Cathepsin L occupies a vacuolar compartment and is a protein maturase within the endo/exocytic system of Toxoplasma gondii

Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.
Molecular Microbiology (Impact Factor: 4.42). 04/2010; 76(6):1340-57. DOI: 10.1111/j.1365-2958.2010.07181.x
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Regulated exocytosis allows the timely delivery of proteins and other macromolecules precisely when they are needed to fulfil their functions. The intracellular parasite Toxoplasma gondii has one of the most extensive regulated exocytic systems among all unicellular organisms, yet the basis of protein trafficking and proteolytic modification in this system is poorly understood. We demonstrate that a parasite cathepsin protease, TgCPL, occupies a newly recognized vacuolar compartment (VAC) that undergoes dynamic fragmentation during T. gondii replication. We also provide evidence that within the VAC or late endosome this protease mediates the proteolytic maturation of proproteins targeted to micronemes, regulated secretory organelles that deliver adhesive proteins to the parasite surface during cell invasion. Our findings suggest that processing of microneme precursors occurs within intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in this human parasite.

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Available from: Vern B Carruthers, Oct 07, 2015
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    • "Recent studies in T. gondii have deciphered some of the machinery involved in trafficking to apical organelles (reviewed in [5]). Early studies noted the existence of an intermediate compartment in the trafficking of micronemal proteins [6], which was subsequently shown to be an endosome-like compartment for the removal of microneme propeptides [7]. Rhoptry and microneme biogenesis in T. gondii occur from the fusion of post-Golgi vesicles, whose scission is likely regulated by a dynamin related protein (DrpB). "
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    ABSTRACT: The human malaria parasite Plasmodium falciparum possesses sophisticated systems of protein secretion to modulate host cell invasion and remodeling. In the present study, we provide insights into the function of the AP-1 complex in P. falciparum. We utilized GFP fusion constructs for live cell imaging, as well as fixed parasites in immunofluorescence analysis, to study adaptor protein mu1 (Pfμ1) mediated protein trafficking in P. falciparum. In trophozoites Pfμ1 showed similar dynamic localization to that of several Golgi/ER markers, indicating Golgi/ER localization. Treatment of transgenic parasites with Brefeldin A altered the localization of Golgi-associated Pfμ1, supporting the localization studies. Co-localization studies showed considerable overlap of Pfμ1 with the resident rhoptry proteins, rhoptry associated protein 1 (RAP1) and Cytoadherence linked asexual gene 3.1 (Clag3.1) in schizont stage. Immunoprecipitation experiments with Pfμ1 and PfRAP1 revealed an interaction, which may be mediated through an intermediate transmembrane cargo receptor. A specific role for Pfμ1 in trafficking was suggested by treatment with AlF4, which resulted in a shift to a predominantly ER-associated compartment and consequent decrease in co-localization with the Golgi marker GRASP. Together, these results suggest a role for the AP-1 complex in rhoptry protein trafficking in P. falciparum. Copyright © 2014. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 01/2015; 1853(3). DOI:10.1016/j.bbamcr.2014.12.030 · 5.02 Impact Factor
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    • "We used overexpression under the strong tubulin promoter to detect the remaining proteins (Fig. 4C and D; Supplementary Fig. S3B and C). Interestingly , TgFYVE1, TgPX1, TgPX2 and TgPX3 were found associated with the recently described vacuolar compartment (VAC), a highly dynamic compartment that houses lysosomal markers such as cathepsin-like cysteine protease (TgCPL) (Fig. 4C) (Parussini et al., 2010). TgPX3 protein partially localized to the VAC compartment but was also detected associated with other unknown subcellular structures (Fig. 4C). "
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    ABSTRACT: Phosphoinositides regulate numerous cellular processes, by recruiting cytosolic effector proteins and acting as membrane signaling 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 endosymbiontic 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 . While 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 the 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 which 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.
    Cellular Microbiology 10/2014; 17(4). DOI:10.1111/cmi.12383 · 4.92 Impact Factor
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    • "The T. gondii VAC contains classically degradative lysosomal-like proteases, such as cathepsins involved in the maturation of MICs (TgMIC2 and TgM2AP). These findings suggest that processing of microneme precursors occurs in intermediate endocytic compartments within the exocytic system, indicating an extensive convergence of the endocytic and exocytic pathways in T. gondii (Parussini et al., 2010). Light microscopy revealed another novel organelle in T. gondii with similarity to the vacuole in plant cells (Miranda et al., 2010; Francia et al., 2011). "
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    ABSTRACT: It is very difficult to define an endocytic system in Toxoplasma gondii. The parasite does not appear to take up exogenous materials via classical endocytosis. The presence of Rab5 and Rab7, classical markers of endocytic compartments, and their decoration of endomembranous structures suggest, however, that an endosomal-like system may operate. Additionally, new findings reveal that dynamin and the transmembrane type-I receptor sortilin are involved in the biogenesis of T. gondii micronemes and rhoptries, unique apical secretory organelles required for parasite migration and host-cell invasion, manipulation and egress. Evidence suggests that the parasite uses an endosomal-like system to traffic and sort proteins to rhoptries and micronemes via the endoplasmic reticulum and Golgi. In this review, I discuss recent findings suggesting that T. gondii and other apicomplexans have reduced their endosomal system and repurposed the evolutionarily conserved regulators of the system to build the apical secretory organelles. This review is also intended to serve as a resource for future investigations of apicomplexan biology and evolution.
    International journal for parasitology 11/2013; 44(2). DOI:10.1016/j.ijpara.2013.10.003 · 3.87 Impact Factor
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