scholarly journals Dually localized proteins found in both the apicoplast and mitochondrion utilize the Golgi-dependent pathway for apicoplast targeting in Toxoplasma gondii

2020 ◽  
Author(s):  
Aparna Prasad ◽  
Pragati Mastud ◽  
Swati Patankar
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Apicomplexan Parasites ◽  
Evolutionary Origins ◽  
Molecular Features ◽  
Experimental Approaches ◽  
Endosymbiotic Organelle ◽  
First Time ◽  
Dependent Pathway

ABSTRACTLike other apicomplexan parasites, Toxoplasma gondii harbours a four-membraned endosymbiotic organelle - the apicoplast. Apicoplast proteins are nuclear-encoded and trafficked to the organelle through the endoplasmic reticulum (ER). From the ER to the apicoplast, two distinct protein trafficking pathways can be used. One such pathway is the cell’s secretory pathway involving the Golgi, while the other is a unique Golgi-independent pathway. Using different experimental approaches, many apicoplast proteins have been shown to utilize the Golgi-independent pathway, while a handful of reports show that a few proteins use the Golgi-dependent pathway. This has led to an emphasis towards the unique Golgi-independent pathway when apicoplast protein trafficking is discussed in the literature. Additionally, the molecular features that drive proteins to each pathway are not known. In this report, we systematically test eight apicoplast proteins, using a C-terminal HDEL sequence to assess the role of the Golgi in their transport. We demonstrate that dually localised proteins of the apicoplast and mitochondrion (TgSOD2, TgTPx1/2 and TgACN) are trafficked through the Golgi while proteins localised exclusively to the apicoplast are trafficked independent of the Golgi. Mutants of the dually localised proteins that localised exclusively to the apicoplast also showed trafficking through the Golgi. Phylogenetic analysis of TgSOD2, TgTPx1/2 and TgACN suggested that the evolutionary origins of TgSOD2, TgTPx1/2 lie in the mitochondrion while TgACN appears to have originated from the apicoplast. Collectively, with these results, for the first time, we establish that the driver of the Golgi-dependent trafficking route to the apicoplast is the dual localisation of the protein to the apicoplast and the mitochondrion.

scholarly journals Multiple Functionally Redundant Signals Mediate Targeting to the Apicoplast in the Apicomplexan Parasite Toxoplasma gondii

2004 ◽  
Vol 3 (3) ◽  
pp. 663-674 ◽  
Author(s):  
Omar S. Harb ◽  
Bithi Chatterjee ◽  
Martin J. Fraunholz ◽  
Michael J. Crawford ◽  
Manami Nishi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Signal Sequence ◽  
Parasitophorous Vacuole ◽  
Transit Peptide ◽  
Functional Domains ◽  
Transit Peptides ◽  
Endosymbiotic Organelle

ABSTRACT 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.

scholarly journals Structural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gaëlle Lentini ◽  
Rouaa Ben Chaabene ◽  
Oscar Vadas ◽  
Chandra Ramakrishnan ◽  
Budhaditya Mukherjee ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Host Cell ◽  
Cell Invasion ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Host Cell Invasion ◽  
Host Cell Membrane ◽  
Apicomplexan Parasites ◽  
Cell Plasma ◽  
Structural Insights

AbstractActive host cell invasion by the obligate intracellular apicomplexan parasites relies on the formation of a moving junction, which connects parasite and host cell plasma membranes during entry. Invading Toxoplasma gondii tachyzoites secrete their rhoptry content and insert a complex of RON proteins on the cytoplasmic side of the host cell membrane providing an anchor to which the parasite tethers. Here we show that a rhoptry-resident kinase RON13 is a key virulence factor that plays a crucial role in host cell entry. Cryo-EM, kinase assays, phosphoproteomics and cellular analyses reveal that RON13 is a secretory pathway kinase of atypical structure that phosphorylates rhoptry proteins including the components of the RON complex. Ultimately, RON13 kinase activity controls host cell invasion by anchoring the moving junction at the parasite-host cell interface.

