Role of the parafusin orthologue, PRP1, in microneme exocytosis and cell invasion in Toxoplasma gondii

Albert Einstein College of Medicine, New York, New York, United States
Cellular Microbiology (Impact Factor: 4.92). 10/2003; 5(9):613-24. DOI: 10.1046/j.1462-5822.2003.00305.x
Source: PubMed


The association of PRP1, a Paramecium parafusin orthologue, with Toxoplasma gondii micronemes, now confirmed by immunoelectron microscopy, has here been studied in relation to exocytosis and cell invasion. PRP1 becomes labelled in vivo by inorganic 32P and is dephosphorylated when ethanol is used to stimulate Ca2+-dependent exocytosis of the micronemes. The ethanol Ca2+-stimulated exocytosis is accompanied by translocation of PRP1 and microneme content protein (MIC3) from the apical end of the parasite. Immunoblotting showed that PRP1 is redistributed inside the parasite, while microneme content is secreted. To study whether similar changes occur during cell invasion, quantitative microscopy was performed during secretion, invasion and exit (egress) from the host cell. Time-course experiments showed that fluorescence intensities of PRP1 and MIC3 immediately after invasion were reduced 10-fold compared to preinvasion levels, indicating that PRP1 translocation and microneme secretion accompanies invasion. MIC3 regained fluorescence intensity and apical distribution after 15 min, while PRP1 recovered after 1 h. Intensity of both proteins then increased throughout the parasite division period until host cell lysis, suggesting the need to secrete microneme proteins to egress. These studies suggest that PRP1 associated with the secretory vesicle scaffold serves an important role in Ca2+-regulated exocytosis and cell invasion.

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Available from: Kami Kim, Mar 06, 2014
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    • "This, however, does not preclude a role in some other phenomena accompanying exocytosis, assuming that a Paramecium cell does not de-and re-phosphorylate an important protein without any need. In fact, a role for pp63/pf in Toxoplasma gondii is reported in host cell penetration [34]. In sum, its role remained open and will be discussed here in the context of present knowledge. "
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    ABSTRACT: Ca2+ signalling governs stimulated exocytosis and exocytosis-coupled endocytosis also in Paramecium cells. Upon stimulation, the < or =10(3) dense-core exocytotic organelles (trichocysts) can be synchronously (80 ms) released, followed by endocytotic membrane resealing (350 ms) and retrieval. Paramecium is the most synchronous dense-core exocytotic system known, allowing to dissect rapidly reversible Ca2+-dependent phenomena. This holds for the reversible de-/re-phosphorylation cycle of a 63 kD phosphoprotein, pp63/parafusin (pf), which we have cloned, immuno-localised, and characterised as phosphoglucomutase, the enzyme funneling glucose into the glycolytic pathway. It was isolated ex vivo, followed by MALDI analysis, while X-ray structure analysis was performed after heterologous expression. We found multiple phosphorylation of superficial Ser/Thr residues. Although present also in exo(-) mutants, pp63/pf is selectively de-phosphorylated only in exo(+) strains during synchronous exocytosis (80 ms) and re-phosphorylated within approximately 20 s, i.e., the time required to re-establish [Ca2+] homeostasis. We have isolated relevant protein phosphatases and kinases and probed their activity on pp63/pf in vitro. We consider Ca2+/calmodulin-activated PP2B (calcineurin, whose subunits have been cloned) relevant for de-phosphorylation. Re-phosphorylation can be achieved by two protein kinases that also have been cloned. One is activated by cGMP (PKG) which in turn is formed by Ca2+-activated guanylate cyclase. Another kinase, casein kinase 2, is inhibited by Ca2+ and, hence, activated with some delay in parallel to decreasing [Ca2+] after exocytosis. In total, several Ca2+-sensitive cycles cooperate whose protein components have been localised to the cell cortex. Regulation of the phosphorylation degree of pp63/pf may affect structure binding on a microscale and/or its enzymatic activity. All this may serve fueling substrate into glycolysis with increased ATP re-formation (compromised in exo(-) mutants) and NADH formation, with effects on Ca2+ signalling including mobilisation from cortical stores (alveolar sacs) and overall effects on ATP and Ca2+ dynamics during synchronous exo- and endocytosis.
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    ABSTRACT: Trichocyst discharge in Paramecium is associated with a calcium-dependent dephosphoglucosylation of the protein parafusin (PFUS), which is believed to be a critical step in the exocytosis cascade. After trichocyst release PFUS dissociates from the empty trichocyst capsule and remains in the cytosol until a new trichocyst develops.The minute tachyzoite of the apicomplexan parasite Toxoplasma contains three types of secretory organelle, the exocytoses of two of which, the micronemes and rhoptries, are essential for invasion of host cells. The discharge of micronemes is a calcium-dependent event, and an ortholog of PFUS, named parafusin-related protein (PRP1), has been found to co-localize with antibodies to microneme contents. The distribution of PRP1 changes upon discharge of micronemes, and localized staining of PRP1 disappeared only to reappear as new micronemes were formed.These results suggest that PFUS, PRP1 and probably other PFUS orthologs have a conserved role in calcium-dependent exocytosis in alveolates.
    No preview · Article · Dec 2003 · European Journal of Protistology
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