator (TgAPT) is an essential metabolic link between the endosymbiont and the parasite cytoplasm. TgAPT is required for fatty acid synthesis in the Apicoplast, but this may not be its most critical function. Further analyses demonstrate that TgAPT also functions to supply the Apicoplast with carbon skeletons for additional pathways and, indirectly, with energy and reduction power. Genetic ablation of the transporter results in rapid death of parasites. The dramatic consequences of loss of its activity suggest that targeting TgAPT could be a viable strategy to identify antiparasitic compounds.
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genetic evidence that an endosymbiont derived endoplasmic reticulum associated protein degradation erad system functions in import of Apicoplast proteins
Journal of Biological Chemistry, 2009Co-Authors: Swati Agrawal, Giel G Van Dooren, Wandy L Beatty, Boris StriepenAbstract:Most apicomplexan parasites harbor a relict chloroplast, the Apicoplast, that is critical for their survival. Whereas the Apicoplast maintains a small genome, the bulk of its proteins are nuclear encoded and imported into the organelle. Several models have been proposed to explain how proteins might cross the four membranes that surround the Apicoplast; however, experimental data discriminating these models are largely missing. Here we present genetic evidence that Apicoplast protein import depends on elements derived from the ER-associated protein degradation (ERAD) system of the endosymbiont. We identified two sets of ERAD components in Toxoplasma gondii, one associated with the ER and cytoplasm and one localized to the membranes of the Apicoplast. We engineered a conditional null mutant in Apicoplast Der1, the putative pore of the Apicoplast ERAD complex, and found that loss of Der1Ap results in loss of Apicoplast protein import and subsequent death of the parasite.
Marilyn Parsons - One of the best experts on this subject based on the ideXlab platform.
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Vesicles Bearing Toxoplasma Apicoplast Membrane Proteins Persist Following Loss of the Relict Plastid or Golgi Body Disruption
PloS one, 2014Co-Authors: Anne Bouchut, Amy E. Derocher, Jennifer A. Geiger, Marilyn ParsonsAbstract: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.
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Apicoplast targeting of a toxoplasma gondii transmembrane protein requires a cytosolic tyrosine based motif
Traffic, 2012Co-Authors: Amy E. Derocher, Marilyn Parsons, Anuradha Karnataki, Pashmi VaneyAbstract:Toxoplasma gondii, like most apicomplexan parasites, possesses an essential relict chloroplast, the Apicoplast. Several Apicoplast membrane proteins lack the bipartite targeting sequences of luminal proteins. Vesicles bearing these membrane proteins are detected during Apicoplast enlargement, but the means of cargo selection remains obscure. We used a combination of deletion mutagenesis, point mutations and protein chimeras to identify a short motif prior to the first transmembrane domain of the T. gondii Apicoplast phosphate transporter 1 (APT1) that is necessary for Apicoplast trafficking. Tyrosine 16 was essential for proper localization; any substitution resulted in misdirection of APT1 to the Golgi body. Glycine 17 was also important, with significant Golgi body accumulation in the alanine mutant. Separation of at least eight amino acids from the transmembrane domain was required for full motif function. Similarly placed YG motifs are present in apicomplexan APT1 orthologs and the corresponding N-terminal domain from Plasmodium vivax was able to route T. gondiiAPT1 to the Apicoplast. Differential permeabilization showed that both the N- and C-termini of APT1 are exposed to the cytosol. We propose that this YG motif facilitates APT1 trafficking via interactions that occur on the cytosolic face of nascent vesicles destined for the Apicoplast.
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a thioredoxin family protein of the Apicoplast periphery identifies abundant candidate transport vesicles in toxoplasma gondii
Eukaryotic Cell, 2008Co-Authors: Amy E. Derocher, Anuradha Karnataki, Isabelle Coppens, Luke A Gilbert, Michael E Rome, Jean E Feagin, Peter J Bradley, Marilyn ParsonsAbstract:Toxoplasma gondii, which causes toxoplasmic encephalitis and birth defects, contains an essential chloroplast-related organelle to which proteins are trafficked via the secretory system. This organelle, the Apicoplast, is bounded by multiple membranes. In this report we identify a novel Apicoplast-associated thioredoxin family protein, ATrx1, which is predominantly soluble or peripherally associated with membranes, and which localizes primarily to the outer compartments of the organelle. As such, it represents the first protein to be identified as residing in the Apicoplast intermembrane spaces. ATrx1 lacks the Apicoplast targeting sequences typical of luminal proteins. However, sequences near the N terminus are required for proper targeting of ATrx1, which is proteolytically processed from a larger precursor to multiple smaller forms. This protein reveals a population of vesicles, hitherto unrecognized as being highly abundant in the cell, which may serve to transport proteins to the Apicoplast.