scholarly journals An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

2019 ◽  
Author(s):  
Pragati Mastud ◽  
Swati Patankar
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Mutational Analysis ◽  
Signal Sequence ◽  
Dual Targeting ◽  
Antioxidant Protein ◽  
N Terminus ◽  
Thioredoxin Peroxidase ◽  
Amino Acid Stretch

AbstractToxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.

scholarly journals An ambiguous N-terminus drives the dual targeting of an antioxidant protein Thioredoxin peroxidase (TgTPx1/2) to endosymbiotic organelles in Toxoplasma gondii

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e7215 ◽  
Author(s):  
Pragati Mastud ◽  
Swati Patankar
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Mutational Analysis ◽  
Signal Sequence ◽  
Dual Targeting ◽  
Antioxidant Protein ◽  
N Terminus ◽  
Thioredoxin Peroxidase ◽  
Amino Acid Stretch

Toxoplasma gondii harbors two endosymbiotic organelles: a relict plastid, the apicoplast, and a mitochondrion. The parasite expresses an antioxidant protein, thioredoxin peroxidase 1/2 (TgTPx1/2), that is dually targeted to these organelles. Nuclear-encoded proteins such as TgTPx1/2 are trafficked to the apicoplast via a secretory route through the endoplasmic reticulum (ER) and to the mitochondrion via a non-secretory pathway comprising of translocon uptake. Given the two distinct trafficking pathways for localization to the two organelles, the signals in TgTPx1/2 for this dual targeting are open areas of investigation. Here we show that the signals for apicoplast and mitochondrial trafficking lie in the N-terminal 50 amino acids of the protein and are overlapping. Interestingly, mutational analysis of the overlapping stretch shows that despite this overlap, the signals for individual organellar uptake can be easily separated. Further, deletions in the N-terminus also reveal a 10 amino acid stretch that is responsible for targeting the protein from punctate structures surrounding the apicoplast into the organelle itself. Collectively, results presented in this report suggest that an ambiguous signal sequence for organellar uptake combined with a hierarchy of recognition by the protein trafficking machinery drives the dual targeting of TgTPx1/2.

scholarly journals Protein kinase TgCDPK7 regulates vesicular trafficking and phospholipid synthesis in Toxoplasma gondii

2021 ◽  
Vol 17 (2) ◽  
pp. e1009325
Author(s):  
Priyanka Bansal ◽  
Neelam Antil ◽  
Manish Kumar ◽  
Yoshiki Yamaryo-Botté ◽  
Rahul Singh Rawat ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Trafficking ◽  
Drug Targets ◽  
Vesicular Trafficking ◽  
Parasite Development ◽  
Apicomplexan Parasites ◽  
Calcium Dependent ◽  
Causative Agents ◽  
Dependent Protein ◽  
Parasite Replication

Apicomplexan parasites are causative agents of major human diseases. Calcium Dependent Protein Kinases (CDPKs) are crucial components for the intracellular development of apicomplexan parasites and are thus considered attractive drug targets. CDPK7 is an atypical member of this family, which initial characterization suggested to be critical for intracellular development of both Apicomplexa Plasmodium falciparum and Toxoplasma gondii. However, the mechanisms via which it regulates parasite replication have remained unknown. We performed quantitative phosphoproteomics of T. gondii lacking TgCDPK7 to identify its parasitic targets. Our analysis lead to the identification of several putative TgCDPK7 substrates implicated in critical processes like phospholipid (PL) synthesis and vesicular trafficking. Strikingly, phosphorylation of TgRab11a via TgCDPK7 was critical for parasite intracellular development and protein trafficking. Lipidomic analysis combined with biochemical and cellular studies confirmed that TgCDPK7 regulates phosphatidylethanolamine (PE) levels in T. gondii. These studies provide novel insights into the regulation of these processes that are critical for parasite development by TgCDPK7.

scholarly journals Untargeted Metabolomics Uncovers the Essential Lysine Transporter in Toxoplasma gondii

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 476
Author(s):  
Joachim Kloehn ◽  
Matteo Lunghi ◽  
Emmanuel Varesio ◽  
David Dubois ◽  
Dominique Soldati-Favre
Keyword(s):  
Amino Acids ◽  
Toxoplasma Gondii ◽  
Rna Degradation ◽  
Major Facilitator Superfamily ◽  
Untargeted Metabolomics ◽  
Apicomplexan Parasites ◽  
Obligate Intracellular ◽  
Intracellular Parasites ◽  
Major Facilitator ◽  
Plasma Membrane Transporter

Apicomplexan parasites are responsible for devastating diseases, including malaria, toxoplasmosis, and cryptosporidiosis. Current treatments are limited by emerging resistance to, as well as the high cost and toxicity of existing drugs. As obligate intracellular parasites, apicomplexans rely on the uptake of many essential metabolites from their host. Toxoplasma gondii, the causative agent of toxoplasmosis, is auxotrophic for several metabolites, including sugars (e.g., myo-inositol), amino acids (e.g., tyrosine), lipidic compounds and lipid precursors (cholesterol, choline), vitamins, cofactors (thiamine) and others. To date, only few apicomplexan metabolite transporters have been characterized and assigned a substrate. Here, we set out to investigate whether untargeted metabolomics can be used to identify the substrate of an uncharacterized transporter. Based on existing genome- and proteome-wide datasets, we have identified an essential plasma membrane transporter of the major facilitator superfamily in T. gondii—previously termed TgApiAT6-1. Using an inducible system based on RNA degradation, TgApiAT6-1 was depleted, and the mutant parasite’s metabolome was compared to that of non-depleted parasites. The most significantly reduced metabolite in parasites depleted in TgApiAT6-1 was identified as the amino acid lysine, for which T. gondii is predicted to be auxotrophic. Using stable isotope-labeled amino acids, we confirmed that TgApiAT6-1 is required for efficient lysine uptake. Our findings highlight untargeted metabolomics as a powerful tool to identify the substrate of orphan transporters.

scholarly journals Toxoplasma gondii myosins B/C

2001 ◽  
Vol 155 (4) ◽  
pp. 613-624 ◽  
Author(s):  
Frédéric Delbac ◽  
Astrid Sänger ◽  
Eva M. Neuhaus ◽  
Rolf Stratmann ◽  
James W. Ajioka ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Daughter Cell ◽  
Gliding Motility ◽  
Apicomplexan Parasites ◽  
Daughter Cells ◽  
Membrane Complex ◽  
Alternatively Spliced ◽  
Butanedione Monoxime ◽  
Inner Membrane Complex

In apicomplexan parasites, actin-disrupting drugs and the inhibitor of myosin heavy chain ATPase, 2,3-butanedione monoxime, have been shown to interfere with host cell invasion by inhibiting parasite gliding motility. We report here that the actomyosin system of Toxoplasma gondii also contributes to the process of cell division by ensuring accurate budding of daughter cells. T. gondii myosins B and C are encoded by alternatively spliced mRNAs and differ only in their COOH-terminal tails. MyoB and MyoC showed distinct subcellular localizations and dissimilar solubilities, which were conferred by their tails. MyoC is the first marker selectively concentrated at the anterior and posterior polar rings of the inner membrane complex, structures that play a key role in cell shape integrity during daughter cell biogenesis. When transiently expressed, MyoB, MyoC, as well as the common motor domain lacking the tail did not distribute evenly between daughter cells, suggesting some impairment in proper segregation. Stable overexpression of MyoB caused a significant defect in parasite cell division, leading to the formation of extensive residual bodies, a substantial delay in replication, and loss of acute virulence in mice. Altogether, these observations suggest that MyoB/C products play a role in proper daughter cell budding and separation.

scholarly journals MHC genes and oxidative stress in sticklebacks: an immuno-ecological approach

2006 ◽  
Vol 273 (1592) ◽  
pp. 1407-1414 ◽  
Author(s):  
Joachim Kurtz ◽  
K. Mathias Wegner ◽  
Martin Kalbe ◽  
Thorsten B.H Reusch ◽  
Helmut Schaschl ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Susceptibility To Infection ◽  
Mhc Genes ◽  
Histocompatibility Complex ◽  
Individual Host ◽  
Experimental Approaches ◽  
Mhc Class Iib ◽  
Class Iib ◽  
First Time ◽  
And Oxidative Stress

Individual variation in the susceptibility to infection may result from the varying ability of hosts to specifically recognize different parasite strains. Alternatively, there could be individual host differences in fitness costs of immune defence. Although, these two explanations are not mutually exclusive, they have so far been treated in separate experimental approaches. To analyse potential relationships, we studied body condition and oxidative stress, which may reflect costs of immunity, in three-spined sticklebacks that had been experimentally exposed to three species of naturally occurring parasite. These sticklebacks differed in a trait, which is crucial to specific parasite defence, i.e. individual genetic diversity at major histocompatibility complex (MHC) class IIB loci. Oxidative stress was quantified as tissue acrolein, a technique that has been applied to questions of immuno-ecology for the first time. We measured gene expression at the MHC and other estimates of immune activation. We found that fish with high levels of MHC expression had poor condition and elevated oxidative stress. These results indicate that MHC-based specific immunity is connected with oxidative stress. They could, thus, also be relevant in the broader context of the evolution of sexually selected signals that are based on carotenoids and are, thus supposed to reflect oxidative stress resistance.

scholarly journals The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

2000 ◽  
Vol 151 (7) ◽  
pp. 1423-1434 ◽  
Author(s):  
Boris Striepen ◽  
Michael J. Crawford ◽  
Michael K. Shaw ◽  
Lewis G. Tilney ◽  
Frank Seeber ◽  
...  
Keyword(s):  
Cell Division ◽  
Toxoplasma Gondii ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Recombinant Expression ◽  
Molecular Genetic ◽  
Time Lapse ◽  
Microtubule Organization ◽  
Apicomplexan Parasites ◽  
Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

scholarly journals Ykt6 membrane-to-cytosol cycling regulates exosomal Wnt secretion

2018 ◽  
Author(s):  
Karen Linnemannstöns ◽  
Pradhipa Karuna M ◽  
Leonie Witte ◽  
Jeanette Clarissa Kittel ◽  
Adi Danieli ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Wnt Signaling ◽  
Signaling Pathways ◽  
Long Range ◽  
Protein Trafficking ◽  
Secretory Pathway ◽  
Multivesicular Bodies ◽  
Phosphorylation Sites ◽  
Wnt Proteins ◽  
C Terminus

Protein trafficking in the secretory pathway, for example the secretion of Wnt proteins, requires tight regulation. These ligands activate Wnt signaling pathways and are crucially involved in development and disease. Wnt is transported to the plasma membrane by its cargo receptor Evi, where Wnt/Evi complexes are endocytosed and sorted onto exosomes for long-range secretion. However, the trafficking steps within the endosomal compartment are not fully understood. The promiscuous SNARE Ykt6 folds into an auto-inhibiting conformation in the cytosol, but a portion associates with membranes by its farnesylated and palmitoylated C-terminus. Here, we demonstrate that membrane detachment of Ykt6 is essential for exosomal Wnt secretion. We identified conserved phosphorylation sites within the SNARE domain of Ykt6, which block Ykt6 cycling from the membrane to the cytosol. In Drosophila, Ykt6-RNAi mediated block of Wg secretion is rescued by wildtype but not phosphomimicking Ykt6. The latter accumulates at membranes, while wildtype Ykt6 regulates Wnt trafficking between the plasma membrane and multivesicular bodies. Taken together, we show that a regulatory switch in Ykt6 fine-tunes sorting of Wnts in endosomes.

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